CAR models: next-generation CAR modifications for enhanced T-cell function

Abstract

T cells genetically targeted with a chimeric antigen receptor (CAR) to B-cell malignancies have demonstrated tremendous clinical outcomes. With the proof in principle for CAR T cells as a therapy for B-cell malignancies being established, current and future research is being focused on adapting CAR technology to other cancers, as well as enhancing its efficacy and/or safety. The modular nature of the CAR, extracellular antigen-binding domain fused to a transmembrane domain and intracellular T-cell signaling domains, allows for optimization by replacement of the various components. These modifications are creating a whole new class of therapeutic CARs. In this review, we discuss the recent major advances in CAR design and how these modifications will impact its clinical application. T cells genetically targeted with a chimeric antigen receptor (CAR) to B-cell malignancies have demonstrated tremendous clinical outcomes. With the proof in principle for CAR T cells as a therapy for B-cell malignancies being established, current and future research is being focused on adapting CAR technology to other cancers, as well as enhancing its efficacy and/or safety. The modular nature of the CAR, extracellular antigen-binding domain fused to a transmembrane domain and intracellular T-cell signaling domains, allows for optimization by replacement of the various components. These modifications are creating a whole new class of therapeutic CARs. In this review, we discuss the recent major advances in CAR design and how these modifications will impact its clinical application. T-cell-based immunotherapies have come of age as a feasible, safe, and efficacious approach to treating cancer. At the same time, the use of highly personalized, living therapeutics poses multiple challenges. Moreover, immunotherapies involving the genetic modification of T cells, such as those involving expression of chimeric antigen receptors (CAR) to modify T-cell specificity, require an additional level of optimization. In this article, we summarize our current understanding of the key aspects of CAR-T-cell design. This is not meant to be an extensive compilation of the full body of knowledge in the field, but rather an overview of the most relevant finding that have driven the field to its current stage, and those that will likely define its forthcoming directions. Antigen-specificity for a T cell is encoded by the T-cell receptor (TCR).1Sadelain M Rivière I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (401) Google Scholar T cells recognize and eradicate infected cells by TCR-mediated detection of microbial antigens, in the form of short amino acids presented by major histocompatibility complex proteins. TCR binding of a specific major histocompatibility complex and peptide combination initiates an intracellular signaling cascade that begins with phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) domains within TCR accessory proteins CD3ζ, CD3σ, CD3/, and CD3ɛ.2Abraham RT Weiss A Jurkat T cells and development of the T-cell receptor signalling paradigm.Nat Rev Immunol. 2004; 4: 301-308Crossref PubMed Scopus (382) Google Scholar,3Irving BA Weiss A The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways.Cell. 1991; 64: 891-901Abstract Full Text PDF PubMed Scopus (626) Google Scholar This signaling cascade terminates in T-cell activation and killing of the target cell. T cells can also target cancerous cells by detection of tumor antigens, which can be novel, or normally expressed only in germ cells, or mutated self-antigens (neo-epitopes).4Restifo NP Dudley ME Rosenberg SA Adoptive immunotherapy for cancer: harnessing the T cell response.Nat Rev Immunol. 2012; 12: 269-281Crossref PubMed Scopus (1213) Google Scholar Investigators have confirmed the power of tumor-reactive T cells by isolating tumor-infiltrating lymphocytes (TILs) from patients with metastatic cancer, expanding them ex vivo, and infusing the TILs back into patients.4Restifo NP Dudley ME Rosenberg SA Adoptive immunotherapy for cancer: harnessing the T cell response.Nat Rev Immunol. 2012; 12: 269-281Crossref PubMed Scopus (1213) Google Scholar,5Stevanović S Draper LM Langhan MM Campbell TE Kwong ML Wunderlich JR et al.Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells.J Clin Oncol. 2015; 33: 1543-1550Crossref PubMed Scopus (385) Google Scholar Some of these patients have achieved durable complete remission (CRs), which is unheard of with standard cytotoxic chemotherapies.6Dudley ME Yang JC Sherry R Hughes MS Royal R Kammula U et al.Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens.J Clin Oncol. 2008; 26: 5233-5239Crossref PubMed Scopus (1084) Google Scholar However, a major impediment to the adaption of TIL therapy to many cancer patients is the laborious and time-consuming (>2–3 months) process required to achieve a sufficient number of tumor-reactive T cells.7Klapper JA Thomasian AA Smith DM Gorgas GC Wunderlich JR Smith FO et al.Single-pass, closed-system rapid expansion of lymphocyte cultures for adoptive cell therapy.J Immunol Methods. 2009; 345: 90-99Crossref PubMed Scopus (39) Google Scholar CARs provided the requisite technological advance and allowed the creation of a large, bulk population of tumor-reactive T cells within as short as 1 week and mediated positive clinical outcomes in many patients with acute or chronic leukemia.8Hollyman D Stefanski J Przybylowski M Bartido S Borquez-Ojeda O Taylor C et al.Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.J Immunother. 2009; 32: 169-180Crossref PubMed Scopus (232) Google Scholar,9Kochenderfer JN Feldman SA Zhao Y Xu H Black MA Morgan RA et al.Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor.J Immunother. 2009; 32: 689-702Crossref PubMed Scopus (280) Google Scholar The CAR is a hybrid antigen receptor, part antibody and part TCR, and is composed of an extracellular antigen-binding domain and intracellular signaling domain(s) (Figure 1a).10Davila ML Sauter C Brentjens R CD19-targeted T cells for hematologic malignancies: clinical experience to date.Cancer J. 2015; 21: 470-474Crossref PubMed Scopus (24) Google Scholar Genetic retargeting of a T cell with a CAR endows a new antigen-specificity through the single-chain variable fragment (scFv), which is derived from a tumor-specific antibody.1Sadelain M Rivière I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (401) Google Scholar The scFv allows the T cell to bind a tumor antigen and T-cell activation is initiated through the intracellular domains, which are derived from CD3ζ ITAM domains.1Sadelain M Rivière I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (401) Google Scholar,3Irving BA Weiss A The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways.Cell. 1991; 64: 891-901Abstract Full Text PDF PubMed Scopus (626) Google Scholar To complete the genetic construct for the CAR, a hinge and a transmembrane domain (TM), commonly from CD8α or immunoglobulin, bridges the extracellular scFv and intracellular CD3ζ ITAM domains. Early in vitro studies demonstrated that T cells gene-targeted with CARs that have an intracellular signaling domain composed of only CD3ζ, supported T-cell activation and target killing, however, these first-generation CAR T cells had very limited persistence and antitumor efficacy in vivo.11Brocker T Karjalainen K Signals through T cell receptor-zeta chain alone are insufficient to prime resting T lymphocytes.J Exp Med. 1995; 181: 1653-1659Crossref PubMed Scopus (197) Google Scholar Since one of the main advantages to the CAR technology is its modular nature it allows continual refinement and modification to T-cell which is how first-generation CARs with CARs. The of a TCR is for only a short peptide amino from a is for to of amino J S J T cell antigen receptor of Rev Immunol. 2015; 33: PubMed Scopus Google Scholar TCR of antigen to T-cell and To this T cells require to The Rev Immunol. PubMed Scopus Google Scholar The signal is through the TCR, but the or is mediated through of by or which are normally expressed on cells J S J T cell antigen receptor of Rev Immunol. 2015; 33: PubMed Scopus Google The Rev Immunol. PubMed Scopus Google Scholar a T cell a peptide expressed on a it is to and T-cell activation is However, are as or and and can 1 and full T-cell target killing, and J S J T cell antigen receptor of Rev Immunol. 2015; 33: PubMed Scopus Google The Rev Immunol. 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