TCR Expression Research Articles

Abstract 2829: Non-viral precision genome engineering enables personalized adoptive neoTCR T cell therapy for cancer including multiple additional edits that improve the activity of neoTCR T cells by enhancing CD4 T cell antigen sensitivity and conferring resistance to TGFβ

Abstract Personalized autologous TCR-T cell therapies targeting neoepitopes (neoE) derived from tumor-specific mutations are a compelling approach for the treatment of patients with solid tumors. Using the ultra-sensitive imPACT Isolation Technology®, antigen-experienced, neoE-specific CD8 T cells are captured from the blood of patients with solid cancers followed by cloning of the cognate, neoE-specific, MHC class I-restricted T cell receptors (neoTCRs). DNA-mediated (non-viral) precision genome engineering technology is then used to engineer autologous CD8 and CD4 T cells to express the neoTCR. We have further built upon this platform to modify neoTCR-T cells to address potential sources for tumor immune evasion. The versatility of this single-step gene editing platform is demonstrated here by the presentation of a neoTCR T cell product with knockout of endogenous TCRα and TCRβ and the simultaneous expression of a neoTCR with CD8 co-receptor (CD8coR) from the same expression cassette along with knockout of TGFβ receptor 2 (TGFBR2). We have previously shown that CD8coR expression augments the activity of CD4 T cells engineered to express MHC class I restricted neoTCRs, by increasing CD4 neoTCR T cell helper and effector function. TGFβ is known to promote tumor growth, metastasis, and epithelial to mesenchymal transition (EMT) and to inhibit effector immune responses while promoting fibrosis and the differentiation of inhibitory cell types such as regulatory T cells. TGFβ is expressed by many tumor types and high TGFβ expression is associated with worse prognosis for several cancer subtypes including colorectal, lung and glioblastoma. TGFBR2 is a critical mediator of TGFβ signaling, resulting in potent, inhibitory effects on T cell function. Our data show that simultaneous ablation of TGFBR2 signaling and expression of CD8coR was achieved with high efficiency, resulting in fully functional CD8 and CD4 neoTCR-T cells. Deletion of TGFBR2 with or without CD8coR expression, together with the expression of a neoE-targeted TCR preserved T cell effector function in the presence of inhibitory concentrations of TGFβ. Importantly, the combination of TGFBR2 knockout and CD8coR expression resulted in additive benefits, providing proof-of-concept for modifying two orthogonal features to improve neoTCR T cell function. Altogether, these results demonstrate the applicability of this versatile, precision genome engineering platform technology to yield enhanced, next-generation neoTCR-T cell therapies to expand the potential for clinical benefit in persons with solid cancers. Citation Format: Charles W. Tran, Kayla Lee, Bhamini Purandare, James Byers, Michael M. Dubreuil, William Lu, Michal Mass, Kyle Jacoby, Stefanie J. Mandl, Barbara Sennino. Non-viral precision genome engineering enables personalized adoptive neoTCR T cell therapy for cancer including multiple additional edits that improve the activity of neoTCR T cells by enhancing CD4 T cell antigen sensitivity and conferring resistance to TGFβ [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 2829.

Abstract 2827: Non-viral gene editing enables multiplex single-step precision genome engineering for adoptive cell therapies

Abstract PACT Pharma has developed a robust single-step, targeted, non-viral method for the manufacturing of personalized adoptive cells therapies for the treatment of solid cancers (NCT03970382). For this, we insert and express a neoepitope-specific T cell receptor (neoTCR) from the endogenous locus while simultaneously abolishing the expression of endogenous TCR. This results in neoTCR-specific T cells in which neoTCR expression is naturally regulated and not impeded by competition for CD3 by the endogenous TCR. We furthermore designed a system to additionally express CD8 coreceptor along with the neoTCR on the same RNA transcript from the native TCR promoter. Both methods, currently in clinical testing, allow for the timely generation of several billion T cells expressing neoTCRs using our GMP cell manufacturing procedure. Nevertheless, the suppressive nature of some tumor microenvironments may require cells engineered with further modifications for long lasting therapeutic outcomes in certain patients. Here we describe how the flexibility of a non-viral system allows for additional complex modifications in a single step. First, we demonstrate an additional gene knock-out in the TGFBR2 gene with simultaneous TCR replacement, making the engineered cells resistant to the immunosuppressive effects of TGF-β while still maintaining neoepitope-specific recognition of the tumor. Second, we demonstrate knock-down in addition to TCR replacement. For some targets, reduction of gene expression may be favored over complete knock-out. Accordingly, we have designed an shRNA expression construct that can efficiently knock-down transcript levels of a given gene in cells expressing the neoTCR without the generation of additional double-stranded breaks, allowing for enhanced T cell function in the absence of additional genomic breaks. Third, we demonstrate a non-viral knock-in strategy to express transgenes with transcriptional regulation independent from the neoTCR. This strategy generates a product from which two genes, the neoTCR plus an additional transgene regulated by its own promoter, can be precision genome engineered into the same cell using homology directed templates that far exceed the size limitations of AAV. This technology can be successfully applied for the expression of intracellular, secreted, and membrane-bound proteins as well as proteins expressed only during the activated T cell state. In conclusion, our single-step non-viral precision genome engineering technology is highly versatile with the ability to knock-out, knock-down, knock-in, and precisely regulate additional genes in a single step. These modifications have the potential to expand the applicability of T cell drug products and are broadly applicable to a variety of other cellular therapies and research models. Citation Format: William Lu, James Byers, Charles W. Tran, Michal Mass, Tanu Shenoy, Shirley Sun, Kyle Jacoby, Stefanie Mandl. Non-viral gene editing enables multiplex single-step precision genome engineering for adoptive cell therapies [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 2827.

