Human Leukocyte Antigen Restriction Research Articles

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

Canonical α/β T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/β TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Discovery of U2AF1 neoantigens in myeloid neoplasms

BackgroundMyelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome...

T cell receptor therapeutics: immunological targeting of the intracellular cancer proteome.

The T cell receptor (TCR) complex is a naturally occurring antigen sensor that detects, amplifies and coordinates cellular immune responses to epitopes derived from cell surface and intracellular proteins. Thus, TCRs enable the targeting of proteins selectively expressed by cancer cells, including neoantigens, cancer germline antigens and viral oncoproteins. As such, TCRs have provided the basis for an emerging class of oncology therapeutics. Herein, we review the current cancer treatment landscape using TCRs and TCR-like molecules. This includes adoptive cell transfer of T cells expressing endogenous or engineered TCRs, TCR bispecific engagers and antibodies specific for human leukocyte antigen (HLA)-bound peptides (TCR mimics). We discuss the unique complexities associated with the clinical development of these therapeutics, such as HLA restriction, TCR retrieval, potency assessment and the potential for cross-reactivity. In addition, we highlight emerging clinical data that establish the antitumour potential of TCR-based therapies, including tumour-infiltrating lymphocytes, for the treatment of diverse human malignancies. Finally, we explore the future of TCR therapeutics, including emerging genome editing methods to safely enhance potency and strategies to streamline patient identification.

“If I Can See It, I Can Kill It”

If one could use the word “elegant” to describe anticancer therapy, that moniker would apply to messenger RNA (mRNA) vaccines. The elegance of mRNA vaccines derives from their apparent ability to overcome one of the major limitations of conventional cancer vaccines: HLA restriction. Most cancer vaccines are composed of glycoproteins that only fit inside the antigen-presenting pockets in human leucocyte antigen (HLA) molecules – our cellular “home address” – a certain percentage of the time. Consequently, anyone treated with a protein-based vaccine is fortunate to achieve an immune response.

Understanding the Role of HLA Class I Molecules in the Immune Response to Influenza Infection and Rational Design of a Peptide-Based Vaccine.

Influenza A virus is a respiratory pathogen that is responsible for regular epidemics and occasional pandemics that result in substantial damage to life and the economy. The yearly reformulation of trivalent or quadrivalent flu vaccines encompassing surface glycoproteins derived from the current circulating strains of the virus does not provide sufficient cross-protection against mismatched strains. Unlike the current vaccines that elicit a predominant humoral response, vaccines that induce CD8+ T cells have demonstrated a capacity to provide cross-protection against different influenza strains, including novel influenza viruses. Immunopeptidomics, the mass spectrometric identification of human-leukocyte-antigen (HLA)-bound peptides isolated from infected cells, has recently provided key insights into viral peptides that can serve as potential T cell epitopes. The critical elements required for a strong and long-living CD8+ T cell response are related to both HLA restriction and the immunogenicity of the viral peptide. This review examines the importance of HLA and the viral immunopeptidome for the design of a universal influenza T-cell-based vaccine.

Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo

BackgroundImmunotherapy based on Bi-specific T Cell Engagers (TCE) represents one of the most attractive strategy to treat cancers resistant to conventional therapies. TCE are antibody-like proteins that simultaneously bind with one arm to a Tumor Associated Antigen (TAA) on cancer cells and with the other one to CD3 complex on a T-cell to form a TCR-independent immune synapse and circumvent Human Leucocyte Antigen restriction. Among them, the tribodies, such as Tb535H, a bi-specific molecule, made up of a Fab and a scFv domain both targeting 5T4 and another scFv targeting CD3, have demonstrated anti-tumor efficacy in preclinical studies.MethodsHere, we generated five novel tri-specific and multi-functional tribodies, called 53X tribodies, composed of a 5T4 binding Fab arm and a CD3 binding scFv, but differently from the parental Tb535H, they contain an additional scFv derived from an antibody specific for an immune checkpoint, such as PD-1, PD-L1 or LAG-3.ResultsCompared with the parental Tb535H bi-specific T cell engager targeting 5T4, the novel 53X tribodies retained similar binding properties of Tb535H tribody, but showed enhanced anti-tumor potency due to the incorporation of the checkpoint inhibitory moiety. In particular, one of them, called 53L10, a tri-specific T cell engager targeting 5T4, CD3 and PD-L1, showed the most promising anti-tumor efficacy in vitro and led to complete tumor regression in vivo.ConclusionsThe novel tribodies have the potential to become strong and safe therapeutic drugs, allowing to reduce also the cost of production as one single molecule contains three different specificities including the anti-TAA, anti-CD3 and anti-IC binding arms.

