Antigen-specific T-cell Receptor Research Articles

Investigation of Antigen-Specific T-Cell Receptor Clusters in Human Cancers.

Cancer antigen-specific T cells are key components in antitumor immune response, yet their identification in the tumor microenvironment remains challenging, as most cancer antigens are unknown. Recent advance in immunology suggests that similar T-cell receptor (TCR) sequences can be clustered to infer shared antigen specificity. This study aims to identify antigen-specific TCRs from the tumor genomics sequencing data. We used the TRUST (Tcr Repertoire Utilities for Solid Tissue) algorithm to assemble the TCR hypervariable CDR3 regions from 9,700 bulk tumor RNA-sequencing (RNA-seq) samples, and developed a computational method, iSMART, to group similar TCRs into antigen-specific clusters. Integrative analysis on the TCR clusters with multi-omics datasets was performed to profile cancer-associated T cells and to uncover novel cancer antigens. Clustered TCRs are associated with signatures of T-cell activation after antigen encounter. We further elucidated the phenotypes of clustered T cells using single-cell RNA-seq data, which revealed a novel subset of tissue-resident memory T-cell population with elevated metabolic status. An exciting application of the TCR clusters is to identify novel cancer antigens, exemplified by our identification of a candidate cancer/testis gene, HSFX1, through integrated analysis of HLA alleles and genomics data. The target was further validated using vaccination of humanized HLA-A*02:01 mice and ELISpot assay. Finally, we showed that clustered tumor-infiltrating TCRs can differentiate patients with early-stage cancer from healthy donors, using blood TCR repertoire sequencing data, suggesting potential applications in noninvasive cancer detection. Our analysis on the antigen-specific TCR clusters provides a unique resource for alternative antigen discovery and biomarker identification for cancer immunotherapies.

Cellular cytotoxicity is a form of immunogenic cell death

BackgroundThe immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic...

T-cells with a single tumor antigen-specific T-cell receptor can be generated in vitro from clinically relevant stem cell sources

ABSTRACT Chimeric antigen receptor (CAR) T-cells have shown great promise in the treatment of B-cell malignancies. For acute myeloid leukemia (AML), however, the optimal target surface antigen has yet to be discovered. Alternatively, T-cell receptor (TCR)-redirected T-cells target intracellular antigens, marking a broader territory of available target antigens. Currently, adoptive TCR T-cell therapy uses peripheral blood lymphocytes for the introduction of a transgenic TCR. However, this can cause graft-versus-host disease, due to mispairing of introduced and endogenous TCR chains. Therefore, we started from hematopoietic stem and progenitor cells (HSPC), that do not express a TCR yet, isolated from healthy donors, patients in remission after chemotherapy and AML patients at diagnosis. Using the OP9-DL1 in vitro co-culture system and agonist selection, TCR-transduced HSPC develop into mature tumor antigen-specific T-cells with only one TCR. We show here that this approach is feasible with adult HSPC from clinically relevant sources, albeit with slower maturation and lower cell yield compared to cord blood HSPC. Moreover, cryopreservation of HSPC does not have an effect on cell numbers or functionality of the generated T-cells. In conclusion, we show here that it is feasible to generate TA-specific T-cells from HSPC from adult healthy donors and patients and we believe these T-cells could be of use as a very valuable form of patient-tailored T-cell immunotherapy.

Single Domain Antibody-Mediated Blockade of Programmed Death-Ligand 1 on Dendritic Cells Enhances CD8 T-cell Activation and Cytokine Production.

Dendritic cell [DC] vaccines can induce durable clinical responses, at least in a fraction of previously treated, late stage cancer patients. Several preclinical studies suggest that shielding programmed death-ligand 1 [PD-L1] on the DC surface may be an attractive strategy to extend such clinical benefits to a larger patient population. In this study, we evaluated the use of single domain antibody [sdAb] K2, a high affinity, antagonistic, PD-L1 specific sdAb, for its ability to enhance DC mediated T-cell activation and benchmarked it against the use of the monoclonal antibodies [mAbs], MIH1, 29E.2A3 and avelumab. Similar to mAbs, sdAb K2 enhanced antigen-specific T-cell receptor signaling in PD-1 positive (PD-1pos) reporter cells activated by DCs. We further showed that the activation and function of antigen-specific CD8 positive (CD8pos) T cells, activated by DCs, was enhanced by inclusion of sdAb K2, but not mAbs. The failure of mAbs to enhance T-cell activation might be explained by their low efficacy to bind PD-L1 on DCs when compared to binding of PD-L1 on non-immune cells, whereas sdAb K2 shows high binding to PD-L1 on immune as well as non-immune cells. These data provide a rationale for the inclusion of sdAb K2 in DC-based immunotherapy strategies.

Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer

BackgroundEfficient identification of neoantigen-specific T-cell responses in epithelial ovarian cancer (EOC) remains a challenge. Existing investigations of spontaneous T-cell response to tumor neoepitope in EOC have taken the approach of comprehensive screening all neoantigen candidates, with a validation rate of 0.5–2%.MethodsWhole-exome and transcriptome sequencing analysis of treatment-naive EOC patients were performed to identify neoantigen candidates, and the immunogenicity of prioritized neoantigens was evaluated by analyzing spontaneous neoantigen-specfic CD4+ and CD8+ T-cell responses in the tumor and/or peripheral blood. The biological relevance of neoantigen-specific T-cell lines and clones were analyzed by evaluating the capacity of autologous ovarian tumor recognition. Genetic transfer of T-cell receptor (TCR) from these neoantigen-specific T-cell clones into peripheral blood T-cells was conducted to generate neoepitope-specific T-cells. The molecular signature associated with positive neoantigen T-cell responses was investigated, and the impacts of expression level and lymphocyte source on neoantigen identification were explored.ResultsUsing a small subset of prioritized neoantigen candidates, we were able to detect spontaneous CD4+ and/or CD8+ T-cell responses against neoepitopes from autologous lymphocytes in half of treatment-naïve EOC patients, with a significantly improved validation rate of 19%. Tumors from patients exhibiting neoantigen-specific T-cell responses exhibited a signature of upregulated antigen processing and presentation machinery, which was also associated with favorable patient survival in the TCGA ovarian cohort. T-cells specific against two mutated cancer-associated genes, NUP214 and JAK1, recognized autologous tumors. Gene-engineering with TCR from these neoantigen-specific T-cell clones conferred neoantigen-reactivity to peripheral T-cells.ConclusionsOur study demonstrated the feasibility of efficiently identifying both CD4+ and CD8+ neoantigen-specific T-cells in EOC. Autologous lymphocytes genetically engineered with tumor antigen-specific TCR can be used to generate cells for use in the personalized adoptive T-cell transfer immunotherapy.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Distinct Surface Expression of Activating Receptor Ly49H Drives Differential Expansion of NK Cell Clones upon Murine Cytomegalovirus Infection

Natural killer (NK) cells show some features of adaptive immunity but have not been studied at the clonal level. Here, we used retrogenic color-barcoding and single-cell adoptive transfers to track clonal immune responses to murine cytomegalovirus (MCMV) infection, derived from individual NK cells expressing activating receptor Ly49H. Clonal expansion of single NK cells varied substantially, and this variation could not be attributed to the additional presence or absence of inhibitory Ly49 receptors. Instead, single-cell-derived variability correlated with distinct surface expression levels of Ly49H itself. Ly49Hhi NK cell clones maintained higher Ly49H expression and expanded more than their Ly49Hlo counterparts in response to MCMV. Thus, akin to adaptive processes shaping an antigen-specific Tcell receptor (TCR) repertoire, the Ly49H+ NK cell population adapts to MCMV infection. This process relies on the clonal maintenance of distinct Ly49H expression levels, generating a repertoire of individual NK cells outfitted with distinct reactivity to MCMV.

Next-Generation Cancer Immunotherapy Targeting Glypican-3.

Glypican-3 (GPC3), a 65 kD protein consisting of 580 amino acids, is a heparan sulfate proteoglycan bound to the cell membrane by glycosylphosphatidylinositol. This protein is expressed in the liver and the kidney of healthy fetuses but is hardly expressed in adults, except in the placenta. Contrarily, GPC3 is specifically expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), yolk sac tumor, and some pediatric cancers. Although the precise function of GPC3 remains unclear, it has been strongly suggested that it is related to the malignant transformation of HCC. We identified GPC3 as a promising target for cancer immunotherapy and have been working on the development of cancer immunotherapeutic agents targeting it through clinical trials. In some trials, it was revealed that the GPC3 peptide vaccines we developed using human leukocyte antigen-A24- and A2-restricted GPC3-derived peptides could induce GPC3-specific cytotoxic T cells in most vaccinated patients and thereby improve their prognosis. To further improve the clinical efficacy of cancer immunotherapy targeting GPC3, we are also developing next-generation therapeutic strategies using T cells engineered to express antigen-specific T-cell receptor or chimeric antigen receptor. In addition, we have successfully monitored the levels of serum full-length GPC3 protein, which is somehow secreted in the blood. The utility of GPC3 as a biomarker for predicting tumor recurrence and treatment efficacy is now being considered. In this review article, we summarize the results of clinical trials carried out by our team and describe the novel agent targeting the cancer-specific shared antigen, GPC3.

