Prospects for gene-engineered T cell immunotherapy for solid cancers

Abstract

Adoptive transfer of receptor-engineered T cells has produced impressive results in treating patients with B cell leukemias and lymphomas. This success has captured public imagination and driven academic and industrial researchers to develop similar 'off-the-shelf' receptors targeting shared antigens on epithelial cancers, the leading cause of cancer-related deaths. However, the successful treatment of large numbers of people with solid cancers using this strategy is unlikely to be straightforward. Receptor-engineered T cells have the potential to cause lethal toxicity from on-target recognition of normal tissues, and there is a paucity of truly tumor-specific antigens shared across tumor types. Here we offer our perspective on how expanding the use of genetically redirected T cells to treat the majority of patients with solid cancers will require major technical, manufacturing and regulatory innovations centered around the development of autologous gene therapies targeting private somatic mutations.

Figure 1

CAR and TCR clinical trials for oncology indications in the US between 1994 and 2014. (a) The number of new CAR (n = 101) and TCR (n = 35) clinical trials for hematologic and solid cancer indications submitted to the US Recombinant DNA Advisory Committee (RAC) as a function of time between 1994 and 2014. (b) Target antigens for CAR and TCR trials submitted to the RAC between 1994 and 2014. The shaded red area represents gene-engineered antigen receptor trials for hematologic cancers, the non-shaded area for solid cancers. Cancer-germline antigens include NY-ESO-1 and MAGE-A3, among others; pigment antigens include MART-1, gp100 and tyrosinase complex. ERRB2, HER-2/neu; CEA, carcinoembryonic antigen; IL-13Ra2, interleukin-13 receptor α2; PSMA, prostate-specific membrane antigen; TAG-72, tumor-associated glycoprotein 72; c-Met, MET proto-oncogene, receptor tyrosine kinase; EGFRvIII, epidermal growth factor receptor variant III; P53, tumor protein p53; MUC1, mucin 1, cell surface associated; L1-CAM, neural cell adhesion molecule L1; 2G1, a non-MHC-restricted TCR recognizing a TRAIL/DR4 complex; VEGFR2, vascular endothelial growth factor receptor 2; HPV-16 E6, human papillomavirus-16 E6 oncoprotein. Data is from the Genetic Modification Clinical Research Information System (http://www.gemcris.od.nih.gov).

Figure 2

Safety and tissue-selectivity mechanisms that may be inserted into gene-engineered T cells. (a) Co-expression of herpes simplex virus-thymidine kinase (HSV-TK) in antigen receptor-modified T cells. Following administration of the anti-viral medication ganciclovir (GCV), HSV-TK catalyzes the generation of GCV-monophosphate (MP) which is subsequently converted to GCV-trisphosphate (TP) by enzymes present in all mammalian cells. GCV-triphosphate inhibits DNA chain elongation in proliferating cells, resulting in lethal toxicity. (b) Co-expression of an inducible caspase-9 (iCasp9) construct in antigen receptor–modified T cells. Administration of a small molecule dimerizer induces dimerization and activation of iCasp9 that subsequently triggers executioner caspases-3, −6 and −7, resulting in apoptosis. (c) Co-expression of a truncated variant of human EGFR (tEGFR) in receptor-modified T cells. Infusion of the EGFR-specific mAb cetuximab results in antibody-dependent cellular cytotoxicity of tEGFR⁺ T cells once the antibody’s Fc domain is engaged by Fc gamma receptors (FcγR) on the surface of immune effector cells. Some reports also suggest that cetuximab might deplete tEGFR⁺ cells though complement fixation, but this mechanism of action remains controversial. MAC, membrane attack complex. (d) RNA electroporation of antigen receptors into T cells. Because of the short half-life of RNA species, receptor expression is self-limited after cell transfer, thereby restricting the potential for uncontrolled on-target but off-tumor toxicities. (e) CARs engineered to deliver a dominant antigen-specific inhibitory signal, termed inhibitory CARs (iCARs), work by attaching the signaling domains of co-inhibitory receptors to an antibody-binding region that recognizes structures on the surface of normal tissues. (f) CARs can be ‘logic-gated’ by co-transducing with two separate CARs: one that provides suboptimal activation when stimulated alone and a second that recognizes a separate antigen, which provides a co-stimulatory signal. Ligation of either receptor alone is insufficient to trigger T-cell activation whereas co-engagement allows T cells to proliferate, acquire effector functions, and exhibit on-target immunity only against tissues expressing both antigens. (g) CARs can be engineered with an ‘on-switch’, whereby the antigen-binding and cytosolic-signaling domains of the receptor are divided into distinct modules. Administration of a small-molecule dimerizer induces heterodimerization of these modules, initiating cellular activation only when both cognate antigen and the small molecule are present. Thus, the duration and intensity of receptor-engineered T cells can be controlled. (h) CARs can be engineered with a ‘masked’ receptor in which the antigen-binding domain is sterically blocked by a peptide mask attached to the receptor by a protease-cleavable linker. Entry of modified cells into tissues enriched in proteases, such as the tumor microenvironment, can cleave the blocking peptide and unmask the binding capacity of the CAR.

Figure 3

A pathway for generating autologous TCR gene therapies targeting neoantigens for patients with advanced epithelial cancers. From a single blood draw, all of the requisite components required to produce this therapy can be procured. Circulating tumor cells (CTCs) can be enriched from the blood using a combination of antibody-mediated isolation based on epithelial marker expression (for example, EpCAM) followed by microfluidic isolation. Subsequent genomic extraction, amplification and whole exome sequencing can identify non-synonymous mutations present within the tumor. Circulating T cells that express PD-1 can be isolated and co-cultured with autologous professional antigen presenting cells (APCs) that have either been pulsed with synthetic long peptides harboring the amino acid change resulting from the mutation or RNA-electroporated with tandem minigenes (TMGs) encoding the amino acid change and flanked on either side by 12 amino acids from the endogenous protein. T cells that upregulate the activation markers 4–1BB and/or OX-40 can then be isolated and the α and β chains of the TCR associated with this cell can be sequenced. The α/β TCR that confers reactivity against a neoantigen can then be cloned into an expression vector, for example an integrating virus or the Sleeping Beauty (SB) transposon/transposase system. T cell subsets isolated from the peripheral blood of the patient can finally be modified with this expression vector, expanded in vitro to numbers sufficient for treatment, and re-infused back into the patient. TL, transmitted light; T_N, T naive; T_SCM, T stem cell memory; T_CM, T central memory; SB11, SB transposase 11.