Competition-Based Cell Assay Employing Soluble T Cell Receptors to Assess MHC Class II Antigen Processing and Presentation.

Accurate assessment of antigen-specific immune responses is critical in the development of safe and efficacious biotherapeutics and vaccines. Endosomal processing of a protein antigen followed by presentation on major histocompatibility complex (MHC) class II constitute necessary steps in the induction of CD4+ T cell immune responses. Current preclinical methods for assessing immunogenicity risk consist of in vitro cell-based assays and computational prediction tools. Cell-based assays are time and labor-intensive while in silico methodologies have limitations. Here, we propose a novel cell-based assay capable of investigating an antigen's endosomal processing and MHC class II presentation capabilities. This novel assay relies on competition between epitopes for MHC class II binding and employs labeled soluble T cell receptors (sTCRs) as detectors of epitope presentation.

Transplantation of T-cell receptor α/ß-depleted allogeneic bone marrow in nonhuman primates.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models. Here we evaluated the outcomes of major histocompatibility complex-matched T-cell receptor α/ß-depleted alloHSCT in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/ß depletion, bone marrow cells were transplanted into major histocompatibility complex-identical MCMs conditioned with total body irradiation. GvHD prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Posttransplant chimerism was determined by sequencing diagnostic single-nucleotide polymorphisms to quantify the amounts of donor and recipient cells present in blood. Animals treated posttransplant with sirolimus developed nearly complete chimerism with acute GvHD. In the cyclophosphamide and tacrolimus treatment group, animals developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. None of the animals developed cytomegalovirus infection. These studies indicate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. Further exploration of this model will provide a platform for elucidating the mechanisms of GvHD and GvHE and for testing novel alloHSCT modalities for HIV infection.

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cells have been recently developed and are producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo 89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET). RESULTS: The 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70%-79%, and cell radiolabeling activity was 98.1-103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Cell proliferation was not significantly different during the early period after radiolabeling, compared with unlabeled cells; however, the proliferative capacity decreased over time (day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance imaging in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by one hour after injection. The cells gradually migrated from the lung to the liver and spleen by day 1, and remained stable in these sites until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed a similar distribution, compared with Jurkat/CAR T-cells, on serial PET images. CAR T cell distribution was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry. CONCLUSION: Real-time in vivo cell trafficking is feasible using PET imaging of 89Zr-DFO-labeled CAR T-cells. This can be used to investigate cellular kinetics, initial in vivo biodistribution, and safety profiles in future CAR T-cell development.

Long-term surviving cancer patients as a source of therapeutic TCR.

We have established a platform for the isolation of tumour-specific TCR from T cells of patients who experienced clinical benefit from cancer vaccination. In this review we will present the rationale behind this strategy and discuss the advantages of working with "natural" wild type TCRs. Indeed, the general trend in the field has been to use various modifications to enhance the affinity of such therapeutic TCRs. This was done to obtain stronger T cell responses, often at the cost of safety. We further describe antigen targets and recent in vitro and in vivo results obtained to validate them. We finally discuss the use of MHC class II-restricted TCR in immunotherapy. Typically cellular anti-tumour immune responses have been attributed to CD8 T cells; however, we isolated mainly CD4 T cells. Importantly, these MHC class II-restricted TCRs have the potential to induce broad, long lasting immune responses that enable cancer control. The use of CD4 T cell-derived TCRs for adoptive immunotherapy has so far been limited and we will here discuss their therapeutic potential.

Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion.

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

Isolation and characterization of NY-ESO-1-specific T cell receptors restricted on various MHC molecules.

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

A humanized TCR retaining authentic specificity and affinity conferred potent anti-tumour cytotoxicity.

