Human TCR Research Articles

Sepsis shapes the human γδ TCR repertoire in an age- and pathogen-dependent manner.

Sepsis affects 25 million children per year globally, leading to 2.9 million deaths and substantial disability in survivors. Extensive characterization of interactions between the host and bacteria in children is required to design novel preventive and therapeutic strategies tailored to this age group. Vγ9Vδ2 T cells are the first T cells generated in humans. These cells are defined by the expression of Vγ9Vδ2 T-cell receptors (TCRs, using the TRGV9 and TRDV2 gene segments), which react strongly against the prototypical bacterial phosphoantigen HMBPP. We investigated this reactivity by analyzing the TCR δ (TRD) repertoire in the blood of 76 children (0-16 years) with blood culture-proven bacterial sepsis caused by HMBPP-positive Escherichia coli or by HMBPP-negative Staphylococcus aureus or by HMBPP-negative Streptococcus pneumoniae. Strikingly, we found that S. aureus, and to a lesser extent E. coli but not S. pneumoniae, shaped the TRDV2 repertoire in young children (<2 years) but not in older children or adults. This dichotomy was due to the selective expansion of a fetal TRDV2 repertoire. Thus, young children possess fetal-derived Vγ9Vδ2 T cells that are highly responsive toward specific bacterial pathogens.

Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3β loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Abstract C007: Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery

Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity, and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B- cells. A new technology we developed that combines profiling of all human TCR and BCR variable regions with phenotypic characterization of immune cells using the same workflow could be particularly useful. The TCR and BCR immunophenotyping method proposed involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, and subsequently determining the expression levels of the most informative T- and B-cell phenotyping genes. Preliminary results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data from the TCR and BCR clonotypes profile combined with RNA expression profiling of the same samples results in a richer data set that includes the identification of major immune cell subtypes and their activation status. Data from human cancer tissues and whole blood samples will be presented. Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Khadija Ghias, Lester Kobzik. Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C007.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies

Chronic stimulation can cause Tcell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function invitro and invivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram Tcell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

T-cell and B-cell receptor repertoire profiling for biomarker discovery

Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B-cells. We developed a new technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in the same workflow. The TCR and BCR immunophenotyping method involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, as well as determining the expression levels of the most informative T- and B-cell phenotyping genes. Results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data allows profiling of the TCR and BCR clonotypes and identifcation of major immune cell subtypes and their activation status. Preliminary data from rheumatoid arthritis blood samples and others will be presented.

Abstract A009: AFNT-111: A novel TCR-engineered T cell therapy targeting the oncogenic driver KRAS G12V