The microbiota is a potential mediator of the crosstalk between γδ T cells and tumors

γδ T cells are one of the immune cell types that express antigen receptors. γδ T cells are able to recognize pathogens or cancer cells independently of human leukocyte antigen restriction, which is an important feature of αβ T cells. Therefore, γδ T cells are considered the bridge between innate and adaptive immunity. These cells exhibit important roles in immune surveillance, exert immune defense against tumors and have become promising effector cells for cancer immunotherapy. However, in particular circumstances, the tumor microenvironment seems to render γδ T cells immunosuppressive and even tumor-promoting, emphasizing the importance of regulating γδ T functions in realizing their translation into practical cancer immunotherapy. In recent years, increasing evidence has demonstrated that the intratumoral and peritumoral microbiota can have complex effects on tumor immunology. Thus, understanding the role of microbiota in the crosstalk between γδ T cells and tumors will provide insights for developing adjuvant immunotherapy with precise regulation of tumor-related microbiota. In the present review, the effects of microbiota on γδ T cell receptor repertoire and the roles of microbiota in some common tumors will be discussed, with implications for future cancer therapy.

RETRACTED ARTICLE: Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

PopCover-2.0. Improved Selection of Peptide Sets With Optimal HLA and Pathogen Diversity Coverage.

The use of minimal peptide sets offers an appealing alternative for design of vaccines and T cell diagnostics compared to conventional whole protein approaches. T cell immunogenicity towards peptides is contingent on binding to human leukocyte antigen (HLA) molecules of the given individual. HLA is highly polymorphic, and each variant typically presents a different repertoire of peptides. This polymorphism combined with pathogen diversity challenges the rational selection of peptide sets with broad immunogenic potential and population coverage. Here we propose PopCover-2.0, a simple yet highly effective method, for resolving this challenge. The method takes as input a set of (predicted) CD8 and/or CD4 T cell epitopes with associated HLA restriction and pathogen strain annotation together with information on HLA allele frequencies, and identifies peptide sets with optimal pathogen and HLA (class I and II) coverage. PopCover-2.0 was benchmarked on historic data in the context of HIV and SARS-CoV-2. Further, the immunogenicity of the selected SARS-CoV-2 peptides was confirmed by experimentally validating the peptide pools for T cell responses in a panel of SARS-CoV-2 infected individuals. In summary, PopCover-2.0 is an effective method for rational selection of peptide subsets with broad HLA and pathogen coverage. The tool is available at https://services.healthtech.dtu.dk/service.php?PopCover-2.0.

Abstract 1515: Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system

Abstract Mutations in critical genes for cell survival and proliferation, e.g., KRAS and TP53, are found as clonal events in multiple tumor types of unrelated people likely due to their importance to the malignant phenotype. T cells recognize products of mutated genes, termed neoantigens, because they are expressed in the tumor but not in the normal tissues; thus, neoantigens are foreign entities from an immunological perspective. T-cell receptors (TCRs) with specificity to the neoantigen in the context of human leukocyte antigen (HLA) on the tumor cell surface can be isolated from the neoantigen-reactive T cell and potentially used for genetically-modified adoptive immunotherapy for any patient with matching mutation and HLA. The purpose of this study was to evaluate the ability of the non-viral Sleeping Beauty transposon/transposase gene transfer system to re-direct the specificity of T cells towards p53 and KRAS neoantigens and to characterize the resultant engineered TCR-T cell populations for specificity and function. Genes encoding the alpha and beta chains of p53 or KRAS neoantigen-specific TCRs were linked by a 2A ribosomal slip site linker and cloned into the clinical Sleeping Beauty transposon. These transposons were co-electroporated into donor peripheral blood leukocytes (PBL) with a DNA plasmid encoding the SB11 transposase and expanded in vitro. Logarithmic proliferation was observed and resulted in large numbers of highly pure TCR-T cells (>80% by introduced TCR expression). The TCR-T cells upregulated 41BB on the T-cell surface and secreted interferon-γ in response to antigen presenting cells with the appropriate HLA molecule pulsed with KRAS or p53 neoantigen peptides but not the cognate wild type peptide, confirming re-directed specificity to mutated KRAS or TP53 genes, respectively. Similarly, TCR-engineered T cells demonstrated cytolysis of tumor cells expressing the neoantigen and appropriate HLA restriction, suggesting that adoptive transfer of these TCR-T cells could mediate anti-tumor responses. In all, we demonstrated that multiple TCRs with unique specificities targeting recurrent p53 and KRAS substitutions in frequent HLA haplotypes could be stably expressed using Sleeping Beauty transposition to re-direct peripheral blood T cells towards tumor cells. Translation of these TCR-T cells into adoptive immunotherapy could result in safe and effective treatments for any cancer patient with matching HLA and KRAS or TP53 hotspot mutations. Citation Format: Ana B. Korngold, Lin-Kin Yong, Ming Zhang, Ugochi Ibekwe, Lenka V. Hurton, Yaoyao Shi, Julissa Simmons, Matthew R. Collinson-Pautz, Eleanor De Groot, Laurence J. Cooper, Drew C. Deniger. Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1515.

