Designer T-cells and T-cell receptors for customised cancer immunotherapies

Cancer immunotherapy, focused on harnessing and empowering the immune system against tumours, has transformed modern oncology. One of the most promising avenues in development involves using genetically engineered T-cells to target cancer antigens via specific T-cell receptors (TCRs). TCRs have a naturally low affinity towards cancer-associated antigens, and therefore show scope for improvement. Here we describe approaches to procure TCRs with enhanced affinity and specificity towards cancer, using protein engineering or selection of natural TCRs from unadulterated repertoires. In particular, we discuss novel methods facilitating the targeting of tumour-specific mutations. Finally, we provide a prospective outlook on the potential development of novel, off-the-shelf immunotherapies by leveraging recent advances in genome editing

The promise of γδ T cells and the γδ T cell receptor for cancer immunotherapy

γδ T cells form an important part of adaptive immune responses against infections and malignant transformation. The molecular targets of human γδ T cell receptors (TCRs) remain largely unknown, but recent studies have confirmed the recognition of phosphorylated prenyl metabolites, lipids in complex with CD1 molecules and markers of cellular stress. All of these molecules are upregulated on various cancer types, highlighting the potential importance of the γδ T cell compartment in cancer immunosurveillance and paving the way for the use of γδ TCRs in cancer therapy. Ligand recognition by the γδ TCR often requires accessory/co-stimulatory stress molecules on both T cells and target cells; this cellular stress context therefore provides a failsafe against harmful self-reactivity. Unlike αβ T cells, γδ T cells recognise their targets irrespective of HLA haplotype and therefore offer exciting possibilities for off-the-shelf, pan-population cancer immunotherapies. Here, we present a review of known ligands of human γδ T cells and discuss the promise of harnessing these cells for cancer treatment

HLA class I-redirected anti-tumour CD4+T-cells require a higher TCR binding affinity for optimal activity than CD8+T-cells

CD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4+ T-cells occur in low frequency, express relatively low affinity T-cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leukocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T-cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the coreceptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T-cells expressing wildtype and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T-cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and, (ii) optimal TCR binding affinity is higher in CD4+ T-cells than CD8+ T-cells. These results indicate that the CD4+ T-cell component of current adoptive therapies using TCRs optimised for CD8+ T-cells is below par and that there is room for substantial improvement. This article is protected by copyright. All rights reserved

The T cell antigen receptor: the Swiss army knife of the immune

The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: ab TCR and gd TCR. The ab TCR is expressed on the majority of peripheral T cells. Most ab T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other ab T cell subsets. These ‘unconventional’ T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). gd T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for gd T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors

Reversible oligonucleotide chain blocking enables bead capture and amplification of T-Cell receptor alpha and beta chain mRNAs

Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3′-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor α and β chain mRNAs. We demonstrate that these beads can be used to capture and pair TCRα and TCRβ sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells

Macrophage-Tropic HIV Induces and Exploits Dendritic Cell Chemotaxis

Immature dendritic cells (iDCs) express the CC chemokine receptor (CCR)5, which promotes chemotaxis toward the CC chemokines regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β. By contrast, mature DCs downregulate CCR5 but upregulate CXC chemokine receptor (CXCR)4, and as a result exhibit enhanced chemotaxis toward stromal cell–derived factor (SDF)-1α. CCR5 and CXCR4 also function as coreceptors for macrophage-tropic (M-tropic) and T cell–tropic (T-tropic) human immunodeficiency virus (HIV)-1, respectively. Here, we demonstrate chemotaxis of iDCs toward M-tropic (R5) but not T-tropic (X4) HIV-1. Furthermore, preexposure to M-tropic HIV-1 or its recombinant envelope protein prevents migration toward CCR5 ligands. The migration of iDCs toward M-tropic HIV-1 may enhance formation of DC–T cell syncytia, thus promoting viral production and destruction of both DC and T helper lymphocytes. Therefore, disturbance of DC chemotaxis by HIV-1 is likely to contribute to immunosuppression in primary infection and AIDS. In addition, migration of iDCs toward HIV-1 may aid the capture of R5 HIV-1 virions by the abundant DC cell surface protein DC-specific intercellular adhesion molecule (ICAM)3-grabbing nonintegrin (DC-SIGN). HIV-1 bound to DC cell–specific DC-SIGN retains the ability to infect replication-permissive T cells in trans for several days. Consequently, recruitment of DC by HIV-1 could combine with the ability of DC-SIGN to capture and transmit the virus to T cells, and so facilitate dissemination of virus within an infected individual