Abstract Mutations in the RAS family are highly prevalent in human cancers, including up to 35% of non-small cell lung, 45% of colorectal, and 95% of pancreatic cancers. Kirsten rat sarcoma viral oncogene homologue (KRAS) is the most frequently mutated RAS oncogene and patients with KRAS mutations have poor responses to standard treatment regimens. Therefore, successful KRAS-targeted therapies represent a high unmet need. The AFNT-111 cellular therapy consists of autologous CD8+ and CD4+ T cells expressing a high affinity TCR specific for the prevalent KRASG12V mutation presented by HLA-A*11:01, one of the most common HLA alleles worldwide. AFNT-111 is engineered to express the CD8α/β coreceptor, enabling a coordinated CD4+/CD8+ tumor response to induce robust T cell activity and persistence while minimizing T cell exhaustion. Lentiviral vector was used to transduce primary healthy human CD4+ and CD8+ T cells with the KRASG12V-specific human TCR and CD8α/β coreceptor. Engineered T cells were assessed against exogenous KRASG12V peptide as well as a panel of tumor cell lines endogenously expressing KRASG12V and probed for in vitro activation, proliferation, cytotoxicity and cytokine secretion. In parallel, in vitro safety studies were performed to evaluate autoantigen cross-reactivity and alloreactivity. In vivo efficacy studies were conducted using established human KRASG12V xenografts in NSG mice. AFNT-111 demonstrated potent functional avidity towards the KRASG12V peptide with no reactivity against KRASWT. Several naturally expressing KRASG12V human tumor cell lines led to significant AFNT-111 T cell activation and proliferation, as well as potent cytotoxicity. The addition of the CD8α/β coreceptor revealed a coordinated and synergistic cytotoxic CD4+ and CD8+ T cell response, enhancing activation of the AFNT-111 cell product. Robust cytotoxicity was associated with increased T cell secretion of IFNγ and TNFα. Durable in vivo anti-tumor efficacy was observed in three established mouse xenograft tumor models. XScan studies using amino acid substitutions of the index KRASG12V peptide revealed a restrictive TCR recognition motif. Further, potentially cross-reactive human self-peptides matching this motif were tested and no significant cross-reactivities were identified. To test alloreactivity, a large lymphoblastoid cell line library was assessed with no alloreactive responses detected. A streamlined manufacturing process has also been developed in which CD4+/CD8+ T cell ratios are controlled, with the final AFNT-111 product retaining a high degree of stem-like properties. AFNT-111 preclinical data demonstrate a highly potent and specific TCR-engineered T cell product that is cytotoxic to KRASG12V-expressing tumor cells both in vitro and in vivo. Cross-reactivity and alloreactivity assessments establish a potentially favorable safety profile of AFNT-111, supporting clinical translation. First-in-human clinical studies will focus on advanced or metastatic pancreatic, colorectal, and lung cancer indications. Citation Format: Michele Hoffmann, Xingyue He, Cheryl Black, Joshua Francis, James Parsons, Christian Roy, Jinsheng Liang, Hongjing Qu, Martin Campbell, Tomasz Sewastianik, Jessica Webb, Aude Chapuis, Thomas Schmitt, Philip Greenberg, Damien Hallet, Markus Vallaster, Piotr Pierog, Gary Shapiro, Hubert Lam, Loic Vincent. AFNT-111: A novel TCR-engineered T cell therapy targeting the oncogenic driver KRAS G12V [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A009.

Abstract LB172: Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery

Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity, and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B- cells. A new technology we developed that combines profiling of all human TCR and BCR variable regions with phenotypic characterization of immune cells using the same workflow could be particularly useful. The TCR and BCR immunophenotyping method proposed involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, and subsequently determining the expression levels of the most informative T- and B-cell phenotyping genes. Preliminary results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data from the TCR and BCR clonotypes profile combined with RNA expression profiling of the same samples results in a richer data set that includes the identification of major immune cell subtypes and their activation status. Data from human cancer tissue and whole blood samples will be presented. Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Paul Diehl, Lester Kobzik. Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB172.