Abstract LB009: A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection

Abstract The approval and commercial launch of multiple first-generation CD19- or BCMA-directed, autologous CAR-T cell products have laid the foundation and opened a path for the development of more advanced cellular therapeutics, including CAR-T cell products with next-generation capabilities. Among these newer designs, allogeneic cell therapies are positioned to unlock the broad potential of engineered immune cells as a leading therapeutic modality. However, expansion, persistence, armoring, and trafficking of allogeneic CAR-T cells are critical to achieving long-term efficacy. Caribou Biosciences is advancing a BCMA-specific allogeneic CAR-T cell product candidate, CB-011, with an immune cloaking approach that includes both the removal of the endogenous B2M protein and the insertion of a B2M-HLA-E-peptide (B2M-HLA-E) fusion protein transgene. This strategy is designed to blunt CAR-T cell rejection by both recipient T cells and NK cells, and CB-011 is in preclinical development for relapsed or refractory multiple myeloma (r/r MM). We used Cas12a chRDNA guides in the manufacture of CB-011 to make four edits, including site-specific insertion of a proprietary humanized anti-BCMA CAR into the TRAC locus with high specificity and efficiency, thus eliminating TCR expression to prevent graft-versus-host disease (GvHD). In addition, we inserted a gene encoding a B2M-HLA-E fusion protein into the native B2M gene locus. This method simultaneously prevents the expression of the native B2M protein and expresses a minor HLA class I antigen HLA-E to blunt both T- and NK-mediated rejection of the CAR-T cells by the recipient’s immune system. B2M is a protein that stabilizes all HLA class I antigens on the cell surface, therefore its knockout eliminates endogenous HLA class I presentation on the surface of the CAR-T cells. To demonstrate that the B2M-HLA-E fusion protein expression protects CB-011 from NK-mediated cell killing, we developed an in vitro competition assay in which CB-011 cells are co-incubated with NK cells. We observed that CAR-T cells expressing the B2M-HLA-E fusion have a survival advantage over cells that do not express the fusion in the presence of NK cells in vitro, indicating that they could resist killing by a recipient’s NK cells and potentially circulate longer. The BCMA-specific CAR leads to long-term survival in mice bearing established orthotopically-engrafted MM tumor cells. Furthermore, mice treated with high doses of CB-011 did not experience symptoms typical of GvHD. This strategy should enable CB-011 CAR-T cells to remain in circulation longer in recipients, providing for increased potential of antitumor activity. Citation Format: Elizabeth Garner, Emilie Degagne, Suparna Roy, Paul Donohoue, Tristan Fowler, Morena Stanaway, Vanina Vicena, Devin Mutha, Benjamin Schilling, McKay Shaw, Mara Bryan, Leslie Edwards, Stephen Smith, Bryan Kohrs, Lynda Banh, Kyle McSweeney, Justin Skoble, Steven Kanner. A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection [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 LB009.

Directing T cell alloreactivity against solid tumors through tumor antigen dependent TCR expression

Abstract A major challenge in T cell therapy against solid tumors is the heterogeneity of tumor antigen (TA) expression. Elimination of TA+ tumor cells often fails to suppress tumor growth due to the expansion of TA− tumor cells. To address this, we developed tumor-activated alloreactive T cells (TAATs) that display alloreactivities only in tumors. TAATs lack endogenous CD3ɛ expression and contain a tumor sensing mechanism that drives the expression of an exogenous copy of CD3ɛ in the presence of TA+ tumor cells. TAATs are therefore inactive in normal tissues due to retention of TCR/CD3 in the ER but become alloreactive in tumors. TAATs attack all HLA+ cells in tumors including tumor cells, stromal cells and cells that suppress immune functions. To generate TAATs, HLA-mismatched allogeneic T cells were stimulated with HLA class I+ breast cancer cells to expand alloreactive T cells. Endogenous CD3ɛ expression was then knocked out using CRISPR/Cas9. The T cells were also modified to express a Her2-specific synthetic Notch receptor that releases an intracellular domain containing a Gal4-VP64 transcription activator upon receptor engagement. In addition, an expression cassette with a CD3ɛ coding sequence driven by a promoter containing Gal4 binding sites was introduced to the T cells. When co-cultured with Her2+ cancer cells, TAATs restored TCR/CD3 expression and were able to degranulate and produce IFNγ in response to anti-CD3 stimulations. TAATs were also able to kill Her2+ cancer cells in co-cultures and kill Her2− cancer cells in the presence of Her2+ cancer cells. Compared with CAR T cells, TAATs may achieve superior tumor suppression through attacking a wider range of cells in tumors and by overcoming the immunosuppressive tumor microenvironment.

Abstract P5-13-30: Analysis of αβ and γδ circulating T cells in the PHERGain randomized phase 2 trial for patients with HER2-positive early breast cancer receiving neoadjuvant trastuzumab and pertuzumab without chemotherapy: LINGain