The affinity of T-cell receptor (TCR) determines the efficacy of TCR-based immunotherapy. By using human leucocyte antigen (HLA)-A*02 transgenic mice, a TCR was generated previously specific for human tumour testis antigen peptide MAGE-A3112-120 (KVAELVHFL) HLA-A*02 complex. We developed an approach to humanize the murine TCR by replacing the mouse framework with sequences of folding optimized human TCR variable domains for retaining binding affinity. The resultant humanized TCR exhibited higher affinity and conferred better anti-tumour activity than its parent murine MAGE-A3 TCR (SRm1). In addition, the affinity of humanized TCR was enhanced further to achieve improved T-cell activation. Our studies demonstrated that the human TCR variable domain frameworks could provide support for complementarity-determining regions from a murine TCR, and retain the original binding activity. It could be used as a generic approach of TCR humanization.

Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system.

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

New Strategies for the Treatment of Solid Tumors with CAR-T Cells.

Recent years, we have witnessed significant progresses in both basic and clinical studies regarding novel therapeutic strategies with genetically engineered T cells. Modification with chimeric antigen receptors (CARs) endows T cells with tumor specific cytotoxicity and thus induce anti-tumor immunity against malignancies. However, targeting solid tumors is more challenging than targeting B-cell malignancies with CAR-T cells because of the histopathological structure features, specific antigens shortage and strong immunosuppressive environment of solid tumors. Meanwhile, the on-target/off-tumor toxicity caused by relative expression of target on normal tissues is another issue that should be reckoned. Optimization of the design of CAR vectors, exploration of new targets, addition of safe switches and combination with other treatments bring new vitality to the CAR-T cell based immunotherapy against solid tumors. In this review, we focus on the major obstacles limiting the application of CAR-T cell therapy toward solid tumors and summarize the measures to refine this new cancer therapeutic modality.

Antigen Selection for Enhanced Affinity T-Cell Receptor-Based Cancer Therapies.

Evidence of adaptive immune responses in the prevention of cancer has been accumulating for decades. Spontaneous T-cell responses occur in multiple indications, bringing the study of de novo expressed cancer antigens to the fore and highlighting their potential as targets for cancer immunotherapy. Circumventing the immune-suppressive mechanisms that maintain tumor tolerance and driving an antitumor cytotoxic T-cell response in cancer patients may eradicate the tumor or block disease progression. Multiple strategies are being pursued to harness the cytotoxic potential of T cells clinically. Highly promising results are now emerging. The focus of this review is the target discovery process for cancer immune therapeutics based on affinity-matured T-cell receptors (TCRs). Target cancer antigens in the context of adoptive cell transfer technologies and soluble biologic agents are discussed. To appreciate the impact of TCR-based technology and understand the TCR discovery process, it is necessary to understand key differences between TCR-based therapy and other immunotherapy approaches. The review first summarizes key advances in the cancer immunotherapy field and then discusses the opportunities that TCR technology provides. The nature and breadth of molecular targets that are tractable to this approach are discussed, together with the challenges associated with finding them.

NKG2D CARs as cell therapy for cancer.

The NKG2D cell receptor and its ligands have attracted considerable interest as a potential strategy to attack tumor cells. NKG2D ligands are expressed on most types of tumors, and they demonstrate relative selectivity of ligand expression on tumor cells compared to healthy cells. Several different variants of NKG2D-based chimeric antigen receptors (CARs) have been developed, and extensive in vivo mechanistic studies performed demonstrated that cytotoxicity and cytokines are important for the efficacy NKG2D CAR adoptive T-cell therapy. NKG2D CARs target tumor cells, and they also target immunosuppressive cells within the tumor microenvironment. Under certain conditions, NKG2D ligand expression can be found on nontumor tissue, so potential off-tumor toxicity remains. In this article, we review the use of NKG2D as a basis for CAR targeting of tumors.

Novel approaches to enhance the specificity and safety of engineered T cells.