Abstract 1780: TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements

Abstract The clinical success of cancer immunotherapy, including engineered T cell therapies, has revolutionized treatment paradigms and patient outcomes. While hematological tumors have benefited most from cell therapy approaches, the treatment of solid tumors remains a challenge in part due to the limited availability of abundant and tumor-specific cell-surface antigens. Peptides derived from intracellular tumor-specific proteins, such as cancer-testis antigens (CTAs), that are presented via HLA (pHLA) enable a therapeutic opportunity to target tumors while sparing normal tissue. Surface-accessible pHLA complexes may be targeted with engineered T cell receptors or TCR mimetic antibodies reformatted to chimeric antigen receptors (CARs). Using MAGE-A4 as a model CTA, we compared engineered human TCR- and CAR-T cells head-to-head to understand how to best deploy these modalities. To this end, we generated fully-human, HLA-A2/MAGE-A4(230-239)-specific TCR and CARs harboring CD28/CD3z or 41BB/CD3z signaling domains. TCR and CAR-T cells demonstrated similar robust on-target reactivity, cytokine release, and target cell lysis in vitro. In vivo, each of these modalities showed potent, dose-dependent anti-tumor efficacy against human xenograft tumors expressing low, endogenous levels (~500 cell-surface copies) of the MAGE-A4 peptide. However, differences emerged when we examined the in vivo kinetics and durability of tumor suppression. MAGE-A4 CD28/z CAR-T demonstrated the most rapid and potent tumor clearance, while the 41BB/z CAR-T showed delayed but ultimately complete efficacy. TCR-T cells induced tumor regressions during the first 2 weeks of treatment, but this response was transient and followed by tumor relapse. These differential responses correlated with early, modest accumulation of CD28/z CAR-T in line with fast tumor clearance. 41BB/z CAR-T showed a remarkable ~800-fold expansion in the tumor versus limited in vivo TCR-T proliferation. Mechanistically, the MAGE-A4 TCR induced strong CD3 proximal signaling associated with a greater induction of T cell dysfunction markers and limited cytotoxic potential in vitro. However, stimulating 41BB signaling pathways in the MAGE-A4 TCR T cells augmented long-term cytotoxicity. These data demonstrate that tumor-specific pHLA complexes can be potently targeted by both TCR and CAR-T cells, and that co-stimulatory signaling is necessary to mediate durable anti-tumor activity. Citation Format: Corinne E. Decker, Jacqueline Idun, Katja Mohrs, Thomas Meagher, Kevin Bray, Iryna Petriv, Jonathon Golas, Timothy Helms, Dharani Ajithdoss, Gavin Thurston, John Lin, Jessica R. Kirshner, David J. DiLillo. TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1780.

Integrative network pharmacology and in silico analyses identify the anti-omicron SARS-CoV-2 potential of eugenol

Eugenol as a natural product is the source of isoniazid, and purified eugenol is extensively used in the cosmetics industry and the productive processes of edible spices. Accumulating evidence suggested that eugenol exerted potent anti-microorganism and anti-inflammation effects. Application of eugenol effectively reduced the risk of atherosclerosis, arterial embolism, and Type 2 diabetes. A previous study confirmed that treatment with eugenol attenuated lung inflammation and improved heart functions in SARS-CoV-2 spike S1-intoxicated mice. In addition to the study, based on a series of public datasets, computational analyses were conducted to characterize the acting targets of eugenol and the functional roles of these targets in COVID-19. The binding capacities of eugenol to conservative sites of SARS-CoV-2 like RNA-dependent RNA polymerase (RdRp) and mutable site as spike (S) protein, were calculated by using molecular docking following the molecular dynamics simulation with RMSD, RMSF, and MM-GBSA methods. The results of network pharmacology indicated that six targets, including PLAT, HMOX1, NUP88, CTSL, ITGB1 andTMPRSS2 were eugenol-SARS-CoV-2 interacting proteins. The omics results of in-silico study further implicated that eugenol increased the expression of SCARB1, HMOX1 and GDF15, especially HMOX1, which were confirmed the potential interacting targets between eugenol and SARS-CoV-2 antigens. Enrichment analyses indicated that eugenol exerted extensive biological effects such as regulating immune infiltration of macrophage, lipid localization, monooxyenase activity, iron ion binding and PPAR signaling. The results of the integrated analysis of eugenol targets and immunotranscription profile of COVID-19 cases shows that eugenol also plays an important role in strengthen of immunologic functions and regulating cytokine signaling. As a complement to the integrated analysis, the results of molecular docking indicated the potential binding interactions between eugenol and four proteins relating to cytokine production/release and the function of T type lymphocytes, including human TLR-4, TCR, NF-κB, JNK and AP-1. Furthermore, results of molecular docking and molecular dynamics (100ns) simulations implicated that stimulated modification of eugenol to the SARS-CoV-2 Omicron Spike-ACE2 complex, especially for human ACE2, and the molecular interaction of eugenol to SARS-CoV-2 RdRp, were no less favorable than two positive controls, molnupiravir and nilotinib. Dynamics (200ns) simulations indicated that the binding capacities and stabilities of eugenol to finger subdomain of RdRp is no less than molnupiravir. However, the simulated binding capacity of eugenol to SARS-CoV-2 wild type RBD and Omicron mutant RBD were less than nilotinib. Eugenol was predicted to have more favor LD50 value and lower cytotoxicity than two positive controls, and eugenol can pass through the blood-brain barrier (BBB). In a brief, eugenol is helpful for attenuating systemic inflammation induced by SARS-CoV-2 infection, due to the direct interaction of eugenol to SARS-CoV-2 proteins and extensive bio-manipulation of pro-inflammatory factors. This study carefully suggests eugenol is a candidate compound of developing drugs and supplement agents against SARS-CoV-2 and its Omicron variants.

