CAR T cells targeting the ganglioside NGcGM3 control ovarian tumors in the absence of toxicity against healthy tissues

Abstract

Chimeric antigen receptor (CAR) T cells have emerged as a powerful immunotherapeutic tool against certain hematological malignancies but a significant proportion of patients either do not respond or they relapse, sometimes as a result of target antigen loss. Moreover, limited clinical benefit has been reported for CAR therapy against epithelial derived solid tumors. A major reason for this is the paucity of solid tumor antigens identified to date that are broadly, homogeneously and stably expressed but not found on healthy tissues. To address this, here we describe the development and evaluation of CAR T cells directed against N-glycoslylated ganglioside monosialic 3 (NGcGM3). NGcGM3 derives from the enzymatic hydroxylation of N-acetylneuraminic acid (NAc) GM3 (NAcGM3) and it is present on the surface of a range of cancers including ovarian, breast, melanoma and lymphoma. However, while NAcGM3 is found on healthy human cells, NGcGM3 is not due to the 7deletion of an exon in the gene encoding for the enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Indeed, unlike for most mammals, in humans NGcGM3 is considered a neoantigen as its presence on tumors is the result of metabolic incorporation from dietary sources. Here, we have generated 3 CARs comprising different single chain variable fragments (scFvs) originating from the well-characterized monoclonal antibody (mAb) 14F7. We show reactivity of the CAR T cells against a range of patient tumor fragments and we demonstrate control of NGcGM3+ SKOV3 ovarian tumors in the absence of toxicity despite the expression of CMAH and presence of NGcGM3+ on healthy tissues in NSG mice. Taken together, our data indicate clinical potential for 14F7-based CAR T cells against a range of cancers, both in terms of efficacy and of patient safety.

Chimeric antigen receptor T cells targeting the GM3(Neu5Gc) ganglioside

“…While that paper utilized CARs based on mouse/human VH/VL 14F7 derived scFv chimeras sourced from the same publication as the murine VH/VL scFv (14F7c1) used in the present study, all scFv variants showed comparable binding affinities (10). Although indications of in vitro CAR T cellmediated cytotoxicity against wild type SKOV3 cells was reported by Cribioli et al, the target-specific nature of these responses were not confirmed due to a lack of non-targeted CAR T cell controls (28). Similar to our results, no delay in tumor outgrowth was seen for wild-type human cancer cell xenografts, although the magnitude of GM3(Neu5Gc) acquisition during in vivo growth was not specifically addressed (28).…”

“…A recent study by Cribioli et al demonstrated that peritumoral injection of 14F7 CAR T cells delays the outgrowth of CMAHexpressing SKOV3 ovarian cancer xenografts injected four days earlier (28). While that paper utilized CARs based on mouse/human VH/VL 14F7 derived scFv chimeras sourced from the same publication as the murine VH/VL scFv (14F7c1) used in the present study, all scFv variants showed comparable binding affinities (10).…”

“…As L1210.Cmah -/cells cannot synthesize endogenous Neu5Gc from Neu5Ac, we explored their ability to incorporate Neu5Gc from external sources. Bovine serum used in cell culture contains millimolar amounts of Neu5Gc in fetal bovine serum (FBS) (30), and previous studies have suggested that this may serve as a source of GM3(Neu5Gc) expression in cells cultured in FBScontaining medium (26,28). In our hands, despite extensive testing, no detectable 14F7 binding (by mAb or scFv staining) or 14F7-28mz CAR T-mediated killing of L1210.Cmah -/cells was observed when cultured in medium supplemented with up to 30% heat-inactivated or non-heat-inactivated fetal bovine serum (Supplementary Figure 1B and data not shown).…”

“…While the precise mechanism for its preferential accumulation in malignant cells remains elusive, it has been suggested that cellular hypoxia might promote the acquisition of Neu5Gc, potentially driven by the upregulation of the sialic acid transport protein, sialin (SLC17A5), under hypoxic conditions (27). A recent study using 14F7-derived mouse/human VH/VL scFvs chimeras indicated that intratumoral administration of anti-GM3 (Neu5Gc) CAR T cells can delay tumor outgrowth in mCmahexpressing ovarian cancer xenografts, but not in mice challenged with wild-type cancer cells (28). In our current investigation, we designed GM3(Neu5Gc)-targeted CAR T cells based on the murine 14F7 mAb to explore their therapeutic potential of 14F7-28z CAR T cells against established tumors of both murine and human origins.…”

Coengineering specificity, safety, and function into T cells for cancer immunotherapy

“…[225][226][227] Like for TCRs, most antigens targeted by CARs are TAA rather than TSA. We recently built CAR T cells against N-glycoslylated ganglioside monosialic 3 (NGcGM3) 228 with previously described scFv derived from the 14F7 monoclonal antibody (mAb) developed at the Center of Molecular Immunology (Havana, Cuba). 229,230 NGcGM3 is present on the surface of a range of cancers including ovarian, breast, melanoma, and lymphoma as a result of metabolic incorporation from dietary sources and it is associated with tumor growth and immune suppression.…”