Dual targeting of hodgkin’s lymphoma by anti‐cd30 car‐t cells co‐transduced with an anti‐pdl1 costimulatory receptor to overcome the immunosuppressive microenvironment

Background: Classic Hodgkin’s lymphoma (cHL) has generally a good prognosis, but patients primary refractory or relapsed after autotransplant have a poor outcome. Clinical trials of CD30 CAR-T cells in relapsed/refractory (r/r) cHL showed inferior results compared to CD19 CAR-T cells in non-Hodgkin lymphomas. CD30 CAR-T cell failure in r/r cHL may be related to PDL1 overexpression by tumor cells and/or the tumor microenvironment causing T cell anergy, exhaustion and apoptosis. To overcome this issue, we propose (Figure 1A) to engineer T cells to coexpress 2nd-generation anti-CD30 CAR (CD28-based) and anti-PDL1 costimulatory chimeric receptor (CCR, 4.1BB-based) devoid of signaling domain. The CD30 CAR would preserve the MHC-independent tumor cytotoxicity while the non-cytotoxic PDL1 CCR would avoid exhaustion signals, competing with the endogenous PD-1 receptor for PD-L1 engagement on tumor and/or immunosuppressive cells, mimicking an anti-PD1 immune checkpoint inhibitor effect. Methods: Single chain fragment variable (scFv) sequences from 5 newly generated anti-CD30 mAbs were selected by surface plasmon resonance to identify the one with the highest affinity. Anti-PDL1 scFv was developed to specifically recognize the extracellular portion which interacts functionally with the cognate PD-1. Single (controls) and double targeting CAR transgenes were cloned in lentiviral vectors. Results: CD30 and PDL1 target recognition was initially optimized exploring the in vitro activity of several CD30 (CD28) and PD-L1 (4.1BB) CARs differing for spacer length (Figure 1B). Untransduced T cells were included as controls together with single-targeting PDL1 CCR to confirm the inability of the PDL1 CCR to induce T cell effector functions per se. We next generated a bicistronic dual-targeting construct with the best spacers (CH3 for CD30 CAR and CD8 for PDL1 CCR) to be compared (Figure 1C) with single-targeting CD30 CAR in long term assays at unfavourable E:T ratio (0.25:1). After 96 h of coculture with the CD30+/PDL1+ HD-LM2 cHL cell line, dual CD30.CAR/PDL1.CCR T cells showed higher cytotoxicity (mean 85.5% vs. 60.2%), enhanced T cell proliferation (mean 66.8% vs. 43%), reduced differentiated T cell memory phenotype (mean central memory population 29.2% vs. 19.1%) and less PD1 expression (mean 22.2% vs. 30,3%), as compared to single CD30.CAR T cells. The research was funded by: Italian Lymphoma Foundation (Bando Giovani Ricercatori 2021 to VMP) Keywords: cellular therapies, Hodgkin lymphoma, targeting the tumor microenvironment No conflicts of interests pertinent to the abstract.