Engineered T cells: the promise and challenges of cancer immunotherapy

Abstract

The immune system evolved to distinguish non-self from self to protect the organism. As cancer is derived from our own cells, immune responses to dysregulated cell growth present a unique challenge. This is compounded by mechanisms of immune evasion and immunosuppression that develop in the tumour microenvironment. The modern genetic toolbox enables the adoptive transfer of engineered T cells to create enhanced anticancer immune functions where natural cancer-specific immune responses have failed. Genetically engineered T cells, so-called 'living drugs', represent a new paradigm in anticancer therapy. Recent clinical trials using T cells engineered to express chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) have produced stunning results in patients with relapsed or refractory haematological malignancies. In this Review we describe some of the most recent and promising advances in engineered T cell therapy with a particular emphasis on what the next generation of T cell therapy is likely to entail.

Figure 1

Comparing basic structure of engineered T cell receptors and chimeric antigen receptors. Endogenous T cell receptors include paired alpha and beta chains associated with delta, epsilon, gamma, and signaling zeta chains. Most transgenic engineered T cell receptors also rely on recruitment of endogenous downstream signaling molecules such as LAT and ZAP70 to transduce the activation signal. Both endogenous and transgenic T cell receptors see intracellularly processed antigens that must be presented in the context of the Major Histocompatibility Complex and require costimulatory signals (not shown) for complete T cell activation. Chimeric antigen receptors, on the other hand, lack alpha and beta chains. The extracellular portion of a chimeric antigen receptor consists of single chain variable fragments derived from antibody heavy and light chain variable domains. Typically these are then fused to a transmembrane domain, an intracellular costimulatory domain and an intracellular zeta chain domain. Again, chimeric antigen receptors must recruit endogenous downstream signaling molecules to transduce activating signal, but costimulation is provided in cis and in response to the same activating signal. Chimeric antigen receptors see surface antigens independent of the MHC and are therefore not tissue type restricted.

Figure 2. CAR Design and Evolution

CARs target surface antigens in an MHC-independent fashion and consist of an extracellular binding domain, hinge domain, transmembrane domain, and intracellular signaling domains. The first clinical trials tested CARs that had a binding domain composed of native CD4 that bound to gp120 on HIV-infected cells^,, with a single signaling domain composed of the CD3ζ chain^–. CAR’s with an extracellular domain composed of antibody single chain fragment variable portions were first reported by Kuwana and later Eshhar and colleagues^,. Second generation CAR’s incorporating CD28 as a costimulatory domain were first developed by Roberts (US Patent 5,686,281) and reported by Finney, and those incorporating 4-1BB as a costimulatory domain by Finney^, Imai, and then others^,. CAR’s incorporating 3 or 4 signaling domains, so called “third and fourth generation”, have also been developed and are beginning clinical trials^,,.

Figure 3. Engineered T Cell Manufacturing

Leukocytes are generally collected by leukapheresis (1) and lymphocytes can be enriched (2) by counterflow centrifugal elutriation or subsets selected (not shown). The enriched lymphocytes are placed in to culture and (3) stimulated with bead-based artificial antigen presenting cells^, and viral vector (4) added. The culture is expanded in a bioreactor for several days (5) and then the T cell bulk product (6) is washed and concentrated, samples removed for quality control release testing (7) and quality assurance review. The final formulation is cryopreserved (8), allowing facile shipment to distant infusion sites, where the final product bag (9) is thawed and infused. Manufacturing time is generally 5 to 10 days, and collection to infusion times can range from 2 to 4 weeks depending on patient clinical status and chemotherapy conditioning regimens.

Figure 4. New CAR Models and Concepts [Au; please also expand the examples here to non-CAR T cells.]

A) T cells redirected for universal cytokine killing (TRUCKs) co-express a CAR and an anti-tumor cytokine. Cytokine expression may be constitutive or induced by T cell activation (eg. IL-12). Targeted by CAR specificity, localized production of pro-inflammatory cytokines recruit endogenous immune cells to tumor sites. B) Universal CAR T cells are engineered to no longer express endogenous TCR and/or HLA molecules preventing GVHD or rejection respectively in the allogeneic setting. C) Self driving CARs co-express a CAR and a chemokine receptor, which binds a tumor ligand (eg. CCR2b-CCL2), thereby enhancing tumor homing. D) CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express a variety of checkpoint molecules (eg. CTLA4, PD1), with a checkpoint switch receptor, or may be administered with a monoclonal antibody checkpoint blockade. E) A self-destructing CAR may be designed by using RNA delivered by electroporation to encode the CAR^,. Alternatively, inducible apoptosis of T cell as shown in the right hand section of panel G may be achieved based on ganciclovir binding to thymidine kinase in gene modified lymphocytes or the more recently described system of activation of human caspase 9 by a small molecule dimerizer^,. F) A Conditional CAR T cell is by default in the “off” position, until the addition of a small molecule to complete the circuit turning the CAR to the “on” position^,. Alternatively, a receptor may be delivered to a T cell that serves as an adaptor to subsequently administered secondary antibodies directed at target antigen. G) Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects. H) A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked scFvs that have different affinities fused to intracellular costimulatory domain(s) and a CD3ζ domain. TanCAR T cell activation is achieved only when target cells co-express both targets. I) A dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3ζ domain and the other CAR includes only the costimulatory domain(s). Dual CAR T cell activation requires co-expression of targets on tumor. J) A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain (eg. CTLA4 or PD1). iCAR T cells co-expressing a standard CAR become activated only when encountering targets cells that possess the standard CAR target but lack the iCAR target.

Figure 4. New CAR Models and Concepts [Au; please also expand the examples here to non-CAR T cells.]

A) T cells redirected for universal cytokine killing (TRUCKs) co-express a CAR and an anti-tumor cytokine. Cytokine expression may be constitutive or induced by T cell activation (eg. IL-12). Targeted by CAR specificity, localized production of pro-inflammatory cytokines recruit endogenous immune cells to tumor sites. B) Universal CAR T cells are engineered to no longer express endogenous TCR and/or HLA molecules preventing GVHD or rejection respectively in the allogeneic setting. C) Self driving CARs co-express a CAR and a chemokine receptor, which binds a tumor ligand (eg. CCR2b-CCL2), thereby enhancing tumor homing. D) CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express a variety of checkpoint molecules (eg. CTLA4, PD1), with a checkpoint switch receptor, or may be administered with a monoclonal antibody checkpoint blockade. E) A self-destructing CAR may be designed by using RNA delivered by electroporation to encode the CAR^,. Alternatively, inducible apoptosis of T cell as shown in the right hand section of panel G may be achieved based on ganciclovir binding to thymidine kinase in gene modified lymphocytes or the more recently described system of activation of human caspase 9 by a small molecule dimerizer^,. F) A Conditional CAR T cell is by default in the “off” position, until the addition of a small molecule to complete the circuit turning the CAR to the “on” position^,. Alternatively, a receptor may be delivered to a T cell that serves as an adaptor to subsequently administered secondary antibodies directed at target antigen. G) Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects. H) A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked scFvs that have different affinities fused to intracellular costimulatory domain(s) and a CD3ζ domain. TanCAR T cell activation is achieved only when target cells co-express both targets. I) A dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3ζ domain and the other CAR includes only the costimulatory domain(s). Dual CAR T cell activation requires co-expression of targets on tumor. J) A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain (eg. CTLA4 or PD1). iCAR T cells co-expressing a standard CAR become activated only when encountering targets cells that possess the standard CAR target but lack the iCAR target.