Adoptive T-cell Immunotherapy Research Articles

Use of Specific T Lymphocytes in Treating Cytomegalovirus Infection in Hematopoietic Cell Transplant Recipients: A Systematic Review

Cytomegalovirus (CMV) poses a significant threat to post-hematopoietic cell transplantation (HCT). Control strategies include letermovir prophylaxis or ganciclovir pre-emptive therapy (PET). Without prophylaxis, 65–90% of seropositive recipients develop a clinically significant CMV infection. Due to PET drawbacks, letermovir prophylaxis is preferable, as it reduces CMV-related events and improves overall survival. However, refractory or resistant CMV-CS remains a challenge, with maribavir showing limited efficacy. This systematic review followed the Cochrane Manual and PRISMA guidelines and was registered in PROSPERO. Searches were conducted in PubMed, Scopus, Embase, and Web of Science. Out of 1895 identified records, 614 duplicates were removed, and subsequent screening excluded 1153 studies. Eleven included studies (2012–2024) involved 255 HCT recipients receiving adoptive immunotherapy (AI), primarily CMV-specific T-cell therapy. GvHD occurred in 1.82% of cases. Adverse events occurred in 4.4% of cases, while mild CRS was observed in 1.3% of patients. Efficacy, evaluated in 299 patients across eleven studies, showed an average response rate of 78.2%. CMV-CS recurrence was observed in 24.4% of 213 patients, and death due to CMV was reported in 9.7% of 307 patients across nine studies. Adoptive hCMV-specific T-cell immunotherapy appears to be a safe, effective alternative for refractory CMV-CS in HCT.

Transient EZH2 suppression by Tazemetostat during in vitro expansion maintains T-cell stemness and improves adoptive T-cell therapy.

The histone methyltransferase enhancer of zeste homolog 2 (EZH2) plays important roles in T-cell differentiation, proliferation and function. Previous studies have demonstrated that genetic deletion of EZH2 in CD8+ or total T cells impairs their antiviral and antitumor activity, cytokine production and ability to expand upon rechallenge. Contrary to the detrimental role of deleting T cell-intrinsic EZH2, here we have demonstrated that transient inhibition of EZH2 in T cells prior to the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetostat, delayed their dysfunctional progression and preserved T-cell stemness and polyfunctionality but had no negative impact on cell proliferation. Tazemetostat induced T-cell epigenetic reprogramming and increased the expression of the self-renewal T-cell transcription factor TCF1 by reducing H3K27 methylation at its promoter preferentially in rapidly dividing T cells. In a murine melanoma model, T cells depleted of EZH2 induced poor tumor control, whereas adoptively transferred T cells pretreated with tazemetostat exhibited superior antitumor immunity, especially when used in combination with anti-PD-1 blockade. Collectively, these data highlight the potential of transient epigenetic reprogramming by EZH2 inhibition to enhance adoptive T-cell immunotherapy.

Biomimetic cascade-released hydrogel scaffolds from microfluidics for efficient T cell recruitment and expansion

Adoptive T-cell immunotherapy has shown powerful therapeutic effects in cancer treatment. Attempts to implement T cell therapy have tended to focus on providing T cells with specific targeting and sufficient activation to enhance their tumor killing ability. Although biomaterial-based scaffolds can provide necessary molecular cues for T cell activation, efficient stimulation necessitates more attention on the reconstruction of natural T cell activation processes. Here, we present a novel biomimetic scaffold with cascade-released capacity for T-cell recruitment and activation. Core-shell structured hydrogel scaffolds are fabricated by single-step microfluidic electrospray, and chemokines and stimulatory antibodies are compartmentalized in the shell and core, respectively. Benefiting from the heterogeneity in the microstructure and swelling degree of the core and shell hydrogels, chemokine peptides are robustly released to recruit tumor-reactive T cells, and stimulatory antibodies are continuously released to support T cell proliferation. Such biomimetic recruitment and activation processes contribute to the formation of T cell clusters, which enhances their proliferation and infiltration in tumor tissues. Owing to the high expression of cytotoxic receptor and chemokine receptor, the activated T cells exhibit potent antitumor activity in vitro and in vivo. These features demonstrate that the cascade-released hydrogel scaffolds are promising for the local recruitment and activation of tumor-reactive T cells.

A strategy to reconstitute immunity without GVHD via adoptive allogeneic Tscm therapy.

Adoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD. The subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence. Our protocol began with the use of 1×107 donor lymphocytes and resulted in 1 ×107 ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the "foreign" antigen without causing GVHD after they were transferred into the recipients. Our strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.

Effects of adoptive T-cell immunotherapy on immune cell profiles and prognosis of patients with unresectable or recurrent cholangiocarcinoma.

The identification of immune cell profiles (ICP) involved in anti-tumor immunity is crucial for immunotherapy. Therefore, we herein investigated cholangiocarcinoma patients (CCA) who received adoptive T-cell immunotherapy (ATI). Eighteen unresectable or recurrent CCA received ATI of αβ T cells alone or combined with chemotherapy. ICP were evaluated by flow cytometry. There were 14 patients with intrahepatic cholangiocarcinoma (iCCA) and four with distal cholangiocarcinoma (dCCA). After one course of treatment, nine iCCA and four dCCA had progressive disease (PD), while five iCCA had stable disease (SD). Median overall survival (OS) was prolonged to 21.9 months. No significant differences were observed in OS between the PD and SD groups of iCCA. The frequency of helper T cells (HT) in iCCA decreased from 70.3% to 65.5% (P = .008), while that of killer T cells (KT) increased from 27.0% to 30.6% (P = .005). dCCA showed no significant changes of immune cells. OS was prolonged in iCCA with increased frequencies of CD3+ T cells (CD3) (P = .039) and αβ T cells (αβ) (P = .039). dCCA showed no immune cells associated with OS. The frequencies of CD3+ T cells and αβ T cells in the PD group for iCCA decreased from 63.5% to 53% (P = .038) and from 61.6% to 52.2% (P = .028), respectively. In the SD group, the frequency of HT decreased from 65.8% to 56.9% (P = .043), whereas that of KT increased from 30.1% to 38.3% (P = .043). In conclusions, ATI affected ICP and prolonged OS. Immune cells involved in treatment effects differed according to the site of cholangiocarcinoma.

Unraveling tumor specific neoantigen immunogenicity prediction: a comprehensive analysis.

Identification of tumor specific neoantigen (TSN) immunogenicity is crucial to develop peptide/mRNA based anti-tumoral vaccines and/or adoptive T-cell immunotherapies; thus, accurate in-silico classification/prioritization proves critical for cost-effective clinical applications. Several methods were proposed as TSNs immunogenicity predictors; however, comprehensive performance comparison is still lacking due to the absence of well documented and adequate TSN databases. Here, by developing a new curated database having 199 TSNs with experimentally-validated MHC-I presentation and positive/negative immune response (ITSNdb), sixteen metrics were evaluated as immunogenicity predictors. In addition, by using a dataset emulating patient derived TSNs and immunotherapy cohorts containing predicted TSNs for tumor neoantigen burden (TNB) with outcome association, the metrics were evaluated as TSNs prioritizers and as immunotherapy response biomarkers. Our results show high performance variability among methods, highlighting the need for substantial improvement. Deep learning predictors were top ranked on ITSNdb but show discrepancy on validation databases. In overall, current predicted TNB did not outperform existing biomarkers. Recommendations for their clinical application and the ITSNdb are presented to promote development and comparison of computational TSNs immunogenicity predictors.

Vaccination strategies and adoptive immunotherapy for ovarian cancer. Literature review

The review considers modern cancer vaccines consisting of autologous whole cells, dendritic cells loaded with autologous tumor-specific antigens, or plasmids, which lead to antigen expression and immune activation after repeated exposure. Each vaccination strategy can be supplemented with various immunomodulatory agents. Engineering autologous immunotherapy of tumor cells is an approach to restoring the effector function of T cells. Other tumor-associated antigens (TAAs) are also of interest in ovarian can cer. Adoptive immunotherapy is based on infusion of autologous or allogeneic tumor-targeted immune cells that have been expanded and/or activated exvivo. Adoptive immunotherapy can be based on antigen-dependent (innate immunity, for example, natural killer (NK) and cytokine-induced killer (CIK) cells) or antigen-independent (adaptive immunity, for instance, TILs, chimeric antigen receptor (CAR) T cells) strategies. NK cells can kill tumor cells without prior sensitization and play an important role in tumor immunosuppression. Adoptive T-cell immunotherapy, CAR-T-cell immunotherapy, allows combining antigen specificity through conjugation of a specific antibody with T-cell activating properties in a single fused molecule. CARs bypass the immune mechanism of cancer cell release as they endow T-lymphocytes with cytotoxic effector properties. In order to reduce mortality and improve prognosis, further study and refinement of immunotherapeutic strategies for the treatment of ovarian cancer is required.