In vitro-generated MART-1-specific CD8 T cells display a broader T-cell receptor repertoire than exvivo naïve and tumor-infiltrating lymphocytes.

The differentiation of human hematopoietic stem cells into CD8 T cells can be achieved invitro with the OP9-DL4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T-cell receptor (TCR) repertoire by expressing tissue-restricted antigens, a distinct repertoire would be generated invitro. To test this notion, we compared the TCR-Vα/Vβ (TRAV/TRBV) gene usage of major histocompatibility complex-restricted antigen (MART-1)-specific T cells generated invitro to that from exvivo naïve T cells and tumor-infiltrating lymphocytes (TILs) using high-throughput DNA sequencing. In contrast to naïve T cells and TILs, which showed the expected narrow TRAV repertoire, invitro-generated MART-1-specific T cells used almost all TRAV gene families and displayed unique CDR3 lengths. Our work demonstrates that the OP9-DL4 system supports the creation of a broad antigen-specific TCR repertoire, suggesting that T cells generated invitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus-derived T cells.

Abstract A034: High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens

Abstract The growing use of immunotherapy to treat advanced cancers has brought about a revolution in techniques to mobilize the immune system to antitumor effect. Chimeric antigen receptor (CAR) T-cells targeting CD19 constitute the first modified T-cell products to garner FDA approval for clinical use. However, CAR technologies can only targeT-cell surface antigens, representing approximately one quarter of potential targets. In contrast, T-cell receptor (TCR) therapies can target peptides presented by the major histocompatibility complex (MHC), including those derived from intracellular antigens. Hitherto, such receptors have generally been identified through low-throughput techniques using cancer patients’ blood, followed by affinity-maturation of TCRs, a step that can decrease safety. Here, we demonstrate a novel pipeline for the identification of potential therapeutic TCRs from the naïve repertoire of healthy individuals. Using a technique called Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA), we input hundreds of antigens of interest, including tumor-associated antigens, viral antigens and neoantigens, and identify thousands of TCRs to these antigens. These TCRs then undergo evaluation for affinity, avidity, cytokine release, cytotoxicity and safety. Cytotoxicity is demonstrated using both peptide-loaded and endogenously presented peptides. Safety is evaluated using alanine-glycine scans; evaluation of reactivity of TCRs againsT-cell lines and primary cells is planned. We have fully characterized several TCRs targeting clinically relevant targets, which demonstrated improved avidity and cytolysis relative to a benchmark TCR, and a promising preliminary safety profile. In the recent year, cancer neoantigens as targets for natural and therapeutic antitumor responses have also gained momentum given their attractive product profile. Hereto, we also show data for TCRs against shared neoantigens. Citation Format: Mark Klinger, Peter Ebert, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins. High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A034.

Abstract B115: Optimization of cancer vaccine development by using Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA)