Abstract Background: The presence of stromal tumor-infiltrating lymphocytes (TILs) represents an independent prognostic factor in HER2-positive early-stage breast cancer (EBC) treated with trastuzumab/pertuzumab-containing regimens. Among distinct subsets of TILs, conventional CD8+ αβ T cells require TCR signaling as a part of adaptive immunity, while γδ T cells display also innate-like activity via the NKG2D receptor contributing to a very rapid tumor immunosurveillance. Specific γδ T cell subsets were associated with remission and improved overall survival of patients with triple-negative breast cancer. However, very little is known about circulating αβ and γδ T cells and their immunological status in HER2-positive breast cancer. In this substudy, we aimed to characterize the αβ and γδ T cell subsets and the association with clinical outcome in peripheral blood of patients with HER2-positive EBC enrolled in the PHERGain trial, which assessed the possibility of chemotherapy de-escalation with neoadjuvant dual HER2 blockade with trastuzumab and pertuzumab using an 18F-fluorodeoxyglucose-PET and pathological response-adapted strategy. Methods: Peripheral blood was obtained from 24 consecutive patients who were assigned to the trastuzumab and pertuzumab group (+/- endocrine therapy as per hormone receptor status) before randomization (baseline) and after 2 treatment cycles (6 weeks). Blood samples were also collected from 48 age-matched healthy donors who represented the control group. Absolute numbers of CD3+, CD3+/CD4+, CD3+/CD8+, and CD3+/CD56+ according to the TCR expression, and annexin V apoptotic rate on αβ and γδ T cells were evaluated by flow cytometry. Subset distribution of T cell differentiation within naïve, central memory, effector memory, and terminally differentiated effector memory cells was also determined. The changes in the frequency of peripheral T cells and rate of apoptotic subsets between timepoints, patients, and healthy donors were compared with Wilcoxon test. The analyses were set at two-sided 0.05 level of significance. Results: Among 24 patients with evaluable blood samples at both timepoints, median age was 50.5 years (IQR 45.8-61), 45.8% had node-positive disease, 79.2% had hormone receptor-positive status, 79.2% had tumors with HER2 IHC 3+ status, and 54.2% achieved a pathological complete response (ypT0/is ypN0) after treatment. At baseline, levels of αβ and γδ T cells in EBC patients were significantly lower than levels in healthy subjects (P ≤0.05). After 6 weeks of study treatment, these levels in EBC patients did not significantly differ from those at baseline. Baseline rates of apoptotic subsets were higher in EBC patients than rates in healthy subjects (P <0.01), but after 6 weeks of study treatment all apoptotic subsets were significantly reduced in EBC patients compared with those at baseline (P ≤0.05). No evidence of association was found between peripheral T cells and pathological complete response in EBC patients. Conclusions: These data suggest a potential involvement of T cell apoptosis on the mechanism of action mediated by dual HER2 blockade with trastuzumab and pertuzumab in patients with HER2-positive EBC. However, further validation is required. Additional data on the subset distribution of T cell differentiation status will be presented at the meeting. Citation Format: Juan Carlos Andreu-Ballester, José Manuel Pérez-García, Begoña Bermejo, Vicente Carañana, Vega Iranzo, Joaquín Gavilà, Ana Santaballa, María Carmen Gómez-Soler, Miguel Sampayo-Cordero, Andrea Malfettone, Javier Cortes, Antonio Llombart-Cussac. Analysis of αβ and γδ circulating T cells in the PHERGain randomized phase 2 trial for patients with HER2-positive early breast cancer receiving neoadjuvant trastuzumab and pertuzumab without chemotherapy: LINGain [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-30.

Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Decoding Clonal Evolution in Relapsed T Cell Acute Lymphoblastic Leukemia at Single Cell Resolution

T-lineage acute lymphoblastic leukemia (T-ALL) comprises approximately 10-15% of pediatric ALL cases with distinct feature in biology and largely inferior outcome compared to B-ALL. Growing evidence has reflected pivotal roles of clonal evolution in T-ALL recurrence, but bulk sequencing may not serve as the perfect model to reliably infer clonal heterogeneities and their immunomodulatory milieu during leukemia development. In this study, single-cell sequencing was applied to uncover leukemic clonal relationships with relapse throughout chemotherapy in T-ALL at a more accurate resolution.We performed bulk whole-exome sequencing for sorted CD7 + BMMCs from 5 pairs of diagnosis-relapse (Dx_Rel) samples, revealing a series of well-reported hotspot mutations in T-ALL. Among those, we observed diagnosis-specific variations and relapse-emerged variations, suggesting the putative correlations with chemo-resistance. Transcriptomic sequencing highlighted additional stemness and metabolic abnormalities underlying leukemic re-occurrence. Incorporated Dx_Rel paired ATAC-seq depicted relapse-specific activated chromatin regions, such as ELK1, ELK4, RUNX1.To dissect clonal diversities within and across the 5 Dx_Rel T-ALL pairs, we carried out high-throughput droplet-based 5'-single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (scTCR-seq). By performing unsupervised clustering of scRNA-seq profiles encompassing 10 samples, we identified 23 distinct T-lineage clusters (Cluster 0-22) based on the two-dimensional UMAP visualization. In 2 out of 5 patients (T593 and T788), diffusion map of T-lineage sub-clusters between diagnostic and relapsed samples appeared to be almost identical, while distinct shifts from diagnosis to relapse in the compositions have been observed in the other 3 out of 5 patients (T956, T723 and T856). Besides, it was noteworthy that two T-cell sub-clusters were concluded as “normal” T cells (Cluster 9 and 12) uniformly presented in both diagnostic and relapsed diffusion of T-cell sub-clusters across 5 Dx_Rel, from which TCR repertoires and expression profiles could well discriminate leukemic cells.Next, we sought to further deconvolute the clonal evolution patterns for T-ALL Dx_Rel pairs. We observed that except in T788 lacking of clonal TCRs, dominant diagnostic clones of the other 4 patients diminished (T593) or vanished (T956, T723, T856) at relapse, sparing newly emerged subclones predominantly substituted at relapse. We clearly depicted two distinct patterns of evolutionary trajectories in these 4 Dx_Rel pairs by comprehensively mapping hierarchical TCR clonotypes onto leukemic clonotypes at single cell levels. Specifically, in T956 and T723, we observed significant outgrowth of incidental diagnostic sub-clones at relapse, whereby surrogate TCR repertoires correspondingly enumerated, suggestive of dynamic shifts in dominant clone over continuous chemo-exposure. Whereas in T593 and T856, expanding clones at relapse were showed up with completely different gene signatures from the diagnostic ones, but dominant clones at diagnosis and relapse were surprisingly presented with identical TCR repertoires. This was undoubtedly informative of leukemic “clonal drift” within which hypothetical intrinsic transformation happened to the same subclones over persistent chemotherapy.Besides, we took advantage of our well-discriminated model to fully delineated the involvement of “normal” T subclusters in leukemic latency and chemo-responsiveness. By analyzing TCR repertoires in combined with expression profiles, we noted that “normal” T cells infiltrated by T-ALL were majorly distributed in CD8-effector sub-clusters compared to those from healthy donor, suggesting a robust leukemic stimulation on effector CD8 signaling in T-ALL microenvironment.Collectively, our presented study accurately distinguished leukemic cells from normal T cells in T-ALL at a single-cell resolution. By tracking transcriptomic profiles within and across Dx_Rel T-ALL pairs, we further identified distinct clonal evolutionary patterns, which may determine diversified fates of leukemic clones in response to therapeutic pressures. In the meantime, we provided a comprehensive phenotypic view on “normal” T cells under leukemic prevalence and re-occurrence, extending significant implications for future precise immunotherapies. DisclosuresNo relevant conflicts of interest to declare.