HLA-Restriction of Human Treg Cells Is Not Required for Therapeutic Efficacy of Low-Dose IL-2 in Humanized Mice.

Regulatory T (Treg) cells are essential to maintain immune homeostasis in the intestine and Treg cell dysfunction is associated with several inflammatory and autoimmune disorders including inflammatory bowel disease (IBD). Efforts using low-dose (LD) interleukin-2 (IL-2) to expand autologous Treg cells show therapeutic efficacy for several inflammatory conditions. Whether LD IL-2 is an effective strategy for treating patients with IBD is unknown. Recently, we demonstrated that LD IL-2 was protective against experimental colitis in immune humanized mice in which human CD4+ T cells were restricted to human leukocyte antigen (HLA). Whether HLA restriction is required for human Treg cells to ameliorate colitis following LD IL-2 therapy has not been demonstrated. Here, we show that treatment with LD IL-2 reduced 2,4,6-trinitrobenzensulfonic acid (TNBS) colitis severity in NOD.PrkdcscidIl2rg-/- (NSG) mice reconstituted with human CD34+ hematopoietic stem cells. These data demonstrate the utility of standard immune humanized NSG mice as a pre-clinical model system to evaluate therapeutics targeting human Treg cells to treat IBD.

223 - Clinical Experience of Tabelecleucel in Patients with Life-Threatening Complications of Epstein–Barr Virus Viremia

Background Epstein-Barr virus (EBV) viremia - a condition of persistent active (vs latent) EBV infection - can be associated with a variety of life-threatening disorders. One such disorder is chronic active EBV (CAEBV), a type of primary immunodeficiency associated with high mortality rates. Another entity is EBV-driven (EBV+) hemophagocytic lymphohistiocytosis (HLH), characterized by a hyperactive immune response with elevated cytokines. Both can be associated with multi-organ dysfunction and failure. In addition, EBV viremia may be associated with complications following allogeneic hematopoietic cell transplantation (HCT) or can herald relapse of EBV+ malignancies. Tabelecleucel, an investigational off-the-shelf, allogeneic EBV-specific T-cell immunotherapy, has previously shown clinical activity in patients with EBV+ post-transplant lymphoproliferative disease (PTLD), EBV+ PTLD with central nervous system involvement and EBV+ leiomyosarcoma (Prockop S et al, JCI 2020; Kurlander et al, ESMO 2018) and is a potential therapy for CAEBV, HLH or EBVviremia in other life-threatening settings since it may control the EBV-based stimuli underlying these distinct diseases (Icheva V et al, J Clin Oncol 2013). Here, we report data in patients with complications of EBV viremia treated with tabelecleucel in an expanded access study (NCT02822495) within two non-overlapping protocols (EAP-201 [2016-2018] and EAP-901 [2018-present]). Methods Eligible patients received tabelecleucel at ~2 x 106 cells/kg/dose on Days 1, 8 and 15, with laboratory assessments on Day ~28 of each 5-week cycle. EAP-201 enrolled patients with persistent EBV viremia (defined as serum EBV DNA assays showing detectable EBV viral load at least 4 weeks apart and known or suspected immunodeficiency or history of prior EBV+ malignancy) as well as patients with EBV+ HLH. EAP-901 includes patients with persistent EBV viremia and known or suspected immunodeficiency. Patients who did not respond could switch to tabelecleucel with a different human leukocyte antigen (HLA) restriction (restriction switch). In EAP-201, response criteria were based on reduction in EBV viral load and/or on HLH response criteria (Table 1). As EAP-901 is intended to provide compassionate access to tabelecleucel, limited patient data are collected compared with EAP-201; however, response is assessed by the investigators based on reduction in EBV viral load to determine whether an HLA restriction switch is needed. In both protocols, patients continued treatment until unacceptable toxicity, maximal response (2 consecutive complete responses [CR] or 3 partial responses [PR]), or up to 4 different HLA restrictions. Both protocols collected serious AEs (SAEs). Results Patient characteristics, treatment exposure, and responses are reported in Table 1; 5 patients with EBV viremia participated in EAP-201 and 4 in EAP-901. Objective response rates were 80% (n=4/5) for EAP-201 and 50% (n=2/4) for EAP-901. For EAP-201, the median time to first response for the 4 patients who responded was 1.7 (min 1.0, max 4.7) months, and for EAP-901 it was approximately 1 month for both patients who responded. Table 2 summarizes the treatment-emergent SAEs (TESAEs) observed with both protocols. EAP-901 reported a fatal SAE of chronic hepatic failure that was assessed as unrelated to treatment. One patient in EAP-901 experienced a grade 3 TESAE of facial nerve disorder that was assessed as possibly related to tabelecleucel; there were no other fatal or treatment-related TESAEs reported in either protocol, and no safety concern has emerged for this patient population. Conclusions Tabelecleucel was well tolerated and showed evidence of clinical activity in patients with life-threatening EBV viremia. Based on these results, further clinical investigation of tabelecleucel in patients with HLH or CAEBV is planned within an upcoming study (ATA129-EBV-205). Download : Download high-res image (743KB) Download : Download full-size image Disclosures Ghobadi: Amgen: Consultancy, Research Funding. Stiff: CRISPR: Consultancy; Gamida-Cell: Research Funding; Atara Biotherapeutics: Research Funding; Amgen: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Macrogenics: Research Funding; Eisai: Research Funding. Reshef: Kite: Consultancy, Research Funding; Atara Biotherapeutics: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Magenta: Consultancy; Incyte: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Pfizer: Consultancy; Celgene: Research Funding; Shire: Research Funding. Navarro: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Gamelin: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Dinavahi: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Sun: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Guzman-Becerra: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Prockop: Mesoblast: Consultancy, Research Funding; Atara Biotherapeutics: Research Funding; Jasper Pharmaceuticals: Research Funding; Memorial Sloan Kettering: Patents & Royalties: IP related to the development of third party viral specific T cells with all of my interests assigned to MSK.