T-cell therapies using engineered T cells show great promise for cancer immunotherapy, as illustrated by the CD19 paradigm. Much of the excitement about this approach, and second-generation CARs in particular, is due to the dramatic clinical results recently reported by a few centers, especially in acute lymphoblastic leukemia, and the applicability of this approach, in principle, to a wide range of cancers. Extending the use of CAR therapies to cancers other than B-cell malignancies will require selective tumor targeting with minimal or acceptable "on-target, off-tumor" effects. The identification of new CAR target antigens is thus one of the next big challenges to address. Recognizing the paucity of currently available tumor-specific targets, we have developed broadly applicable approaches to enhance the tumor selectivity and safety of engineered T cells. Here, we review 2 promising concepts. One is to improve tumor targeting based on combinatorial antigen recognition. The other uses receptors that provide antigen-specific inhibition, which we named iCARs, to divert T cells from the normal tissues one wants to protect.

MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.

Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+) T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.

Recognition of a natural WT1 epitope by a modified WT1 peptide-specific T-cell receptor.

Wilms' tumor gene WT1 is highly expressed in leukemia and in various types of solid tumors and exerts an oncogenic function. Thus, WT1 protein is a most promising tumor-associated antigen. We have been successfully performing WT1 vaccination with a 9-mer modified WT1(235) peptide, which has one amino acid substitution (M→Y) at position 2 of 9-mer natural WT1(235) peptide (235-243 a.a.), for close to 700 HLA-A*24:02-positive patients with leukemia or solid tumors. Although vaccination of modified WT1(235) peptide induced natural WT1(235) peptide-recognizing cytotoxic T-lymphocytes (CTLs) and exerted cytotoxic activity towards leukemia and solid tumor cells that expressed the natural WT1(235) peptide (epitope) but not the vaccinated modified WT1(235) peptide (epitope), the molecular basis has remained unclear. In this study, we established a modified WT1(235) peptide-specific CTL clone, we isolated T-cell receptor (TCR) genes from it and transduced the TCR genes into CD8(+) T-cells. The TCR-transduced CD8(+) T-cells produced interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in response to stimulation not only with the modified WT1(235) peptide but also with the natural WT1(235) peptide and lysed modified or natural WT1(235) peptide-pulsed target cells and endogenously WT1-expressing leukemia cells in a HLA-A*24:02-restriction manner. These results provided us, for the first time at molecular basis, with a proof-of-concept of modified WT1(235) peptide-based immunotherapy for natural WT1(235) peptide-expressing malignancies.

T cell receptor engineering.

T lymphocytes express on their surface a heterodimeric αß receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibody-directed target antigens, antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex. In the past 10 years, there have been significant efforts to engineer TCRs in various formats, which would allow improved recognition and destruction of virus-infected cells or cancer. The proposed therapeutic approaches involve either the use of engineered, high-affinity TCRs in soluble forms, analogous to antibody-directed therapies, or the use of engineered TCRs whose genes are reintroduced into autologous T cells and transferred back into patients (T cell adoptive therapies). This chapter describes three methods associated with the engineering of TCRs for these therapeutic purposes: (1) use of a yeast display system to engineer higher affinity single-chain VαVß TCRs, called scTv; (2) use of a T cell display system to engineer higher affinity full-length TCRs; and (3) expression, purification, and characterization of soluble TCRs in an Escherichia coli system.

Platypus TCRµ provides insight into the origins and evolution of a uniquely mammalian TCR locus.

TCRµ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and nonmammals. In this study, we show that TCRµ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRµ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRµ is expressed in a form containing double V domains. These V domains more closely resemble Ab V than that of conventional TCR. Platypus TCRµ differs from its marsupial homolog by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRµ but it has been lost from this lineage.

Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer.

BACKGROUND: Infection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. METHODS: HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. RESULTS: Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. CONCLUSION: Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies.

Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA.

BACKGROUND: Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs. RESULTS: The effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Valphabeta domain orientation was far superior to the Vbetaalpha domain orientation regarding monomeric yield of functionally folded molecules. CONCLUSION: The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.

Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR). Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.