Chimeric antigen receptor T-cells: Properties, production, and quality control.

Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive T-cell immunotherapy for haematological malignancies. First introduced into clinical practice in 2017, CAR T-cell therapy is now finding its place in the management of lymphoid malignancies, primarily of B-cell lineage, including lymphoblastic leukaemia, non-Hodgkin lymphoma and plasma cell myeloma, with remarkable therapeutic outcomes. CAR T-cells are a customised therapeutic product for each patient. Manufacture commences with collection of autologous T-cells, which are then genetically engineered ex vivo to express transmembrane CARs. These chimeric proteins consist of an antibody-like extracellular antigen-binding domain, to recognise specific antigens on the surface of tumour cells (e.g. CD19), linked to the intracellular co-stimulatory signalling domains of a T-cell receptor (e.g. CD137). The latter is required for in vivo CAR T-cell proliferation, survival, and durable efficacy. Following reinfusion, CAR T-cells harness the cytotoxic capacity of a patient's immune system. They overcome major mechanisms of tumour immuno-evasion and have potential to generate robust cytotoxic anti-tumour responses. This review discusses the background to CAR T-cell therapies, including their molecular design, mechanisms of action, methods of production, clinical applications and established and emerging technologies for CAR T-cell evaluation. It highlights the need for standardisation, quality control and monitoring of CAR T-cell therapies, to ensure their safety and efficacy in clinical management.

Recombinant human cyclophilin A in combination with adoptive T cell therapy improves the efficacy of cancer immunotherapy in experimental models in vivot;

Adoptive T-cell therapy (ACT) is successfully applied in cancer treatment, however, its efficiency can be limited by a low viability, short persistence time, and loss of functional activity of T cells after adoptive transfer. The search for novel immunomodulators that can improve the viability, expansion, and functions of T cells after their infusion with the minimal side effects could contribute to the development of more efficient and safe ACT strategies. Recombinant human cyclophilin A (rhCypA) is of particular interest in this respect, as it exhibits pleiotropic immunomodulatory activity and stimulates both innate and adaptive anti-tumor immunity. Here, we evaluated the effect of rhCypA on the efficacy of ACT in the mouse EL4 lymphoma model. Lymphocytes from transgenic 1D1a mice with an inborn pool of EL4-specific T cells were used as a source of tumor-specific T cells for ACT. In models of immunocompetent and immunodeficient transgenic mice, the course (3 days) rhCypA administration was shown to significantly stimulate EL4 rejection and prolong the overall survival of tumor-bearing mice after adoptive transfer of lowered doses of transgenic 1D1a cells. Our studies showed that rhCypA significantly improved the efficacy of ACT by enhancing the effector functions of tumor-specific cytotoxic T cells. These findings open up the prospects for the development of innovative strategies of adoptive T-cell immunotherapy for cancer using rhCypA as an alternative to existing cytokine therapies.

A close look at current γδ T-cell immunotherapy.

Owing to their antitumor and major histocompatibility complex (MHC)-independent capacities, γδ T cells have gained popularity in adoptive T-cell immunotherapy in recent years. However, many unknowns still exist regarding γδ T cells, and few clinical data have been collected. Therefore, this review aims to describe all the main features of the applications of γδ T cells and provide a systematic view of current γδ T-cell immunotherapy. Specifically, this review will focus on how γδ T cells performed in treating cancers in clinics, on the γδ T-cell clinical trials that have been conducted to date, and the role of γδ T cells in the pharmaceutical industry.

Immunotherapy for HER-2 positive breast cancer.

Immunotherapy is a developing treatment for advanced breast cancer. Immunotherapy has clinical significance for the treatment of triple-negative breast cancers and human epidermal growth factor receptor-2 positive (HER2+) breast cancers. As a proved effective passive immunotherapy, clinical application of the monoclonal antibodies trastuzumab, pertuzumab and T-DM1 (ado-trastuzumab emtansine) has significantly improved the survival of patients with HER2+ breast cancers. Immune checkpoint inhibitors that block programmed death receptor-1 and its ligand (PD-1/PD-L1) have also shown benefits for breast cancer in various clinical trials. Adoptive T-cell immunotherapies and tumor vaccines are emerging as novel approaches to treating breast cancer, but require further study. This article reviews recent advances in immunotherapy for HER2+ breast cancers.