Targeting the recurrent Rac1P29S neoepitope in melanoma with heterologous high-affinity T cell receptors.

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope FSGEYIPTV carries the Rac1P29S amino acid change caused by a c.85C>T missense mutation, which is the third most common hotspot mutation in melanoma. Here, we isolated and characterized TCRs to target this HLA-A*02:01-binding neoepitope by adoptive T cell therapy. Peptide immunization elicited immune responses in transgenic mice expressing a diverse human TCR repertoire restricted to HLA-A*02:01, which enabled isolation of high-affinity TCRs. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cells and we observed regression of Rac1P29S expressing tumors in vivo after adoptive T cell therapy (ATT). Here we found that a TCR raised against a heterologous mutation with higher peptide-MHC affinity (Rac2P29L) more efficiently targeted the common melanoma mutation Rac1P29S. Overall, our study provides evidence for the therapeutic potential of Rac1P29S-specific TCR-transduced T cells and reveal a novel strategy by generating more efficient TCRs by heterologous peptides.

OR24-2 Cutting-edge translational strategies in type 1 diabetes

Abstract Background and purpose of the study The overall goal of this study is to develop precision-targeted therapies for type 1 diabetes (T1D) by blocking the presentation of pancreatic antigens to T cells using an innovative translational approach. There is currently no curative therapy for T1D and the only available treatment is insulin replacement, which – although being life-saving – remains problematic and can be associated with often fatal high or low glucose levels. We hypothesize that the presentation of pathogenic pancreatic peptides to T cells within the HLA-DQ8 pocket is a key trigger in T1D, and that blocking the peptide binding to this pocket can be harnessed to treat/prevent the autoimmune response targeting beta cells in T1D. We propose to block antigen presentation to treat/prevent autoimmunity in T1D using retro-inverso D-amino acid peptides (RIPs). To test our hypothesis, we used the following tools: recombinant human HLA-DQ8, human B cells homozygous for HLA-DQ8, humanized mice expressing human HLA-DQ8, NOD mice, and peripheral blood mononuclear cells (PBMCs) isolated from new onset HLA-DQ8 T1D patients. All of them represent exquisite translational instruments to evaluate in vitro, ex vivo, and in vivo the functional role of our RIPs in preventing beta cell destruction. Summary of results We have identified a RIP (RI-CT) that inhibits InsB: 9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to HLA-DQ8 expressed on a human B-cell line. Specifically, RI-CT averted T-cell activation in a mixed lymphocyte reaction containing human DQ8 cells loaded with InsB: 9-23 peptide and murine T-cells expressing a human TCR specific for the InsB: 9-23–DQ8 complex. These results were also confirmed in humanized transgenic B6-DQ8 mice (KO for murine MHC-II) both ex vivo and in vivo, as shown by decreased production of pro-inflammatory cytokines (including IL-2 and IFN-γ) and reduced lymphocyte proliferation. Moreover, RI-CT prevents autoimmune diabetes in NOD mice as demonstrated by a significant decrease in pancreatic immune cell infiltration in treated mice versus controls. Importantly, at 25 weeks of age 70% of RI-CT treated mice were protected from the development of diabetes, while as expected 80% of control NOD mice had developed the disease. Interestingly, RI-CT injection also altered T-cell subpopulations in NOD mice significantly decreasing CD8 expression and significantly increasing Foxp3 levels in treated mice. Of note, RI-CT significantly inhibits InsB: 9-23-mediated lymphocyte activation in peripheral blood mononuclear cells isolated from new onset DQ8-T1D patients. Conclusions In summary, we discovered a RIP that blocks InsB: 9-23 binding to HLA-DQ8 and its presentation to T-cells averting beta cells destruction, and that delays T1D in NOD mice. These data set the stage for using our approach of blocking antigen presentation by RIP as a novel therapeutic approach for autoimmune diseases in general. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.

