Abstract Cell therapy using T cell receptors (TCRs) and chimeric antigen receptors (CARs) represents a new wave of immunotherapies garnering considerable attention and investment. Further progress in this area of medicine depends in part on improving the functional capabilities of the engineered components, while maintaining the overall size of recombinant constructs to ensure their compatibility with existing gene delivery vehicles. We describe a single-variable-domain TCR (svd TCR) that utilizes only the variable domain of the β chain (Vβ). This Vβ module not only works in TCR and CAR formats, but also can be used to create single-chain bispecific CARs and TCRs. Comparison of individual ligand-binding Vβ domains in different formats suggests that the lone Vβ sequence controls the sensitivity and a major part of the specificity of the CAR or TCR construct, regardless of signaling format, in Jurkat and primary T cells.
We describe an approach to cancer therapy based on exploitation of common losses of genetic material in tumor cells (loss of heterozygosity) (Basilion et al., 1999; Beroukhim et al., 2010). This therapeutic concept addresses the fundamental problem of discrimination between tumor and normal cells and can be applied in principle to the large majority of tumors. It utilizes modular activator/blocker elements that integrate signals related to the presence and absence of ligands displayed on the cell surface (Fedorov et al., 2013). We show that the targeting system works robustly in vitro and in a mouse cancer model where absence of the HLA-A*02 allele releases a brake on engineered T cells activated by the CD19 surface antigen. This therapeutic approach potentially opens a route toward a large, new source of cancer targets.
Claudin18.2 (CLDN18.2) is an attractive target for the treatment of gastric and gastroesophageal junction cancers (GC/GEJC). CLDN18.2, a tight-junction protein in gastric cells, is exposed during malignant transformation. Recent third-party Phase I clinical trials have reported efficacy of CLDN18.2 CAR T cells against GC/GEJC but improving CAR persistence in the tumor microenvironment (TME) remains a challenge. Here we engineered a bispecific CAR, IMPT-601, that combines both CLDN18.2-specific targeting to bind tumor cells and TGF-β-specific targeting to convert the inhibitory TGF-β signal into an activation signal by CAR T cells in the TME.
Methods
CD62L+ T cells were isolated from donor leukapheresis and transduced with lentivirus to manufacture CAR T cells. IMPT-601 was evaluated using 2D cytotoxic T lymphocyte (CTL) assays, 3D tumor spheroid CTL assays, and repeated antigen challenges. In vivo efficacy and preliminary toxicity were assessed in MHC I/II double knock-out (DKO) NSG implanted with CLDN18.2 tumor cells.
Results
IMPT-601 was potent in CTL assays against CLDN18.2-expressing cell lines. No off-target toxicity was observed after co-culture with CLDN18.1-expressing cell lines. When tested in long-term repeated antigen challenges (RACs) with tumor cells that express CLDN18.2 and secrete TGF-β, IMPT-601 exhibited persistent and robust eradication of tumor cells while retaining T-cell fitness, with a marked reduction in exhaustion markers and regulatory T cells (Tregs) compared to single-input CLDN18.2 CAR T cells. To test efficacy in a 3D spheroid model, IMPT-601 cells were co-cultured with tumor cells endogenously expressing both CLDN18.2 and TGF-β. IMPT-601 displayed complete clearance of spheroids, lower exhaustion markers, and a reduction in Tregs. IMPT-601, which cross-reacts with human and murine CLDN18.2 and human and murine TGF-β, effectively cleared CLDN18.2+ and TGF-β–secreting gastric tumors in MHC I/II DKO NSG mice. Animals treated with IMPT-601 showed higher numbers of lymphocytes in the tumor compared to the benchmark single-input CLDN18.2 CAR T cells. Additionally, animals treated with IMPT-601 showed a reduction in the percentage of Tregs in the tumor tissue compared to benchmark single-input CLDN18.2 CAR T cells. Animals treated with IMPT-601 maintained stable body weights throughout the study, suggesting IMPT-601 is well tolerated in these animals.
Conclusions
IMPT-601 is a potentially promising CAR T cell therapy candidate for the treatment of GC/GEJC by enabling the conversion of the immunosuppressive TGF-β into an activation signal, potentiating CAR T cells to accumulate in the immunosuppressive TME while retaining T cell fitness and reducing the frequency of regulatory T cells by dual targeting CLDN18.2 and TGF-β.
Cell therapy, with all its promise as a powerful solid-tumor modality, is still hampered by the fundamental obstacle of cancer therapy: the acute shortage of truly tumor-specific targets. It is well known that an average tumor contains loss of heterozygosity (LOH) at an astonishing frequency: ~20% genome wide. These losses are irreversible and absolutely distinguish the cancer from normal cells.
Methods
We describe a novel approach to cancer immunotherapy that draws on LOH as a large, so far untapped source of cancer targets. To exploit such allelic losses, we focus on polymorphic loci and target the remaining allelic product of a locus that has LOH. We engineer T cells with a modular signal-integration circuit designed to be activated only by tumor cells that have lost expression of one specific allele on their surface.
Results
We use the HLA locus which undergoes LOH at a frequency of 13%, and the HLA-A*02 allele specifically, as proof of concept. We present a large body of quantitative in vitro data, along with in vivo data, that support the use of a synthetic signal-integration circuit called Tmod as a cancer therapy. We also describe Tmod's mechanistic properties, including thorough structure/function analysis of its components.
Conclusions
LOH is a rich source of new targets, provided a system of sufficient power can be devised to exploit them. Our Tmod signal integration system confers on engineered T cells the capacity to discriminate effectively between normal and tumor cells that contain specific allelic losses.
Ethics Approval
The animal study was approved by Explora BioLabs' Ethics Board, protocol number EB17-010-059
Overcoming the suppressive tumor microenvironment (TME) remains an important unmet challenge for chimeric antigen receptor (CAR) T cell therapies. A key suppressive factor, transforming growth factor β (TGF-β), is a primary driver of T cell suppression reducing T cell receptor (TCR)-mediated cytotoxicity and driving the differentiation of immunosuppressive regulatory T cells (Tregs) in the TME.
Methods
To overcome TGF-β-specific immunosuppression, we employed a TGF-β-targeting CAR co-expressed in T cells with a human papillomavirus type 16 (HPV16) E711-19-specific, HLA-A*02:01-restricted TCR (E7 TCR).
Results
In comparison to T cells expressing E7 TCR alone or E7 TCR with an irrelevant CD19-targeting CAR, T cells co-transduced with E7 TCR and TGF-β CAR showed enhanced proliferation and cytokine production, while maintained cytotoxicity throughout repeat antigen challenge assays with HPV16+ Ca Ski tumor cells. The inclusion of TGF-β CAR also reduced PD1+ expression and Treg differentiation after repeat antigen challenges. Transcriptional analysis further confirmed reduced FOXP3 expression as well as enhanced proinflammatory genes such as TNF and IFNG.
Conclusions
In combination, these data clearly show that a TGF-β CAR can enhance TCR function and limit Treg differentiation and is therefore likely to improve the function and persistence of TCR therapies in the TME.