Memory Phenotype in Allogeneic Anti-BCMA CAR-T Cell Therapy (P-BCMA-ALLO1) Correlates with In Vivo Tumor Control

The emergence of CAR-T cell therapy has transformed the treatment of the previously refractory/relapsed multiple myeloma (MM). Yet, autologous CAR-T cells suffer from inconsistent manufacturing, long manufacturing timelines, and high cost, which can limit patient accessibility. To address these issues, we engineered a fully allogeneic anti-BCMA CAR-T cell candidate for MM from healthy donors (P-BCMA-ALLO1). Herein, we demonstrate that P-BCMA-ALLO1 maintains a T stem cell memory phenotype (T SCM) through genetic editing, which correlates with antitumor efficacy.Using Poseida's proprietary non-viral piggyBac® (PB) DNA Delivery System, in combination with the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System and a proprietary “booster molecule”, we generated P-BCMA-ALLO1 from healthy donor T cells. We used CC to eliminate surface expression of TCR and MHC class I to make fully allogeneic CAR-T cells. In addition to the CAR molecule, PB enables the delivery of a selectable marker allowing the generation of a final cell product that is >95% CAR-positive. The inclusion of the “booster molecule” in the manufacturing process improves the expansion of gene-edited cells without compromising memory phenotype or function. This process can produce up to hundreds of patient doses from a single manufacturing run using one healthy donor, thereby significantly reducing manufacturing cost per dose. We characterized the phenotype and functionality of P-BCMA-ALLO1 using flow cytometry and Nanostring to assess their memory phenotype at both the protein and mRNA levels. Also analyzed was antitumor toxicity and proliferative capacity through multiple rounds of activation using in vitro co-culture assays and serial restimulation, respectively. The relationship of all characterizations with in vivo efficacy was then determined, as defined by control of tumor growth in an immunodeficient RPMI-8226 subcutaneous murine tumor model.We found that P-BCMA-ALLO1 is comprised of a high frequency of T SCM after editing (Fig. 1), and the maintenance of that memory phenotype correlates with antitumor efficacy. In vivo, these CAR-T cells are potent in controlling tumor growth, comparable to or better than autologous anti-BCMA CAR-T cells. Our analysis revealed that the expression of memory markers at the surface protein level (CD27, CD62L, CD127, CCR7) and mRNA level significantly correlate with in vivo tumor control. Conversely, suboptimal research products with worse in vivo outcomes express a more exhausted gene expression profile. We reveal from our analysis that the most effective P-BCMA-ALLO1 in vivo share similar characteristics: (1) these products were a result of efficient manufacturing, with >90% CAR+ and >99% TCR-; (2) they carry a memory phenotype, with 50-70% T scm and high proliferative capacity after multiple rounds of restimulation; (3) they are >90% viable; and (4) they show strong antitumor efficacy both in vitro and in vivo.We demonstrate that Tscm percentage in the final product correlates with antitumor activity. P-BCMA-ALLO1 is advancing rapidly towards the clinic (NCT04960579) to positively impact the outcomes of CAR-T therapy for MM patients.Figure 1: Memory composition of P-BCMA-ALLO1 research products. P-BCMA-ALLO1 consists mostly of stem cell memory (T scm) and central memory (T cm) T cells that are CD62L + as opposed to effector memory (T em) and effector (T eff) T cells. [Display omitted] DisclosuresTseng: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhang: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cranert: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Richter: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marquez: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Qiu: Poseida Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Cho: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tong: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Domingo: Poseida Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Argus: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sparks: Poseida Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Ostertag: Poseida: Current Employment, Current equity holder in publicly-traded company. Coronella: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shedlock: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company.

ERAP1 Controls the Autoimmune Response against Melanocytes in Psoriasis by Generating the Melanocyte Autoantigen and Regulating Its Amount for HLA-C*06:02 Presentation.

Autoimmune diseases develop when autoantigens activate previously quiescent self-reactive lymphocytes. Gene-gene interaction between certain HLA class I risk alleles and variants of the endoplasmic reticulum aminopeptidase ERAP1 controls the risk for common immune-mediated diseases, including psoriasis, ankylosing spondylitis, and Behçet disease. The functional mechanisms underlying this statistical association are unknown. In psoriasis, HLA-C*06:02 mediates an autoimmune response against melanocytes by autoantigen presentation. Using various genetically modified cell lines together with an autoreactive psoriatic TCR in a TCR activation assay, we demonstrate in this study that in psoriasis, ERAP1 generates the causative melanocyte autoantigen through trimming N-terminal elongated peptide precursors to the appropriate length for presentation by HLA-C*06:02. An ERAP1 risk haplotype for psoriasis produced the autoantigen much more efficiently and increased HLA-C expression and stimulation of the psoriatic TCR by melanocytes significantly more than a protective haplotype. Compared with the overall HLA class I molecules, cell surface expression of HLA-C decreased significantly more upon ERAP1 knockout. The combined upregulation of ERAP1 and HLA-C on melanocytes in psoriasis lesions emphasizes the pathogenic relevance of their interaction in patients. We conclude that in psoriasis pathogenesis, the increased generation of an ERAP1-dependent autoantigen by an ERAP1 risk haplotype enhances the likelihood that autoantigen presentation by HLA-C*06:02 will exceed the threshold for activation of potentially autoreactive T cells, thereby triggering CD8+ T cell-mediated autoimmune disease. These data identify ERAP1 function as a central checkpoint and promising therapeutic target in psoriasis and possibly other HLA class I-associated diseases with a similar genetic predisposition.