The past, present, and future of immunotherapy for colorectal cancer

Colorectal cancer is prevalent worldwide, with various factors influencing the survival rate of late-stage metastatic cases. Current standard treatments include surgical removal, adjuvant chemotherapy, and neoadjuvant chemotherapy. Novel immunotherapy research shows promising results for various cancer types, including colorectal cancer. Current immunotherapy options are limited to specific molecular subtypes of colorectal cancer, while the remaining are limited to standard protocol. This review article summarizes approved, developing, and potential sources for novel colorectal cancer immunotherapy treatment through active-specific, checkpoint inhibitor, cytokine, cytotoxic, and adoptive T-cell immunotherapy. Such a study would be beneficial to patients with colorectal cancer.

HLA-haploidentical hematopoietic stem cells transplantation with regulatory and conventional T-cell adoptive immunotherapy in pediatric patients with very high-risk acute leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is still needed for many children with very high-risk acute leukemia. An HLA-haploidentical family donor is a suitable option for those without an HLA-matched donor. Here we present outcomes of a novel HLA-haploidentical HSCT (haplo-HSCT) strategy with adoptive immunotherapy with thymic-derived CD4+CD25+ FoxP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) performed between January 2017 and July 2021 in 20 children with high-risk leukemia. Median age was 14.5 years (range, 4-21), 15 had acute lymphoblastic leukemia, 5 acute myeloid leukemia. The conditioning regimen included total body irradiation (TBI), thiotepa, fludarabine, cyclophosphamide. Grafts contained a megadose of CD34+ cells (mean 12.4 × 106/Kg), Tregs (2 × 106/Kg) and Tcons (0.5-1 × 106/Kg). All patients achieved primary, sustained full-donor engraftment. Only one patient relapsed (5%). The incidence of non-relapse mortality was 15% (3/20 patients). Five/20 patients developed ≥ grade 2 acute Graft versus Host Disease (aGvHD). It resolved in 4 who are alive and disease-free; 1 patient developed chronic GvHD (cGvHD). The probability of GRFS was 60 ± 0.5% (95% CI: 2.1-4.2) (Fig.6), CRFS was 79 ± 0.9% (95% CI: 3.2-4.9) as 16/20 patients are alive and leukemia-free. The median follow-up was 2.1 years (range 0.5 months-5.1 years). This innovative approach was associated with very promising outcomes of HSCT strategy in pediatric patients.

IMMU-13. PROMOTER DEMETHYLATION OF NY-ESO-1 PRIMES GLIOBLASTOMA FOR IMMUNOTHERAPEUTIC TARGETING

Abstract INTRODUCTION New York-esophageal squamous cell carcinoma (NY-ESO-1) specific adoptive T-cell immunotherapy is an appealing strategy for the treatment of gliomas because it elicits a strong antibody and T-cell response and is downregulated in most normal adult tissues which produces an effective and specific antitumor response. However, NY-ESO-1 is often poorly expressed in GBM, presumably through promoter methylation. Neither the prevalence of NY-ESO-1 downregulation in glioblastoma (GBM) patient tumors nor the presumed mechanism of downregulation by promoter methylation have been formally established. METHODS We characterized baseline CpG methylation of NY-ESO-1 in patient GBM samples using bisulfite sequencing. We induced NY-ESO-1 expression in vitro in human GBM cell lines (U251) and patient-derived gliomaspheres using the demethylating agent decitabine (DAC). We investigated the epigenetic response of DAC treated GBM cell lines with bisulfite sequencing and NY-ESO-1 expression with quantitative real time PCR. Lastly, we performed single cell RNA sequencing on DAC treated and untreated U251 and PGDSC to evaluate tumor subpopulations that upregulate NY-ESO-1 and co-expressed cancer testis antigens after DAC treatment. RESULTS NY-ESO-1 expression is associated with promoter methylation in the majority of GBM. Prolonged low dose of DAC explicitly demethylated the promoter region of NY-ESO-1 and resulted in robust mRNA expression. DAC treatment co-expresses NY-ESO-1 with other cancer testis antigens CTAG2 and MAGEA4 as demonstrated by single cell RNA sequencing. CONCLUSION These results reveal a feasible strategy of cancer testis antigen promoter demethylation which creates an inducible tumor antigen for immunotherapeutic targeting. We speculate that DAC induced upregulation of NY-ESO-1 and co-expressed cancer testis antigens renders tumor recognition by NY-ESO-1 specific T cells.