Cellular-level versus receptor-level response threshold hierarchies in T-Cell activation

Peptide-MHC (pMHC) ligand engagement by T-cell receptors (TCRs) elicits a variety of cellular responses, some of which require substantially more TCR-mediated stimulation than others. This threshold hierarchy could reside at the receptor level, where different response pathways branch off at different stages of the TCR/CD3 triggering cascade, or at the cellular level, where the cumulative TCR signal registered by the T-cell is compared to different threshold values. Alternatively, dual-level thresholds could exist. In this study, we show that the cellular hypothesis provides the most parsimonious explanation consistent with data obtained from an in-depth analysis of distinct functional responses elicited in a clonal T-cell system by a spectrum of biophysically defined altered peptide ligands across a range of concentrations. Further, we derive a mathematical model that describes how ligand density, affinity, and off-rate all affect signaling in distinct ways. However, under the kinetic regime prevailing in the experiments reported here, the TCR/pMHC class I (pMHCI) dissociation rate was found to be the main governing factor. The CD8 coreceptor modulated the TCR/pMHCI interaction and altered peptide ligand potency. Collectively, these findings elucidate the relationship between TCR/pMHCI kinetics and cellular function, thereby providing an integrated mechanistic understanding of T-cell response profiles

Cytomegalovirus-mediated T cell receptor repertoire perturbation is present in early life

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus, particularly in sub-Saharan Africa, where it is endemic from infancy. The T cell response against CMV is important in keeping the virus in check, with CD8 T cells playing a major role in the control of CMV viraemia. Human leukocyte antigen (HLA) B*44:03-positive individuals raise a robust response against the NEGVKAAW (NW8) epitope, derived from the immediate-early-2 (IE-2) protein. We previously showed that the T cell receptor (TCR) repertoire raised against the NW8-HLA-B*44:03 complex was oligoclonal and characterised by superdominant clones, which were shared amongst unrelated individuals (i.e., “public”). Here, we address the question of how stable the CMV-specific TCR repertoire is over the course of infection, and whether substantial differences are evident in TCR repertoires in children, compared with adults. We present a longitudinal study of four HIV/CMV co-infected mother-child pairs, who in each case express HLA-B*44:03 and make responses to the NW8 epitope, and analyse their TCR repertoire over a period spanning more than 10 years. Using high-throughput sequencing, the paediatric CMV-specific repertoire was found to be highly diverse. In addition, paediatric repertoires were remarkably similar to adults, with public TCR responses being shared amongst children and adults alike. The CMV-specific repertoire in both adults and children displayed strong fluctuations in TCR clonality and repertoire architecture over time. Previously characterised superdominant clonotypes were readily identifiable in the children at high frequency, suggesting that the distortion of the CMV-specific repertoire is incurred as a direct result of CMV infection rather than a product of age-related “memory inflation.” Early distortion of the TCR repertoire was particularly apparent in the case of the TCR-β chain, where oligoclonality was low in children and positively correlated with age, a feature we did not observe for TCR-α. This discrepancy between TCR-α and -β chain repertoire may reflect differential contribution to NW8 recognition. Altogether, the results of the present study provide insight into the formation of the TCR repertoire in early life and pave the way to better understanding of CD8 T cell responses to CMV at the molecular level

Clonotypically similar hybrid ab T cell receptors can exhibit markedly different surface expression, antigen specificity and cross-reactivity

Emerging data indicate that particular major histocompatibility complex (MHC)-bound antigenic peptides can be recognized by identical or nearidentical ab T cell receptors (TCRs) in different individuals. To establish the functional relevance of this phenomenon, we artificially paired a and b chains from closely related TCRs specific for the human leucocyte antigen (HLA)-B*35:01-restricted HIV-1 negative regulatory factor (Nef)- derived epitope VY8 (VPLRPMTY, residues 74–81). Several hybrid TCRs generated in this manner failed to express at the cell surface, despite near homology with naturally isolated ab chain combinations. Moreover, a substantial proportion of those ab TCRs that did express lost specificity for the index VY8 peptide sequence. One such hybrid ab pair gained neo-variant specificity in the context of the VY8 backbone. Collectively, these data show that clonotypically similar TCRs can display profound differences in surface expression, antigen specificity and cross-reactivity with potential relevance for the control of mutable viruses