Abstract Therapeutic vaccines have been in development for decades with the goal of priming and boosting an immune response against specific antigens in cancer patients. More recently, vaccination with mutation-derived neoantigens has emerged as is an attractive approach to treat cancer patients. However, appropriately selecting immunogenic antigens and neoepitopes that successfully prime the immune system of cancer patents remains a challenge. In fact, despite extensive efforts throughout the years, many vaccines have proven to be ineffective in mediating a clinically relevant anti-tumor immune response in humans. With the advent of emerging new technologies, including the combination of next-generation sequencing and improved bioinformatics tools, a new era of vaccine development is underway. Hereto, we present a novel, highly sensitive, multiplex approach, known as Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA), that combines high-throughput TCR repertoire sequencing with conventional immune monitoring techniques to assess T-cell specificity to large numbers of query antigens. MIRA maps antigen specificity to TCR sequence at an unprecedented sensitivity (1 in ~10 million T-cells) and scale (more than 400 antigens at a time). To validate this approach, we assessed the response of more than 10 million naïve human T-cells from each of 50 healthy donors against a panel of 366 neoepitopes. These include post-translationally modified peptides, derived from over 130 prevalent mutations in greater than 50 cancer indications. From three billion total input T-cells, we identified tens of thousands of TCRB sequences from responsive T-cell clones, of which at least one TCR was yielded for the majority of queried neoepitopes. The TCRs identified were skewed toward a small number of query antigens, with 12 neoepitopes accounting for over 50% of the TCRs identified. This suggests a higher precursor frequency of specific T-cells in the naïve repertoire or a higher immunogenicity of these epitopes or both. By performing MIRA experiments in parallel using transgenes versus peptides, we can further validate which of the neoepitopes were also presented via cells’ natural antigen presentation machinery. Of note, we also paired 52% of the TCRB sequences with their cognate TCRA subunit sequence thus permitting reconstitution of individual TCRs. Generating immunogenicity data to antigens of interest at scale facilitates an opportunity to better inform antigen selection in vaccine programs and improve vaccine design. Once a vaccine has been designed and administered to a patient, MIRA offers a unique solution to assess peptides based on their ability to elicit robust T responses. By creating a post-vaccination, patient-specific MIRA, a permanent record or signature of that patient’s response to the vaccine is created. This information can then be leveraged to identify early biomarkers of response to the vaccine and monitor this response longitudinally by simply using single chain TCRB deep sequencing from a small amount of a patient’s peripheral blood. Citation Format: Peter Ebert, Mark Klinger, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins. Optimization of cancer vaccine development by using Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA) [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B115.

A rare population of tumor antigen-specific CD4+CD8+ double-positive \u03b1\u03b2 T lymphocytes uniquely provide CD8-independent TCR genes for engineering therapeutic T cells

BackgroundHigh-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. However, discovery of suitable therapeutic TCR genes is hampered by the fact that naturally occurring tumor antigen-specific TCRs are generally of low-affinity, and artificial modification of TCRs can mediate cross-reactivity to other antigens expressed in normal tissues. Here, we discovered a naturally occurring T-cell clone which expressed high-affinity HLA-A*02:01 (A*02)-restricted TCR against NY-ESO-1 from a patient who had NY-ESO-1-expressing ovarian tumor.MethodsA*02-restricted NY-ESO-1-specific T-cell clones were established from peripheral blood of patients who had NY-ESO-1-expressing ovarian tumors. TCR α and β chain genes were retrovirally transduced into polyclonally activated T cells. Phenotype and function of the parental and TCR-transduced T cells were analyzed by flow cytometry, ELISA and cytotoxicity assay. In vivo therapeutic efficacy was investigated in a xenograft model using NOD/SCID/IL-2Rγ-deficient (NSG) mice.ResultsA rare population of NY-ESO-1-specific T cells, which we named 19305DP, expressed cell surface CD4, CD8α, and CD8β but not CD56 and recognized A*02+NY-ESO-1+ cancer cell lines in a CD4- and CD8-independent manner. 19305DP showed a gene expression profile that is consistent with a mixed profile of CD4+ and CD8+ single-positive T cells. Both CD4+ and CD8+ T cells that were retrovirally transduced with 19305DP-derived TCR gene (19305DP-TCR) showed strong reactivity against A*02+NY-ESO-1+ cancer cells, whereas TCR genes from the conventional A*02-restricted NY-ESO-1-specific CD8+ single-positive T-cell clones functioned only in CD8+ T cells. Both 19305DP-TCR gene-engineered CD4+ and CD8+ T cells eliminated A*02+NY-ESO-1+ tumor xenografts in NSG mice. Finally, based on reactivity against a series of alanine-substituted peptides and a panel of normal human tissue-derived primary cells, 19305DP-TCR was predicted to have no cross-reactivity against any human non-NY-ESO-1 proteins.ConclusionTogether, our results indicate that the naturally occurring 19305DP-TCR derived from CD4+CD8+ double-positive αβ T cells, is a promising therapeutic TCR gene for effective and safe adoptive T-cell therapy in A*02+ patients with NY-ESO-1-expressing tumor.