Two types of human TCR differentially regulate reactivity to self and non-self antigens

SummaryBased on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are reported as disease-associated. Thus, 10%–30% of most frequent cord blood TCRs are associated with common pathogens and autoantigens. TDT-dependent TCRs present distinct structural features and are less shared among subjects. TDT-dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes.

B.1.1.7 (Alpha) variant is the most antigenic compared to Wuhan strain, B.1.351, B.1.1.28/triple mutant and B.1.429 variants.

The rapid spread of the SARS-CoV-2 virus and its variants has created a catastrophic impact worldwide. Several variants have emerged, including B.1.351 (Beta), B.1.1.28/triple mutant (P.1), B.1.1.7 (Alpha), and B.1.429 (Epsilon). We performed comparative and comprehensive antigenicity mapping of the total S-glycoprotein using the Wuhan strain and the other variants and identified 9-mer, 15-mer, and 20-mer CTL epitopes through in silico analysis. The study found that 9-mer CTL epitope regions in the B.1.1.7 variant had the highest antigenicity and an average of the three epitope types. Cluster analysis of the 9-mer CTL epitopes depicted one significant cluster at the 70% level with two nodes (KGFNCYFPL and EGFNCYFPL). The phage-displayed peptides showed mimic 9-mer CTL epitopes with three clusters. CD spectra analysis showed the same band pattern of S-glycoprotein of Wuhan strain and all variants other than B.1.429. The developed 3D model of the superantigen (SAg)-like regions found an interaction pattern with the human TCR, indicating that the SAg-like component might interact with the TCR beta chain. The present study identified another partial SAg-like region (ANQFNSAIGKI) from the S-glycoprotein. Future research should examine the molecular mechanism of antigen processing for CD8+ T cells, especially all the variants’ antigens of S-glycoprotein.

Abstract 625: Immunophenotyping of TCR and BCR clonotypes

Abstract TCR and BCR repertoire profiling holds great potential for understanding disease mechanisms and for development of new therapeutics in infectious diseases, autoimmunity and in immuno-oncology. However, this potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T and B cells. To facilitate these studies, we developed a novel technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells. The developed TCR/BCR immunophenotyping method involves multiplex RT-PCR amplification and sequencing of CDR3 regions of TCR and BCR genes and a set of the most informative T- and B-cell phenotyping genes. Bioinformatic analysis of NGS data allows profiling of TCR/BCR clonotypes, and identification of major immune cell subtypes and their activation status. Data will be presented showing how combined TCR/BCR clonotype analysis combined with targeted expression profiling of immune cells can be applied for large-scale discovery of novel cell typing and activation biomarkers in several immune-responsive model systems. Preliminary studies indicate the assay has unparalleled throughput, sensitivity, and improved cost-effectiveness for high-throughput immunity biomarker discovery applications. Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Lester Kobzik. Immunophenotyping of TCR and BCR clonotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 625.