Téloméropathies de recrutement hématologique : étude de 15 cas

IntroductionTelomeres are composed of a repeated sequence of double-stranded nucleotides TTAGGG and numerous proteins including the Shelterin complex. Their main role is to maintain the stability of the genome during cell replication through a mechanism of copying the repeted sequence by the telomerase complexe. All the diseases involving a deregulation of this complex are now grouped together under the term telomeropathies. They are difficult to diagnose and manage. Our objective was to describe the clinico-biological characteristics and treatments used, in patients affected by telomeropathies previously seen by an hematologist followed at the Lille University Hospital Center. MethodsThis is a retrospective, single-center study carried out within the department of internal medicine-clinical immunology, Reference center for rare autoimmune and systemic diseases at Lille University Hospital Center between 2005 and 2020 including all patients followed for telomeropathy. ResultsProbands and relatives were included. Fifteen patients were studied from 10 independant families. Sixty percent had an heterozygous TERC gene mutation. Sixty seven percent had haematological diseases including macrocytosis, anemia and/or thrombocytopenia, 20 % had a fibrotic hepatic disease, 27 % had a fibrotic pulmonary disease. Lymphocyte immunophenotyping showed a double negative T lymphocyte population with γδ TCR expression in 5 (33 %) patients. Forty-seven percent of the patients had not received any treatment. Twenty-seven percent were on androgen therapy. Twenty percent had received cyclosporine and 13 % anti-lymphocyte serum in the context of initial misdiagnosis. ConclusionIt is important to be aware of the complexity of telomeropathies, a differential diagnosis of immune aplastic anemia, in order to optimize management and avoid inappropriate treatments. Allografting of hematopoietic stem cells is the only potentially curative treatment. Our analysis found particularities in immunophenotyping lymphocyte not previously described to our knowledge, whose physiopathological imputability remains to be demonstrated.

Monitoring malignant T-cell clones by direct TCR expression assay in patients with leukemic cutaneous T-cell lymphoma during extracorporeal photopheresis.

Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vβ usages after adoptive immunotherapy. This study was performed to test the effectiveness of DTEA in assessing malignant T-cell clones in L-CTCL patients. Total RNAs extracted from peripheral blood mononuclear cells of patients before starting extracorporeal photopheresis (ECP) (n=15) and during therapy at 3months and 6 months (n=12) were used for DTEA, with customized probes for 45 TCR-Vα and 46 TCR-Vβ family members. At baseline, DTEA detected TCR-Vβ clones in all 15 patients (100%) compared to flow cytometry that detected TCR-Vβ clones in 9 of 13 patients (69.2%). In addition to predominant TCR-Vβ clones, DTEA also detected additional TCR-Vβ clones in 8 of 15 patients (53.3%). Furthermore, DTEA simultaneously identified clonal TCR-Vα usages, which allowed us to pair TCR-Vα and TCRVβ usages by malignant T-cells and identify diversified clonotypes. Changes in the relative frequencies of clonal TCR-Vβ and TCRVα usages over therapy were consistent with patients' clinical responses. Our results indicate that DTEA can effectively assess malignant T-cell clones by detecting clonal TCR-Vα and TCR-Vβ usages. By providing a global view of TCR repertoires, DTEA may also help us understand the origin(s) of malignant T-cells and pathogenesis of CTCL.

Brief homogeneous TCR signals instruct common iNKT progenitors whose effector diversification is characterized by subsequent cytokine signaling

Innate-like Tcell populations expressing conserved TCRs play critical roles in immunity through diverse developmentally acquired effector functions. Focusing on the prototypical lineage of invariant natural killer T (iNKT) cells, we sought to dissect the mechanisms and timing of fate decisions and functional effector differentiation. Utilizing induced expression of the semi-invariant NKT cell TCR on double positive thymocytes, an initially highly synchronous wave of iNKT cell development was triggered by brief homogeneous TCR signaling. After reaching a uniform progenitor state characterized by IL-4 production potential and proliferation, effector subsets emerged simultaneously, but then diverged toward different fates. While NKT17 specification was quickly completed, NKT1 cells slowly differentiated and expanded. NKT2 cells resembled maturing progenitors, which gradually diminished in numbers. Thus, iNKT subset diversification occurs in dividing progenitor cells without acute TCR input but utilizes multiple active cytokine signaling pathways. These data imply a two-step model of iNKT effector differentiation.