High-throughput Tcell receptor engineering by functional screening identifies candidates with enhanced potency and specificity.

A major challenge in adoptive Tcell immunotherapy is the discovery of natural Tcell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human Tcell line at high throughput. We applied TCR-Engine to successfully engineer synthetic TCRs for increased potency and specificity to a clinically relevant tumor-associated antigen (MAGE-A3) and validated their translational potential through multiple invitro and invivo assessments of safety and efficacy. Thus, TCR-Engine represents a valuable technology for engineering of safe and potent synthetic TCRs for immunotherapy applications.

Importance of CAR-T cell therapy monitoring using high-throughput assays

Advances in the development of chimeric antigen receptor (CAR)-T cells have undermined the paramount importance of this technology for the success of adoptive T-cell immunotherapy. The bespoke production of autologous CAR-T cells is a lengthy and costly process. Thus, the development of more cost-effective allogeneic 'off-the-shelf' CAR-T cells provides a more readily available treatment option. The exploration of methods to reduce costs and to determine which CAR-T cells are the most effective is key for providing this breakthrough treatment to most patients. The process from the design and development of CAR-T cells, through pre-clinical and clinical testing and manufacturing, to patient monitoring involves a variety of high-throughput tools that enable the monitoring of all processes to ensure the safety and efficacy of the treatment.

P574: CPX-351 COMBINED WITH HEMATOPOIETIC CELL TRANSPLANTATION WITH REGULATORY AND CONVENTIONAL T CELL IMMUNOTHERAPY FOR HIGH-RISK ACUTE MYELOID LEUKEMIA

Background: Patients with newly diagnosed acute myeloid leukemia with myelodysplasia-related changes (MRC-AML) or therapy-related AML (t-AML) have an extremely poor prognosis. CPX-351, a dual-drug liposomal encapsulation of cytarabine and daunorubicin, improved significantly rates of remission and overall survival (Lancet JE et al, JCO 2018). Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative strategy for such patients, but incidence of post-transplant relapse is still unacceptably high. We developed a novel HSCT strategy that combines an age-adapted myeloablative irradiation-based conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy (Treg/Tcon HSCT). HLA-haploidentical Treg/Tcon HSCT ensured low relapse rate (4%) and resulted in an unprecedented chronic graft versus host disease/relapse free survival (CRFS, 75%) in AML patients (Pierini et al., Blood Adv. 2021). Aims: We report preliminary outcomes of the combination therapy with CPX-351 and Treg/Tcon HSCT for MRC-AML and t-AML at our center. Methods: We included in the study all patients with MRC-AML or t-AML who were referred at our center from September 2019 to January 2022 and could be treated with CPX-351. Treg/Tcon HSCT consisted of a myeloablative conditioning regimen with total marrow/lymphoid irradiation, thiotepa, fludarabine and cyclophosphamide followed by an infusion of 2x106/Kg donor Tregs on day -4, 1x106/Kg Tcons on day -1 and a megadose (>6x106/Kg) of purified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GvHD prophylaxis was given post-transplant (clinicaltrials.gov: #NCT03977103). Results: Seventeen patients with a median age of 57.5 years (46-72) were treated with CPX-351: 13 with newly diagnosed AML (11 MRC-AML and 2 t-AML) and 4 patients who relapsed after a previous HSCT and who acquired novel cytogenetic abnormalities (2 complex karyotypes, CK). 9/13 patients with newly diagnosed AML had karyotype abnormalities: 4 monosomy 7, 1 CK, 1 t(3;3), 1 isolated del(20q) and 2 (both t-AML) t(11;20) which resulted in NUP98-TOP1 fusion. Nine patients achieved complete remission (CR), 2 partial response (PR) and 2 had persistent disease. No patient experienced grade>3 extrahematologic toxicity. Ten/13 patients underwent a first HSCT (8 in CR, 2 in PR). Eight underwent Treg/Tcon HSCT (3 from HLA-matched, 5 from HLA-haploidentical donor), while 2 unmanipulated haploidentical HSCT followed by post-transplant cyclophosphamide (PT-Cy). All patients achieved full-donor engraftment. No patients experienced non-relapse mortality (NRM). Three/10 patients had a grade ≥ III acute GvHD; all of them are alive and off immunosuppressive therapy. At a median follow up of 16.2 months (range 9-29 months), 9/10 patients are alive and in CR with no chronic GvHD. No relapse occurred in the 8 patients who underwent Treg/Tcon HSCT, while one patient who underwent HSCT followed by PT-CY relapsed. The 4 patients who were treated after one previous HSCT received one single induction cycle with CPX-351, achieved CR and experienced no grade>3 extra-hematologic toxicity. All of them could undergo a second HSCT. NRM occurred in 1 patient (septic shock), while 3 patients are alive to date. Summary/Conclusion: CPX-351 allowed most of MRC-AML and t-AML patients to reach HSCT in CR despite high risk genetics and even when used in patients who relapsed after a 1st HSCT. Combination of CPX-351 with Treg-Tcon HSCT is safe and exert powerful antileukemic activity. Thus, it appears to be a promising strategy to ensure high rate of CRFS in very high-risk AML.