Humanization of T cell–mediated immunity in mice

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαβ variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting “VelociT” mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αβ T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I– and MHC-II–restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

In silico screening and epitope mapping of leptospiral outer membrane protein—Lsa46

Leptospirosis is one of the neglected diseases caused by the spirochete, Leptospira interrogans. Leptospiral surface adhesion (Lsa) proteins are surface exposed outer membrane proteins present in the pathogen. It acts as laminin and plasminogen binding proteins which enable them to infect host cells. The major target for the development of vaccine in the current era focuses on surface exposed outer membrane proteins, as they can induce strong and fast immune response in hosts. Therefore, the present study mapped the potential epitopes of the Leptospiral outer membrane proteins, mainly the surface adhesion proteins. Protein sequence analysis of Lsa proteins was done by in silico methods. The primary protein sequence analysis revealed Lsa46 as a suitable target which can be a potent Leptospiral vaccine candidate. Its structure was modelled by threading based method in I-TASSER server and validated by Ramachandran plot. The predicted epitope’s interactions with human IgG, IgM(Fab) and T-cell receptor TCR(αβ) were performed by molecular docking studies using Biovia Discovery studio 2018. One of the predicted B-cell epitopes and the IgG showed desirable binding interactions, while four of the predicted B-cell epitopes and T-cell epitopes showed desirable binding interactions with IgM and TCR respectively. The molecular dynamic simulation studies carried out with the molecular docked complexes gave minimized energies indicating stable interactions. The structural analysis of the entire simulated complex showed a stable nature except for one of the Epitope-IgM complex. Further the binding free energy calculation of eight receptor-ligand complex predicted them energetically stable. The results of the study help in elucidating the structural and functional characterization of Lsa46 for epitope-based vaccine design. Communicated by Ramaswamy H. Sarma.

69 Immunophenotyping of TCR and BCR clonotypes

BackgroundT-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling holds great potential for understanding disease mechanisms and for the development of new therapeutics in infectious diseases, autoimmunity and in immuno-oncology. However, this potential could be greatly improved by combining information about receptor clonotypes with immuno-phenotypes of T and B cells.MethodsTo facilitate these studies, we developed a novel technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in bulk and at the single-cell level in PBMC and immune cell fraction samples. The developed TCR/BCR Immunophenotyping method involves multiplex RT-PCR amplification and sequencing of CDR3 regions of TCR and BCR genes and a set of the most informative T- and B-cell phenotyping genes. Bioinformatics analysis of NGS data allows profiling of TCR/BCR clonotypes, and identification of major immune cell subtypes and their activation status.ResultsData will be presented showing how combined TCR/BCR clonotype analysis combined with targeted expression profiling of immune cells can be applied for large-scale discovery of novel cell typing and activation biomarkers in several immune-responsive model systems.ConclusionsPreliminary studies demonstrate the assay has unparalleled throughput, sensitivity, and improved cost-effectiveness for high-throughput immunity biomarker discovery applications.

Erratum for the Research Article: "Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes" by Han et al.

The Research Article, “Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes,” by Han et al. has been corrected to list Alp Yurter as an additional author. He was responsible for the generation of the human HPV-specific TCR transgenic lines utilized in Figs. 4B/4D, 6A/6I/6J and carried out the T cell assay experiments necessary to generate the HPV-related portions of Figs. 4 and 6.

Generation of a HuTCR mouse platform-derived MAGE-A1-directed high-affinity TCR with superior potency versus human-derived TCRs.

e14515 Background: As cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are suppressed via negative thymic selection. Therefore, patient- or donor-derived TCRs are typically of low affinity and result in a reduced antitumor effect. Using our proprietary HuTCR platform, which consists of mouse lines carrying the full human TCR α/β loci in combination with common human HLA alleles, we have isolated high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development. Furthermore, we validated MAGE-A1 as a potential cancer therapy target by using immunohistochemistry to evaluate expression in several major tumor types and healthy tissue. Methods: Using scRNAseq, TCRs were isolated from HuTCR mice. Human-derived MAGE-A1-specific TCR sequences were obtained from publicly available databases. All TCRs were expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional avidity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope peptides and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by co-culture with a panel of tumor cell lines varying in MAGE-A1 and/or MHC-class-I expression. MAGE-A1 expression on protein level was evaluated by immunohistochemistry. Results: Immunization of HuTCR mice with the antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR affinities ranging from 0.3 nM to 3 nM. By comparison, human-derived TCRs exhibited generally lower functional avidity from 3 nM to 60 nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cell lines expressing low MAGE-A1 and/or HLA-A2. Immunohistochemical analysis of MAGE-A1 expression in healthy tissues demonstrated highly selective expression of MAGE-A1 in testis, only. Screening for expression confirmed that a significant proportion of several major cancer types expresses MAGE-A1 as reported by various other groups [reviewed in Curr Opin Cell Biol. 2015 December; 37: 1–8]. Conclusions: The HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. In addition, it was confirmed that MAGE-A1 has a highly selective expression pattern in healthy tissues (testis, only), but shows distinct expression in several major human tumor types.