Production of Universal Anti-Bcma CAR-T Cells with Reduced Alloreactivity, but Potent Effector Function for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR)-T therapy has recently shown great promise against multiple myeloma (MM) by targeting the B cell maturation antigen (BCMA). However, the production of autologous CAR-T cells on an individualized basis has significant challenges such as manufacture time, reproducibility, consistency and cost. In addition, viral-vector-produced CAR-T cells tend to be comprised of differentiated T cell subsets, which correlate with poor in vivo durability. Using the high-fidelity NextGEN™ (NG) CRISPR gene editing system in combination with the non-viral piggyBac™ (PB) DNA transposition technology, which produces CAR-T cells predominantly in a highly desirable stem cell memory T cell subset, we have developed P-BCMA-ALLO1, an off-the-shelf allogeneic CAR-T cell product that overcomes these limitations and can be given to any MM patient.The NG system was used to disrupt both TCR and MHCI expression in P-BCMA-ALLO1 to reduce possible alloreactivities. The DNA cleavage specificity is guaranteed by a truly dimeric system requiring the simultaneous binding of two obligate homodimers. High-throughput DNA sequencing analysis was performed on human T cells with gene disruption of TCRα, β2M or PD-1. Indel frequencies of amplified genomic on-target and off-target sites were evaluated from T cells that had been edited with either single or dual guide RNAs (gRNA)s. Herein, an average of 100,000 sequences were analyzed per site and non-edited T cells were included as a background reference. For these targets, the NG system demonstrated high-efficiency gene editing (>84% for TCRα, >64% for β2M, and >50% for PD-1) that was performed in resting T cells. No indel events were detected at any off-target site, proving the exquisite specificity of this system.P-BCMA-ALLO1 was incapable of mediating allo-responses such as graft-vs-host disease (GvHD) and graft rejection. Using a clinically-scalable purification method, P-BCMA-ALLO1 cells were enriched to be >99.5% TCR/MHCI double-negative and alloreactivities were evaluated by standard mixed lymphocyte reaction for either cell proliferation or IFNγ production in two separate assays: a 7-12 day CFSE-based cell proliferation assay and a 20-hour IFNγ-ELISPOT assay. Compared to non-edited CAR-T cells, P-BCMA-ALLO1 did not exhibit any detectable response to allogeneic PBMC stimulation, nor did it trigger any allogeneic T cell proliferative response above background level of cell proliferation or IFNγ production.Pre-clinical evaluation of P-BCMA-ALLO1 demonstrated optimal T cell phenotype and potent in vitro and in vivo efficacy. More than 95% of the P-BCMA-ALLO1 cells were CAR+ and greater than 50% of the product were CD45RA+CD62L+ stem cell memory T cells (TSCM), a highly desirable early memory T cell subset that exhibits the greatest capacity for self-renewal and the multipotent capacity to the entire spectrum of memory and effector T cell subsets. In addition, the CAR-T cells did not express the exhaustion markers PD-1, TIM-3 and LAG-3, nor did they exhibit any evidence of tonic signaling as IFNγ production in the absence of target antigen. In a short-term 24-hour in vitro co-culture assay with BCMA+ MM cell lines, P-BCMA-ALLO1 secreted high levels of IFNγ and mediated robust (70-90%) killing of cell targets. In addition, more than 80% of the cells proliferated in a longer 72-hour CFSE-based proliferation assay. P-BCMA-ALLO1 also showed strong potency in a NSG mouse model engrafted with MM.1S cells, an aggressive human MM-derived tumor model. While all control animals showed rapid tumor growth by bioluminescent imaging (BLI) and succumbed to disease within five weeks, all treated mice survived for the duration of the study. After P-BCMA-ALLO1 administration, tumor burden was rapidly reduced to the limit of detection by BLI within 7-10 days. Lastly, several occurrences of tumor relapse were observed in some of these animals, but were subsequently controlled without re-administration of the product. Altogether, these data demonstrate that P-BCMA-ALLO1 exhibited significantly reduced alloreactivity yet retained potent effector function, suggesting its safe and effective use for treating any MM patient. DisclosuresWang:Poseida Therapuetics: Employment. Barnett:Poseida Therapeutics, Inc.: Employment. Martin:Poseida Therapeutics, Inc.: Employment. Hermanson:Poseida Therapeutics: Employment. Li:Poseida Therapuetics: Employment. Smith:Poseida Therapeutics, Inc.: Employment. Wu:Poseida Therapuetics: Employment. Cranert:Poseida Therapuetics: Employment. Rengarajan:Poseida Therapeutics, Inc.: Employment. Tan:Poseida Therapeutics, Inc.: Employment. Tong:Poseida Therapuetics: Employment. Codde:Poseida Therapeutics, Inc.: Employment. Down:Poseida Therapeutics, Inc.: Employment. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment.

Chromatin assembly factor 1B critically controls the early development but not function acquisition of invariant natural killer T cells in mice.

CD4+ CD8+ double-positive thymocytes give rise to both conventional TCRαβ+ T cells and invariant natural killer T cells (iNKT cells), but these two kinds of cells display different characteristics. The molecular mechanism underlying iNKT cell lineage development and function acquisition remain to be elucidated. We show that the loss of chromatin assembly factor 1B (CHAF1b) maintains the normal development of conventional TCRαβ+ T cells but severely impairs early development of iNKT cells. This dysregulation is accompanied by the impairment in chromatin activation and gene transcription at Vα14-Jα18 locus. Notably, ectopic expression of a Vα14-Jα18 TCR rescues Chaf1b-deficient iNKT cell developmental defects. Moreover, cytokine secretion and antitumor activity are substantially maintained in Vα14-Jα18 TCR transgene-rescued Chaf1b-deficient iNKT cells. Our study identifies CHAF1b as a critical factor that controls the early development but not function acquisition of iNKT cells via lineage- and stage-specific regulation.

TetTCRII-SeqHD: High-dimensional analysis of antigen-specific ex vivo CD4+ T cells

Abstract Objective: We present a method for high-throughput and inexpensive generation of DRB1 tetramers to assess CD4+ T cell specificity with special applications in oncoimmunology. Methods: Peptides were generated by in vitro transcription and translation (IVTT) of dsDNA. These peptides were then loaded onto DRB1 MHC molecules via chemical exchange and tetramerized using DNA barcoded streptavidin as previously described. As a proof of concept, we generated 250 DNA-tetramers bearing peptide segments from a variety of naïve and experienced antigens. These tetramers were pooled and used to stain primary CD4+ T cells from healthy donors. These cells were then labeled with Abseq antibodies (BD Biosciences), sorted and loaded onto the BD Rhapsody. Results: We analyzed ~5000 CD4+ T cells from healthy peripheral blood and assessed their phenotype, specificity and TCR sequence. We demonstrated that this technique robustly identifies and characterizes antigen-specific CD4+ T cells. It replicates previous findings in low-throughput systems in terms of clonality and phenotype distribution across memory and naïve T cell phenotypes. Given these results, we then analyzed tumor infiltrating lymphocytes and identified foreign specific cells within this compartment. We also found explicit differences in the phenotypes of antigen specific cells within the tumor and a paired healthy tissue sample. Conclusions: In conclusion, we validate the use of a high-throughput technology for assessing the specificity, phenotype, TCR, and gene expression patterns in antigen-specific CD4+ T cells in peripheral blood and tumor infiltrating lymphocytes.