532 - A Multicenter, Multicohort, Open-Label, Single Arm per Cohort, Phase II Study to Assess the Efficacy and Safety of Tabelecleucel in Patients with EBV-Associated Diseases Using an Adaptive Two-Stage Study Design

Background Epstein-Barr virus infection is associated with a variety of life-threatening malignant and non-malignant diseases. In immunocompromised individuals where T-cell function is impaired, loss of immune control of Epstein-Barr virus (EBV) can lead to proliferation of EBV-transformed B cells and result in immunodeficiency-associated lymphoproliferative diseases (IA-LPD). EBV-driven IA-LPDs are a heterogenous group of diseases with variable clinical and pathologic features, including primary immunodeficiency-associated LPD (EBV+ PID-LPD), acquired immunodeficiency-associated LPD (EBV+ AID-LPD), and post-transplant lymphoproliferative disorder (EBV+ PTLD). In addition, EBV-infection of mesenchymal cells can cause sarcomas, including leiomyosarcoma (EBV+ sarcoma). In rare instances, EBV viremia (ie, persistent EBV infection) can lead to non-malignant yet life-threatening complications such as chronic active EBV (CAEBV) or hemophagocytic lymphohistiocytosis (HLH). During the transition from primary EBV infection to latent state, only a subset of viral genes is expressed. The resulting latency proteins are a target of CD8+ T cells, making EBV+ malignancies good candidates for treatment with EBV-targeted cytotoxic lymphocytes. Tabelecleucel (tab-cel) is an off-the-shelf, partially human leukocyte antigen (HLA)-matched, allogeneic EBV-specific T-cell immunotherapy generated from screened EBV+ donors. Tabelecleucel is characterized by both HLA genotype and HLA restriction, the HLA allele(s) through which tab-cel exerts cytotoxic activity. Tab-cel is selected for each patient from an inventory based on the HLA restriction(s) and partial HLA matching. Initial safety and efficacy data support that tab-cel was well tolerated and showed clinical activity in patients with each of the EBV-associated diseases being proposed in study 205 [Nikiforow S et al. ESMO 2020; Kurlander LS et al. Ann Oncol 2018; Prockop S et al. JCI 2020]. These data encourage investigation to further evaluate the safety and efficacy of tab-cel in these populations. Study design and objectives This is a multicenter, multicohort, open-label, single-arm per cohort, phase II study to assess the efficacy and safety of tab-cel in patients with EBV-associated diseases (ATA129-EBV-205). The primary objective of the study is to assess the clinical benefit of tab-cel as measured by objective response rate. This study will also evaluate clinically relevant disease-specific outcomes and will characterize the safety profile of tab-cel in these patient populations. This study will enroll patients who are relapsed/refractory or newly diagnosed and ineligible for first-line therapy in the following cohorts: EBV+ AID-LPD, EBV+ PID-LPD, EBV+ sarcoma, including LMS, CAEBV/HLH and EBV+ PTLD with central nervous system [CNS]-involvement. In addition, a cohort of patients with previously untreated EBV+ PTLD where first-line therapy is not appropriate is planned to start enrollment in 2021. A Phase III study of tab-cel for solid organ or allogeneic hematopoietic cell transplant patients with EBV+ PTLD after failure of rituximab or rituximab plus chemotherapy (NCT03394365) is ongoing. Patients in each cohort will receive tab-cel in 35-day cycles. During each cycle, patients will receive intravenous tab-cel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by an observation period through day 35 (Figure 1). An adaptive two-stage design will be used for each cohort in this study, and for each, a maximum of eight patients will be enrolled in stage 1. The decision to move to stage 2 enrollment for any given cohort will be based on an analysis of the first eight evaluable patients in the cohort using investigator's assessment (per defined radiologic, clinical, and/or laboratory response criteria). If at least two among the first eight evaluable patients in stage 1 are responders W(ie, complete response or partial response), additional patients may be enrolled in stage 2 of that cohort. The number of patients enrolled in stage 2 will depend on the number of observed responders in stage 1 (Table 1). This design will allow for minimization of the expected number of patients required to evaluate the efficacy of tab-cel in each disease cohort independently. Figure 1. Study design schema Download : Download high-res image (589KB) Download : Download full-size image Disclosures Prockop: Jasper Pharmaceuticals: Research Funding; Mesoblast: Consultancy, Research Funding; Atara Biotherapeutics: Research Funding; Memorial Sloan Kettering: Patents & Royalties: IP related to the development of third party viral specific T cells with all of my interests assigned to MSK. Dinavahi: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Navarro: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Guzman-Becerra: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Sun: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Gamelin: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