Identification of Specificity Groups in Myeloma Patients T Cell Receptor (TCR) Repertoire through Single Cell TCR Sequencing

Background: A renewed interest in immune and cellular based therapeutics in multiple myeloma was recently fueled by the development of CD38 targeting monoclonal antibodies as well as the introduction of engineered CAR-T cells. Daratumumab treatment in myeloma patients was demonstrated to expand clonal CD8+T cells and T cell clonality was correlated with the depth of response consistent with a daratumumab mediated cytotoxic T cell effect. However the mechanisms behind this adaptive immune response and the identity of the T cell receptor (TCR) interacting with MHC presented tumoral peptide (pMHC) remains elusive. The large diversity of TCR combinations generated though somatic recombination of the VDJ gene sequences (~ 1015combinations) represents a major challenge for the accurate characterization of the antigen specific TCR. The aim of this study was to define the identity of the adaptive immune repertoire of the bone marrow infiltrating T lymphocytes (single cell TCR α/β paired sequencing) in myeloma patients treated to daratumumab and IMiDs based therapies.Methods and Results: BM aspirates from patients (n=24) treated with daratumumab single agent or in combination with pomalidomide or lenalidomide (MM014, MM3008 and MMY3012 trials) were collected post initiation of therapy (cycle 3, day 1) followed by magnetic beads sorting of CD3pos T cells from Ficoll generated mononuclear cell fractions. Using the 10x Genomics Single cell VDJ solution which combines single cell droplet microfluidics with 5' molecular barcoding, T cells from each patient were partitioned into droplets containing individual cells with primers specific for the constant region of the V(D)J locus allowing the PCR amplification and enrichment of α and β TCR individual cell barcoded cDNA. Paired-end sequencing was performed on Illumina NEXTseq platform. Cell Ranger VDJ pipeline was used for sample de-multiplexing, barcode processing and grouping of T cells into clonotypes with shared TCR α/β sequences. Of note, generated sequences span the full length of V-J genes (including CDR3) allowing faithful reconstruction of TCR transcripts. Consistent with the known high TCR diverse repertoire, we identified 32322 individual clonotypes corresponding to an average of 1346 clonotypes with paired α/β TCR sequences per patient. Clonotypes proportion (> 2%) and number of individual clonotypes did correlate with the depth of response (≥VGPR vs PR vs PD). Analysis of clonotype TCRs and CDR3 sequences identified 11 clonotypes with the exact paired CDR3 αβ sequences that were shared by at least 2 patients. Of interest the CDR3 sequence of one shared clonotype is predicted (https://vdjdb.cdr3.net) to bind an epitope derived for CD317 (also know as BST2 or HM1.24) previously demonstrated to be highly expressed on myeloma cells (Jalili A et al. Blood 2005). Both patients harboring this CD317 reactive T cell clone are in sCR for more than 2 years. Of note, TCRs that recognize the same peptide-MHC complexes do not always share the exact CDR3 sequences but rather have conserved CDR3 sequence features, rendering possible to predictively model epitope specificity. Indeed, recent studies demonstrated that similarity in CDR3 sequences (CDR3 differing by up to one amino acid) or shared CDR3 motifs of 2-4 amino acids in length, define the TCR clusters that are often contact points with the antigenic peptides. Such features in CDR3 sequences facilitates T cell target antigen discovery. Therefore, we applied the GLIPH algorithm (https://github.com/immunoengineer/gliph) to cluster the sequenced TCRs based on their high probability of sharing pMHC specificity owing to both conserved CDR3 motifs and global similarity in their CDR3 sequences. GLIPH grouped the TCRs sequences identified in our study into 171 unique clusters (with a minimum of 3 clones each) that are predicted to recognize the same pMHC ligands. Furthermore, it identified 26 CDR3 motifs that are elevated at least 10-fold over expected frequency in a naïve TCR reference pool (p <0.001). Functional validation of this myeloma-targeting TCR clusters through pMHC tetramer binding is ongoing.Conclusion: Single cell TCR profiling identified unique clonotypes that are highly enriched in marrow infiltrating T cells and are predicted to be reactive with myeloma peptides. This work facilitates the future development of TCR engineered T cells targeting myeloma neoepitopes. DisclosuresNeri:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. McCulloch:Takeda: Other: Travel expenses; Celgene: Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector.

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs. Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between naïve and effector cells.