Abstract PO030: A Novel T-cell Population Expressing the Ectoenzymes CD38 and CD39 is Associated with Melanoma Patient Non-Response to Immunotherapy

Abstract Cancer immunotherapy has emerged as one of the most potent therapeutic tools in treating melanoma and a variety of other malignancies. Immunotherapy includes inhibition of the modulators of T cell antitumor function, the immune checkpoints, by using antibodies against them. In advanced melanoma patients, immune checkpoint inhibitors ipilimumab (IPI) and nivolumab (NIVO) have had unprecedented efficacy. Although many patients respond well to this therapy, many patients remain unresponsive. The mechanism underlying treatment failures remains poorly understood. Using a novel high-dimensional flow cytometry analysis methodology, CytoBrute, we found that in the cohort of advanced melanoma patients receiving sequential NIVO-IPI, high baseline levels of a novel immunophenotype, CD4+CD38+CD39+CD127-GARP- T-cells (median frequency 0.3%), was associated with poor outcomes (disease progression p=0.008, and decreased overall survival p<0.0001 HR = 6.8). Both CD38 and CD39 are ectoenzymes with CD38 hydrolyzing NAD+ to cyclic-ADP ribose and CD39 converting extracellular ATP to immunosuppressive adenosine nucleosides. Both of these proteins are being evaluated as targets to increase the efficacy of immunotherapies. The association of this novel phenotype, which we have termed T-cells expressing ectoenzymes (Tee), was validated in two independent cohorts of melanoma patients treated with NIVO monotherapy, with progressors showing a higher frequency of this immunophenotype at baseline (p=0.047). In IPI+NIVO adjuvant treated melanoma patients, an increase in this population of cells was observed during treatment in relapsing patients, leading to a higher frequency of these cells in relapsing patients relative to patients with no evidence of disease (p=002). Preliminary data obtained from single cell RNA sequencing from the ectoenzyme expressing peripheral blood T cell population, demonstrated predominant expression of IL-32 and CD99 (a membrane glycoprotein associated with lymphocyte diapedesis) in this population, along with several additional clusters distinguished by expression of specific TcR V beta genes. Collectively, we have identified a novel population of ectoenzyme expressing T-cells (CD3+CD4+CD38+CD39+CD127-GARP-) associated with poor melanoma patient outcomes in response to checkpoint immunotherapy. Further investigation of the mechanisms by which this novel T-cell phenotype is associated with poor patient outcomes is under way. Citation Format: Ankita Mitra, David M. Woods, Anjali Rao, Andressa S. Laino, Aidan Winters, Itai Yanai, Pratip K. Chattopadhyay, Jeffrey S. Weber. A Novel T-cell Population Expressing the Ectoenzymes CD38 and CD39 is Associated with Melanoma Patient Non-Response to Immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO030.

Gene Augmentation and Editing to Improve TCR Engineered T Cell Therapy against Solid Tumors

Recent developments in gene engineering technologies have drastically improved the therapeutic treatment options for cancer patients. The use of effective chimeric antigen receptor T (CAR-T) cells and recombinant T cell receptor engineered T (rTCR-T) cells has entered the clinic for treatment of hematological malignancies with promising results. However, further fine-tuning, to improve functionality and safety, is necessary to apply these strategies for the treatment of solid tumors. The immunosuppressive microenvironment, the surrounding stroma, and the tumor heterogeneity often results in poor T cell reactivity, functionality, and a diminished infiltration rates, hampering the efficacy of the treatment. The focus of this review is on recent advances in rTCR-T cell therapy, to improve both functionality and safety, for potential treatment of solid tumors and provides an overview of ongoing clinical trials. Besides selection of the appropriate tumor associated antigen, efficient delivery of an optimized recombinant TCR transgene into the T cells, in combination with gene editing techniques eliminating the endogenous TCR expression and disrupting specific inhibitory pathways could improve adoptively transferred T cells. Armoring the rTCR-T cells with specific cytokines and/or chemokines and their receptors, or targeting the tumor stroma, can increase the infiltration rate of the immune cells within the solid tumors. On the other hand, clinical “off-tumor/on-target” toxicities are still a major potential risk and can lead to severe adverse events. Incorporation of safety switches in rTCR-T cells can guarantee additional safety. Recent clinical trials provide encouraging data and emphasize the relevance of gene therapy and gene editing tools for potential treatment of solid tumors.