Clinical Experience of Tabelecleucel in Patients with Life-Threatening Complications of Epstein-Barr Virus Viremia

Background Epstein-Barr virus (EBV) viremia - a condition of persistent active (vs latent) EBV infection - can be associated with a variety of life-threatening disorders. One such disorder is chronic active EBV (CAEBV), a type of primary immunodeficiency associated with high mortality rates. Another entity is EBV-driven (EBV+) hemophagocytic lymphohistiocytosis (HLH), characterized by a hyperactive immune response with elevated cytokines. Both can be associated with multi-organ dysfunction and failure. In addition, EBV viremia may be associated with complications following allogeneic hematopoietic cell transplantation (HCT) or can herald relapse of EBV+ malignancies. Tabelecleucel, an investigational off-the-shelf, allogeneic EBV-specific T-cell immunotherapy, has previously shown clinical activity in patients with EBV+ post-transplant lymphoproliferative disease (PTLD), EBV+ PTLD with central nervous system involvement and EBV+ leiomyosarcoma (Prockop S et al, JCI 2020; Kurlander et al, ESMO 2018) and is a potential therapy for CAEBV, HLH or EBVviremia in other life-threatening settings since it may control the EBV-based stimuli underlying these distinct diseases (Icheva V et al, J Clin Oncol 2013). Here, we report data in patients with complications of EBV viremia treated with tabelecleucel in an expanded access study (NCT02822495) within two non-overlapping protocols (EAP-201 [2016-2018] and EAP-901 [2018-present]). Methods Eligible patients received tabelecleucel at ~2 x 106 cells/kg/dose on Days 1, 8 and 15, with laboratory assessments on Day ~28 of each 5-week cycle. EAP-201 enrolled patients with persistent EBV viremia (defined as serum EBV DNA assays showing detectable EBV viral load at least 4 weeks apart and known or suspected immunodeficiency or history of prior EBV+ malignancy) as well as patients with EBV+ HLH. EAP-901 includes patients with persistent EBV viremia and known or suspected immunodeficiency. Patients who did not respond could switch to tabelecleucel with a different human leukocyte antigen (HLA) restriction (restriction switch). In EAP-201, response criteria were based on reduction in EBV viral load and/or on HLH response criteria (Table 1). As EAP-901 is intended to provide compassionate access to tabelecleucel, limited patient data are collected compared with EAP-201; however, response is assessed by the investigators based on reduction in EBV viral load to determine whether an HLA restriction switch is needed. In both protocols, patients continued treatment until unacceptable toxicity, maximal response (2 consecutive complete responses [CR] or 3 partial responses [PR]), or up to 4 different HLA restrictions. Both protocols collected serious AEs (SAEs). Results Patient characteristics, treatment exposure, and responses are reported in Table 1; 5 patients with EBV viremia participated in EAP-201 and 4 in EAP-901. Objective response rates were 80% (n=4/5) for EAP-201 and 50% (n=2/4) for EAP-901. For EAP-201, the median time to first response for the 4 patients who responded was 1.7 (min 1.0, max 4.7) months, and for EAP-901 it was approximately 1 month for both patients who responded. Table 2 summarizes the treatment-emergent SAEs (TESAEs) observed with both protocols. EAP-901 reported a fatal SAE of chronic hepatic failure that was assessed as unrelated to treatment. One patient in EAP-901 experienced a grade 3 TESAE of facial nerve disorder that was assessed as possibly related to tabelecleucel; there were no other fatal or treatment-related TESAEs reported in either protocol, and no safety concern has emerged for this patient population. Conclusions Tabelecleucel was well tolerated and showed evidence of clinical activity in patients with life-threatening EBV viremia. Based on these results, further clinical investigation of tabelecleucel in patients with HLH or CAEBV is planned within an upcoming study (ATA129-EBV-205). Disclosures Ghobadi: Amgen: Consultancy, Research Funding. Stiff:CRISPR: Consultancy; Gamida-Cell: Research Funding; Atara Biotherapeutics: Research Funding; Amgen: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Macrogenics: Research Funding; Eisai: Research Funding. Reshef:Kite: Consultancy, Research Funding; Atara Biotherapeutics: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Magenta: Consultancy; Incyte: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Pfizer: Consultancy; Celgene: Research Funding; Shire: Research Funding. Navarro:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Gamelin:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Dinavahi:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Sun:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Guzman-Becerra:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Prockop:Mesoblast: Consultancy, Research Funding; Atara Biotherapeutics: Research Funding; Jasper Pharmaceuticals: Research Funding; Memorial Sloan Kettering: Patents & Royalties: IP related to the development of third party viral specific T cells with all of my interests assigned to MSK.