Combining CD34+ stem cell selection with prophylactic pathogen and leukemia directed T-cell immunotherapy to simultaneously reduce graft versus host disease, infection, and leukemia recurrence after allogeneic stem cell transplant.

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34+ stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8+ and to a lesser extent CD4+ T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

In-situ scalable manufacturing of Epstein\u2013Barr virus-specific T-cells using bioreactor with an expandable culture area (BECA)

The ex-vivo expansion of antigen-specific T-cells for adoptive T-cell immunotherapy requires active interaction between T-cells and antigen-presenting cells therefore culture density and environment become important variables to control. Maintenance of culture density in a static environment is traditionally performed by the expansion of the culture area through splitting of culture from a single vessel into multiple vessels—a highly laborious process. This study aims to validate the use and efficacy of a novel bioreactor, bioreactor with an expandable culture area—dual chamber (BECA-D), that was designed and developed with a cell chamber with expandable culture area (12–108 cm2) and a separate media chamber to allow for in-situ scaling of culture with maintenance of optimum culture density and improved nutrient and gas exchange while minimizing disturbance to the culture. The performance of BECA-D in the culture of Epstein–Barr virus-specific T-cells (EBVSTs) was compared to the 24-well plate. BECA-D had 0.9–9.7 times the average culture yield of the 24-well plates across 5 donor sets. BECA-D was able to maintain the culture environment with relatively stable glucose and lactate levels as the culture expanded. This study concludes that BECA-D can support the culture of ex-vivo EBVSTs with lower manufacturing labour and time requirements compared to the use of the 24-well plate. BECA-D and its adaptation into a closed system with an automated platform (currently being developed) provides cell therapy manufacturers and developers with a closed scale-out solution to producing adoptive cell therapy for clinical use.

SARS-CoV-2-specific T cells generated for adoptive immunotherapy are capable of recognizing multiple SARS-CoV-2 variants

Adoptive T-cell immunotherapy has provided promising results in the treatment of viral complications in humans, particularly in the context of immunocompromised patients who have exhausted all other clinical options. The capacity to expand T cells from healthy immune individuals is providing a new approach to anti-viral immunotherapy, offering rapid off-the-shelf treatment with tailor-made human leukocyte antigen (HLA)-matched T cells. While most of this research has focused on the treatment of latent viral infections, emerging evidence that SARS-CoV-2-specific T cells play an important role in protection against COVID-19 suggests that the transfer of HLA-matched allogeneic off-the-shelf virus-specific T cells could provide a treatment option for patients with active COVID-19 or at risk of developing COVID-19. We initially screened 60 convalescent individuals and based on HLA typing and T-cell response profile, 12 individuals were selected for the development of a SARS-CoV-2-specific T-cell bank. We demonstrate that these T cells are specific for up to four SARS-CoV-2 antigens presented by a broad range of both HLA class I and class II alleles. These T cells show consistent functional and phenotypic properties, display cytotoxic potential against HLA-matched targets and can recognize HLA-matched cells infected with different SARS-CoV-2 variants. These observations demonstrate a robust approach for the production of SARS-CoV-2-specific T cells and provide the impetus for the development of a T-cell repository for clinical assessment.