Enhancing T Cell Receptor Stability in Rejuvenated iPSC-Derived T Cells Improves Their Use in Cancer Immunotherapy

Limited Tcell availability and proliferative exhaustion present major barriers to successful Tcell-based immunotherapies and may potentially be overcome through the use of "rejuvenated" induced pluripotent stem cells derived from antigen-specific Tcells (T-iPSCs). However, strict antigen specificity is essential for safe and efficient Tcell immunotherapy. Here, we report that CD8αβ Tcells from human T-iPSCs lose their antigen specificity through additional rearrangement of the Tcell receptor (TCR) α chain gene during the CD4/CD8 double positive stage of invitro differentiation. CRISPR knockout ofa recombinase gene in the T-iPSCs preventedthisadditional TCR rearrangement. Moreover, whenCD8αβ Tcells were differentiated from monocyte-derived iPSCs that were transduced with an antigen-specific TCR, they showed monoclonal expression of the transduced TCR. TCR-stabilized, regenerated CD8αβ Tcells effectively inhibit tumorgrowth in xenograft cancer models. These approaches could contribute to safe and effective regenerative Tcell immunotherapies.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

Interleukin-6 signalling mediates Galectin-8 co-stimulatory activity of antigen-specific CD4 T-cell response.

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-6-deficient antigen-presenting cells. In addition, exogenous IL-6 fully restored Gal-8-induced co-stimulation. Taken together, our results demonstrate that IL-6 signalling mediates the Gal-8 immune-stimulatory effect.

Detecting neoepitope-specific intra-tumoral T cell responses in a glioblastoma patient treated with personal neoantigen vaccine

Abstract Recent advances in single cell T cell receptor (TCR) sequencing have allowed detailed analysis of TCRαβ pairs. However, efficient methods for probing the specificity of discovered TCRs remain limited. We developed a streamlined approach for cloning and expressing TCRs and screening against candidate antigens. A key innovation was the establishment of a plasmid library to encode all variable (V) regions of the TCR, from which any TCR of interest can be assembled with only custom synthesis of short CDR3 regions. A reporter cell system allows rapid detection of TCR antigen specificity and assessment of functional avidity based on cytokine production. We applied this pipeline to study the intra-tumoral TCR repertoire of a patient with glioblastoma treated with personal neoantigen vaccine. The vaccine consisted of up to 20 synthetic long peptides containing predicted neoepitopes and poly-ICLC adjuvant. Single cell TCR sequencing of CD3+ tumor infiltrating lymphocytes (TILs) and peripheral T cells in vitro expanded against immunizing peptides showed that 4 CD4+ and 2 CD8+ T cell clonotypes in peripheral blood were identical to TILs. The cloning and expression system was used to test their specificity. We identified a TCR from a CD4+ TIL specific for ARHGAP35, a neoantigen targeted by vaccination, and capable of discriminating between mutant and wildtype peptide. This result provides the first demonstration that neoepitope-sensitized T cells can traffic from the periphery to an intracranial GBM. Overall, we demonstrated our ability to identify TCRαβ pairs, express them on demand, and probe antigen specificity. Ongoing studies are characterizing the functional avidity of neoepitope-specific TCRs and reactivity against autologous tumor.

Rapid Construction of Antitumor T-cell Receptor Vectors from Frozen Tumors for Engineered T-cell Therapy.

T cells genetically engineered with tumor antigen-specific T-cell receptor (TCR) genes have demonstrated therapeutic potential in patients with solid tumors. In order to achieve broader application, an efficient method to identify TCR genes for an array of tumor antigens and HLA restriction elements is required. Here, we have developed a method to construct a TCR-expression library from specimens, including frozen tumor biopsies, that contain antigen-specific T cells. TCR-expressing cassettes were constructed and cloned in a retroviral plasmid vector within 24 hours by unbiased PCR amplification of TCR α and β chain variable regions assembled with TCR constant regions. The method was validated by constructing TCR-expressing vectors from tumor antigen-specific T-cell clones and functionally assessing TCR gene-transduced T cells. We applied this method to frozen ovarian tumor specimens that were infiltrated by tumor antigen-specific T cells. The tumor-derived TCR libraries were expressed in peripheral T cells from healthy volunteers and screened for tumor antigen-specific TCR pairs with the use of an MHC/peptide tetramer reagent. Tumor antigen-specific TCR-expressing transgenes were recovered from isolated tetramer-positive T cells. Peripheral T cells that were engineered with library-derived TCR gene showed potent therapeutic antitumor effect in a tumor xenograft model. Our method can efficiently and rapidly provide tumor-specific TCR-expressing viral vectors for the manufacture of therapeutic and personalized antitumor T-cell products. Cancer Immunol Res; 6(5); 594-604. ©2018 AACR.

Characterization of Postinfusion Phenotypic Differences in Fresh Versus Cryopreserved TCR Engineered Adoptive Cell Therapy Products

Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort’s superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.