A Multicenter, Multicohort, Open-Label, Single-Arm per Cohort, Phase II Study to Assess the Efficacy and Safety of Tabelecleucel in Patients with EBV-Associated Diseases Using an Adaptive Two-Stage Study Design

Background Epstein-Barr virus infection is associated with a variety of life-threatening malignant and non-malignant diseases. In immunocompromised individuals where T-cell function is impaired, loss of immune control of Epstein-Barr virus (EBV) can lead to proliferation of EBV-transformed B cells and result in immunodeficiency-associated lymphoproliferative diseases (IA-LPD). EBV-driven IA-LPDs are a heterogenous group of diseases with variable clinical and pathologic features, including primary immunodeficiency-associated LPD (EBV+ PID-LPD), acquired immunodeficiency-associated LPD (EBV+ AID-LPD), and post-transplant lymphoproliferative disorder (EBV+ PTLD). In addition, EBV-infection of mesenchymal cells can cause sarcomas, including leiomyosarcoma (EBV+ sarcoma). In rare instances, EBV viremia (ie, persistent EBV infection) can lead to non-malignant yet life-threatening complications such as chronic active EBV (CAEBV) or hemophagocytic lymphohistiocytosis (HLH). During the transition from primary EBV infection to latent state, only a subset of viral genes is expressed. The resulting latency proteins are a target of CD8+ T cells, making EBV+ malignancies good candidates for treatment with EBV-targeted cytotoxic lymphocytes. Tabelecleucel (tab-cel) is an off-the-shelf, partially human leukocyte antigen (HLA)-matched, allogeneic EBV-specific T-cell immunotherapy generated from screened EBV+ donors. Tabelecleucel is characterized by both HLA genotype and HLA restriction, the HLA allele(s) through which tab-cel exerts cytotoxic activity. Tab-cel is selected for each patient from an inventory based on the HLA restriction(s) and partial HLA matching. Initial safety and efficacy data support that tab-cel was well tolerated and showed clinical activity in patients with each of the EBV-associated diseases being proposed in study 205 [Nikiforow S et al. ESMO 2020; Kurlander LS et al. Ann Oncol 2018; Prockop S et al.JCI 2020]. These data encourage investigation to further evaluate the safety and efficacy of tab-cel in these populations. Study design and objectives This is a multicenter, multicohort, open-label, single-arm per cohort, phase II study to assess the efficacy and safety of tab-cel in patients with EBV-associated diseases (ATA129-EBV-205). The primary objective of the study is to assess the clinical benefit of tab-cel as measured by objective response rate. This study will also evaluate clinically relevant disease-specific outcomes and will characterize the safety profile of tab-cel in these patient populations. This study will enroll patients who are relapsed/refractory or newly diagnosed and ineligible for first-line therapy in the following cohorts: EBV+ AID-LPD, EBV+ PID-LPD, EBV+ sarcoma, including LMS, CAEBV/HLH and EBV+ PTLD with central nervous system [CNS]-involvement. In addition, a cohort of patients with previously untreated EBV+ PTLD where first-line therapy is not appropriate is planned to start enrollment in 2021. A Phase III study of tab-cel for solid organ or allogeneic hematopoietic cell transplant patients with EBV+ PTLD after failure of rituximab or rituximab plus chemotherapy (NCT03394365) is ongoing. Patients in each cohort will receive tab-cel in 35-day cycles. During each cycle, patients will receive intravenous tab-cel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by an observation period through day 35 (Figure 1). An adaptive two-stage design will be used for each cohort in this study, and for each, a maximum of eight patients will be enrolled in stage 1. The decision to move to stage 2 enrollment for any given cohort will be based on an analysis of the first eight evaluable patients in the cohort using investigator's assessment (per defined radiologic, clinical, and/or laboratory response criteria). If at least two among the first eight evaluable patients in stage 1 are responders W(ie, complete response or partial response), additional patients may be enrolled in stage 2 of that cohort. The number of patients enrolled in stage 2 will depend on the number of observed responders in stage 1 (Table 1). This design will allow for minimization of the expected number of patients required to evaluate the efficacy of tab-cel in each disease cohort independently. Figure 1. Study design schema Disclosures Prockop: Jasper Pharmaceuticals: Research Funding; Mesoblast: Consultancy, Research Funding; Atara Biotherapeutics: Research Funding; Memorial Sloan Kettering: Patents & Royalties: IP related to the development of third party viral specific T cells with all of my interests assigned to MSK. Dinavahi:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Navarro:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Guzman-Becerra:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Sun:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Gamelin:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

