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, 70^VEGF and Meso^VEGF respectively).

Results 70^VEGF and Meso^VEGF 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). 70^VEGF and Meso^VEGF 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. 70^VEGF and Meso^VEGF 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 (70^19,VEGFKO) nor exogenous administration of anti-VEGF scFv alone were sufficient to phenocopy the proliferative advantage of 70^VEGF when co-cultured with tumor cells; however, when exogenous anti-VEGF scFv was added to 70^19,VEGFKO CAR-T cells the performance was similar to 70^VEGF, arguing that a combination of intracrine and exogenous VEGF modulate CAR-T cell function. Nanostring gene expression profiling revealed distinct expression programs between Meso^VEGF 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

References

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.