A Rare Case of Chronic Lymphoproliferative Disorder of NK Cells with an Unusual Expression of CD57

Abstract Introduction/Objective Natural Killer cells (NK-cells) malignancies are extremely rare and the two NK-cell disorders involving the peripheral blood and bone marrow include chronic lymphoproliferative disorder of NK-cells (CLPDNK) and aggressive NK-cell leukemia (ANKL). ANKL is EBV associated with a prevalence in Asian adults and a fulminant clinical course leading to death within 2 months. Methods A 76-year-old female with a history of iron deficiency anemia presented for evaluation of lymphocytosis (78 TH/mm) and negative EBV with extreme fatigue, weight loss, and worsening weakness from the past 8 months. Results The peripheral and bone marrow aspirate showed an increase in granular lymphocytes with bland nuclei and abundant pale-staining cytoplasm containing fine to coarse azurophilic granules with no evidence of nuclear atypia. Flow cytometry demonstrated an atypical lymphocytic cell population comprising >50% of total marrow and peripheral blood lymphocytes demonstrating loss of CD56 and diminished CD7 expression. The analysis showed atypical lymphocytes with the following immunophenotype: CD2, CD8 (the majority, at least 75%),CD7 (dim), CD38, CD11c (subset) with no expression of CD3, CD5, CD4, CD56, or CD11c. Flow cytometry is important as it helps in the phenotypic detection of aberrancy. A broad panel of immunohistochemical stains revealed scattered positivity for CD3, CD4, CD7, CD8, TIA-1, granzyme, perforin, and tryptase and negative for CD56 and CD57. Our top differential with a similar clinical course included T-cell large granular lymphocytic leukemia which is characterized by a clonal proliferation of mature cytotoxic T cells and also has an indolent course. They show CD3 positivity by flow cytometry along with co-expression of NK cell-associated markers (CD16 and CD57), and variable expression of other pan T cell markers such as CD2, CD5, CD7. It is found incidentally in patients having cytopenias with a persistent increase in circulating mature NK-cells. Of note, the PET scan revealed splenomegaly with no other significant findings.The patient was started on steroids and is currently doing well. Conclusion Thus, overall morphologic, immunophenotypic, and molecular results with negative expression of surface or cytoplasmic CD3 as well as the absence of TCR expression were most consistent with CLPD-NK with an unusual expression of CD57 expression detected in flow cytometry.

Abstract 2183: Targeting TRBC1 and 2 for the treatment of T cell lymphomas

Abstract Background: Mature T cell lymphomas are aggressive, treatment resistant cancers that are associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy T cells. Unlike B cell depletion, pan T cell aplasia is prohibitively toxic. The mutually exclusive expression of TCR β chains (TRBC) 1 or 2 allows targeting the malignant T cell population while preserving T cell function. Notably, a two amino acid inversion is the only exposed and antibody-accessible feature differentiating the two isoforms, making the development of therapeutics targeting TRBC1 and TRBC2 challenging. We have previously described a TRBC1 specific antibody which has been incorporated into a Chimeric Antigen Receptor (CAR) (Maciocia et al, Nat Med, 2017). Here we describe the derivation and characterization of a TRBC2 specific binder and CAR. In addition, we investigate use of these selective binders as Antibody Drug Conjugates (ADCs) for the treatment of HTLV-1 associated leukemia/lymphoma for which CAR T cell therapy may not be well suited. Methods: Anti TRBC2 antibodies were derived through crystallography and structural engineering of the previously described TRBC1 specific antibody. Briefly, TRBC2 binders were obtained by screening of small libraries of the TRBC1 binder with randomizations in key residues identified by crystallographic data and in silico design. TRBC2 binder candidates were first optimized for CAR function. Subsequently, TRBC2 specific CAR T cells were evaluated in vitro and in vivo for anti-tumor activity and selectivity. TRBC1/TRBC2 targeting antibodies, were further characterized as ADCs for biophysical properties, antibody internalization and cytotoxic function. Results: In vitro testing of TRBC2 CARs showed comparable efficacy to the previously described TRBC1 CAR. TRBC2 CAR T cells were effective at killing TRBC2 target cells while sparing TRBC1 positive cells. In vivo mouse models demonstrated that both TRBC1 and TRBC2 directed CARs could target their respective antigens with a high degree of specificity. TRBC1 and TRBC2 specific antibodies were used to generate ADC molecules as a proof of concept. Anti-TRBC1/TRBC2 antibodies were internalized and showed potential as ADCs. Interestingly, we demonstrate that an optimal affinity window facilitates improved antibody uptake and have further engineered both our TRBC1 and TRBC2 antibodies to take advantage of this particularity. Conclusions: Following on from structural and library-based approaches to generate CAR T cells capable of specifically targeting TRBC2, we have further characterized TRBC2 specific CAR T cells in vitro and in vivo. We have shown that highly specific antibodies, engineered as targeting moieties for TRBC1 and TRBC2 CAR T cells, show promising characteristics for utility potentially also as ADCs, offering another modality through which this targeting paradigm can be exploited for the treatment of peripheral T cell lymphomas. Citation Format: Mathieu Ferrari, Vania Baldan, Priyanka Ghongane, Alex Nicholson, Reyisa Bughda, Zulaikha Akbar, Patrycja Wawrzyniecka, Paul Maciocia, Shaun Cordoba, Simon Thomas, Shimobi Onuoha, Martin Pule. Targeting TRBC1 and 2 for the treatment of T cell lymphomas [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 2183.

Investigating of type IV pili to the pathogenicity of Aeromonas schubertii

Aeromonas schubertii is associated with multi-organ necrosis disease that frequently infects cultured snakehead fish. We examined whether type IV pili were important in the pathogenicity of A. schubertii. A Tap pilus mutant (Δpili) was constructed by insertional mutagenesis of the tapM/N/O/P/Q gene cluster. Compared with the wild-type (WT) strain, the proliferation was not changed, but the sensitivity to oxidative stress and low pH were different. The mutant displayed increased survival within macrophages compared with the WT strain. In contrast, cell and tissue adhesion of the mutant was significantly lower than the WT strain. Fish challenged by immersion resulted in reduced cumulative mortality of the mutant compared with the WT strain although intraperitoneal infection was identical for both strains. Expression of IFN-γ, IL-1, TCR, CC22, CD4 and CD40 in infected fish were similar between the two bacterial strains. Together these data indicated that tapM/N/O/P/Q is necessary for Tap pilus biogenesis in A. schubertii and adhesion is a key function for pathogenicity that is mediated by Tap pili.