Determining the impact of HLA type and chronic viral infection on peripheral T-cell receptor sharing between unrelated individuals

Abstract T cells generate highly diverse T-cell receptors (TCRs) to recognize antigens, most of which are private to each individual. TCRs with low junctional complexity have a higher likelihood of being generated and shared across individuals. Additional factors that may further limit diversity and increase the likelihood that particular TCRs are public include: (a) thymus selection for maturation of TCRs that bind antigens presented by an individual’s human leukocyte antigen (HLA) proteins, and (b) T-cell proliferation upon exposure to cognate antigens, which remain in the periphery as memory T cells. Numerous public TCRs have been associated with particular HLA restrictions and antigen specificities1. Chowell et al. also showed that HLA diversity is important for mounting an effective T-cell response against solid tumors2. However, little is known about the impact of these forces on the diversity of the TCR repertoire and expected TCR sharing across individuals. Here, we leverage the TCR repertoires of 666 healthy individuals sequenced with the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle, WA) to address these questions. We investigate (a) how HLA restriction affects immune repertoires by examining the effect of HLA heterozygosity and divergence on the diversity of the TCR repertoire; (b) how the similarity of HLA alleles between individuals impacts the number of TCRs shared between individuals and (c) the impact of shared antigen exposure by determining how age and CMV status impacts clone sharing between individuals. These data show that HLA type, age, and CMV exposure shape the immune repertoire and influence the sharing of clones between individuals.

Antigen Experienced T Cells from Peripheral Blood Recognize p53 Neoantigens.

The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens. PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol. T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen-reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation-specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens. PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses.See related commentary by Olivera et al., p. 1203.

A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disorder (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy (ALLELE)

Background In immunocompromised individuals where T-cell function is impaired, EBV-transformed B-cells can proliferate resulting in lymphoproliferative disorders or lymphoma (EBV+LPD). EBV+LPD following solid organ transplant (SOT) is known as EBV+ post-transplant lymphoproliferative disorder (EBV+PTLD). Treatments for EBV+PTLD following SOT include reduction in immunosuppression and off-label rituximab ± multi-agent chemotherapy (CT). Limitations of current EBV+PTLD treatments include lack of response, relapse, potential for graft rejection, short- and long-term toxicity and high mortality, highlighting a clear unmet medical. Tabelecleucel (tab-cel) is an off-the-shelf allogeneic EBV-specific T cell immunotherapy generated from healthy donors that is selected for each patient (pt) from a library of T cells using the most appropriate human leukocyte antigen (HLA) restriction to address the patient's disease. Tab-cel has been evaluated in several clinical studies; One published interim analysis showed that in ALLELE-eligible patients with EBV+PTLD following SOT, the objective response rate (ORR) was 83% (5/6 patients) (Prockop et al, ASH 2017). Here we describe the design of an ongoing study of tab-cel for SOT subjects with EBV+PTLD after failure of rituximab ± CT. Study Design and Methods ALLELE is a multicenter, open-label, single arm, phase 3 study evaluating the clinical benefit of tab-cel in pts with EBV+PTLD following SOT (n=33) after failure of (1) rituximab monotherapy or (2) rituximab plus CT (NCT03394365). Key inclusion criteria are a diagnosis of locally assessed, biopsy-proven EBV+PTLD and failure of rituximab or rituximab plus concurrent/sequential CT. Exclusion criteria include untreated or in-progress treatment of central nervous system PTLD and suspected or confirmed grade >2 graft-vs-host disease. Tab-cel is partially matched to each patient from a library that is HLA-characterized using 1 EBV HLA restriction allele and at least 1 other matched HLA allele. Tab-cel is administered in cycles lasting 5 weeks (35 days). During each cycle, subjects receive intravenous (IV) tab-cel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by observation through day 35. Response per clinical and radiographic assessment are evaluated at the end of each cycle by the investigator and independent review using modified Lugano Classification criteria. Up to 2 different HLA restrictions are allowed per protocol. Treatment is continued until maximal response, unacceptable toxicity, initiation of non-protocol therapy, or failure of the maximum allowable HLA restrictions. After treatment is completed or discontinued, subjects are assessed every 3 months, up to 24 months and every 6 months thereafter up to 5 years for survival status. The primary endpoint of the study is overall response rate (ORR) following the administration of tab-cel.