Abstract LB-085: ALPN-202 combines checkpoint inhibition with conditional T cell costimulation to overcome T cell suppression by M2c macrophages and improve the durability of engineered T cell anti-tumor responses

Abstract Background: Despite significant clinical benefit of checkpoint inhibitors (CPI) in some settings, unfortunately, the majority of patients still fail to respond and/or develop resistance. This is likely due to a complex set of factors including, but not limited to, the presence of immunosuppressive myeloid cells and/or T cell exhaustion due to chronic TCR activation in the absence of sufficient costimulation. ALPN-202, a variant CD80 vIgD™-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and blocks PD-L1 and CTLA-4, was designed to overcome several of these suppressive mechanisms. The objective of these studies was to measure effects of ALPN-202 on the suppression of T cell activation by M2c macrophages and to determine in vivo effects of ALPN-202 on T cell exhaustion in an adoptively transferred human TCR transgenic tumor model. Methods: M2c macrophages differentiated from primary monocytes with M-CSF and IL-10 were cocultured for 72hr with T cells, anti-CD3 and a titration of ALPN-202, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies. Cytokine concentrations in the culture media were measured at 24 and 72 hrs, and T cells and macrophages characterized at 72 hrs by flow cytometry. To measure its anti-tumor activity and effect on T cell exhaustion in vivo, ALPN-202 was evaluated in a humanized model using anti-HPV E6 TCR-transduced human T cells transferred into immunodeficient NSG mice bearing HPV+ SCC152 squamous cell tumors stably expressing PD-L1. Tumor volume was measured twice weekly and on day 38 tumors were harvested, digested, and intratumoral T cells characterized for expression of exhaustion markers. Results: In the in vitro coculture assay, ALPN-202 increased T cell proliferation and production of IL-2, IFNγ, TNFα, GM-CSF, IL-6, and IL-21 significantly more potently than CPI alone. Additionally, the M2c macrophages in the presence of ALPN-202 displayed a dose-dependent elevation of MHC II, CD80, and CD86, indicative of a more pro-inflammatory, M1-like phenotype. In the SCC152 tumor model, ALPN-202 induced a more robust anti-tumor response than CPI while the intratumoral T cells expressed lower levels of exhaustion markers. Conclusions: As a dual checkpoint inhibitor and conditional CD28-costimulator, ALPN-202 induced robust and selective T cell costimulation in vitro which overcame M2c macrophage-mediated suppression more potently than CPI alone. Intriguingly, not only were T cells vigorously activated, but M2c macrophages transitioned to a more M1-like proinflammatory phenotype. In a humanized tumor model, ALPN-202 treatment resulted in potent anti-tumor activity with reduced signs of T cell exhaustion in the TME. The data suggest that by combining CD28 costimulation with CPI, ALPN-202 may provide a more robust and persistent anti-tumor T cell response compared to CPI alone. Citation Format: Mark F. Maurer, Siddarth Chandrasekaran, Katherine Lewis, Sherri Mudri, Kayla Kleist, Fariha Ahmed-Qadri, Chelsea Gudgeon, Steven D. Levin, Stacey R. Dillon, Kristine M. Swiderek, Stanford L. Peng. ALPN-202 combines checkpoint inhibition with conditional T cell costimulation to overcome T cell suppression by M2c macrophages and improve the durability of engineered T cell anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-085.