Long-Term Outcomes of Patients with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease Following Solid Organ Transplant or Allogeneic Hematopoietic Cell Transplant Treated with Tabelecleucel in a Multicenter Expanded Access Program Study

Background Patients (pts) undergoing a solid organ (SOT) or allogeneic hematopoietic cell transplant (HCT) are at risk for developing Epstein-Barr virus-driven post-transplant lymphoproliferative disease (EBV+PTLD). There is a need for effective and well-tolerated therapies in pts with EBV+PTLD who have failed rituximab ± chemotherapy (CT). Tabelecleucel (tab-cel) is an investigational, off-the-shelf, allogeneic EBV-specific T-cell immunotherapy generated from healthy donors that is selected for a given pt from an existing library, based on human leukocyte antigen (HLA) restriction and matching. Objective Here, we report long-term outcomes using tab-cel through an ongoing US, multicenter Expanded Access Program (EAP) study (EBV-CTL-201, NCT02822495) in pts with relapsed/refractory (R/R) EBV+PTLD following HCT (n = 14) and SOT (n = 12). Methods Pts received tab-cel matched by ≥2/10 HLA alleles, including ≥1 HLA allele through which tab-cel exerts cytotoxicity (HLA restriction). In each 5-week cycle, pts received tab-cel 1.6–2 × 106 cells/kg intravenously on days 1, 8, and 15, with an imaging-based response assessment around day 28. Pts were treated until maximal response (with up to 4 tab-cel restrictions), unacceptable toxicity, or withdrawal of consent. Results herein reflect a data snapshot from June 3, 2019. Results All pts had received prior rituximab and 7/12 SOT pts received prior CT. Intermediate/high risk PTLD-prognostic index (PTLD-IPI; Choquet et al, Ann Hematol 2007) was noted in 79% and 42% of HCT and SOT pts, respectively. The results are presented in figure 1. While the median follow-up time in HCT pts was short, 3 pts were followed for over 12 months including 2 who were followed for more than 24 months. In pts responding to tab-cel, 1-year OS was 85.7% in HCT and 100% in SOT, and no deaths were attributable to EBV+PTLD progression. In a subset of study pts (HCT: n=11; SOT: n=11) with adequate ECOG, no CNS disease, and no PTLD-related ventilatory support who would have likely been eligible for Atara's ongoing Phase 3 studies, the ORR was 55% (HCT) and 82% (SOT), with a 2-yr OS of 79% (HCT) and 81% (SOT). The safety profile of tab-cel was consistent with previously published data. At the data snapshot for this abstract, no tab-cel-related adverse events led to treatment discontinuation or death. In addition, no cytokine release syndrome, organ rejection or tumor flare adverse events were reported in the EBV+PTLD pts treated with tab-cel in this EAP study. Conclusions The data demonstrate a high response rate for tab-cel in EBV+PTLD in both HCT and SOT after initial treatment failure. Longer term follow-up shows a favorable 2-year OS in this predominantly high-risk population for whom there are no approved alternative therapies. Similar outcomes were observed in the subset of pts potentially eligible for ongoing Phase 3 studies of tab-cel in R/R EBV+PTLD following SOT or HCT.

A simple and efficient method of generating HCMV pp65-specific T cells using overlapping peptides.

The methods for expansion of human cytomegalovirus (HCMV)-specific T lymphocytes are limited due to the complex culture process, long culture duration, and human leukocyte antigen (HLA) restriction. Here, we report that in vitro stimulation with pp65kDa phosphoprotein (pp65)-derived overlapping synthetic peptides rapidly generates large numbers of HCMV-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells (PBMCs) regardless of HLA type. Treatment of PBMCs from healthy volunteers expressing HLA-A*02:01 or HLA-A*24:02 with 138 pp65 overlapping peptides (OLP) resulted in an expansion of HCMV pp65 NLVPMVATV (NLV) pentamer-specific CD8+ T lymphocytes that expressed interferon (IFN)-γ, but the pp65 NLV peptide did not generate HCMV-specific CD8+ T lymphocytes in PBMCs obtained from an HLA-A*24:02 donor due to HLA restriction. The OLP-induced T lymphocytes specific for HCMV derived from PBMCs of HLA-A*02:01- and HLA-A*24:02-expressing donors showed effective cytolytic responses against target cells loaded with OLP or the NLV epitope, but pp65 NLV peptide-induced T lymphocytes did not. Phenotypic analyses demonstrated that OLP increased the frequency of CD3+ CD8+ cells, but not CD3+ CD4+, CD14+, or CD56+ cells, in donor PBMCs. Thus, this study provides evidence that in vitro stimulation with OLP efficiently generates sufficient numbers of HCMV pp65-specific cytotoxic T lymphocytes for adoptive cell therapy. Keywords: human cytomegalovirus; cytotoxic T lymphocyte; overlapping peptides; pp65; cytotoxicity.