Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma

Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte–associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4+CD25+ T reg cell depletion has failed to consistently enhance immune-based therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25+ effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infiltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are significantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors

Donor whole blood DNA methylation is not a strong predictor of acute graft versus host disease in unrelated donor allogeneic haematopoietic cell transplantation

Allogeneic hematopoietic cell transplantation (HCT) is used to treat many blood-based disorders and malignancies. While this is an effective treatment, it can result in serious adverse events, such as the development of acute graft-versus-host disease (aGVHD). This study aimed to develop a donor-specific epigenetic classifier that could be used in donor selection in HCT to reduce the incidence of aGVHD. The discovery cohort of the study consisted of 288 donors from a population receiving HLA-A, -B, -C and -DRB1 matched unrelated donor HCT with T cell replete peripheral blood stem cell grafts for treatment of acute leukaemia or myelodysplastic syndromes after myeloablative conditioning. Donors were selected based on recipient aGVHD outcome; this cohort consisted of 144 cases with aGVHD grades III-IV and 144 controls with no aGVHD that survived at least 100 days post-HCT matched for sex, age, disease and GVHD prophylaxis. Genome-wide DNA methylation was assessed using the Infinium Methylation EPIC BeadChip (Illumina), measuring CpG methylation at >850,000 sites across the genome. Following quality control, pre-processing and exploratory analyses, we applied a machine learning algorithm (Random Forest) to identify CpG sites predictive of aGVHD. Receiver operating characteristic (ROC) curve analysis of these sites resulted in a classifier with an encouraging area under the ROC curve (AUC) of 0.91. To test this classifier, we used an independent validation cohort (n=288) selected using the same criteria as the discovery cohort. Different attempts to validate the classifier using the independent validation cohort failed with the AUC falling to 0.51. These results indicate that donor DNA methylation may not be a suitable predictor of aGVHD in an HCT setting involving unrelated donors, despite the initial promising results in the discovery cohort. Our work highlights the importance of independent validation of machine learning classifiers, particularly when developing classifiers intended for clinical use

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Misalignment evaluation of superconducting magnets in the relativistic heavy ion collider

In this paper, we first present the procedures for aligning and installing RHIC superconducting magnets, and then discuss the effects and compensation methods for the remaining misalignments

NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Relapse is a major cause of treatment failure after allogeneic hematopoietic stem cell transplantation (alloHSCT). Treatment options for relapse have been inadequate, and the majority of patients ultimately die of their disease. There is no standard approach to treating relapse after alloHSCT. Withdrawal of immune suppression and donor lymphocyte infusions are commonly used for all diseases; although these interventions are remarkably effective for relapsed chronic myelogenous leukemia, they have limited efficacy in other hematologic malignancies. Conventional and novel chemotherapy, monoclonal antibody therapy, targeted therapies, and second transplants have been utilized in a variety of relapsed diseases, but reports on these therapies are generally anecdotal and retrospective. As such, there is an immediate need for well-designed, disease-specific trials for treatment of relapse after alloHSCT. This report summarizes current treatment options under investigation for relapse after alloHSCT in a disease-specific manner. In addition, recommendations are provided for specific areas of research necessary in the treatment of relapse after alloHSCT

Tumor-reactive CD4+ T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts

Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II–restricted recognition of tumors by tumor-reactive CD4+ T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation

Immunotherapy with CD25/CD71-allodepleted T cells to improve T-cell reconstitution after matched unrelated donor hematopoietic stem cell transplant: a randomized trial

BACKGROUND AND AIMS: Delayed immune reconstitution is a major challenge after matched unrelated donor (MUD) stem cell transplant (SCT). In this randomized phase 2 multi-center trial, Adoptive Immunotherapy with CD25/71 allodepleted donor T cells to improve immunity after unrelated donor stem cell transplant (NCT01827579), the authors tested whether allodepleted donor T cells (ADTs) can safely be used to improve immune reconstitution after alemtuzumab-based MUD SCT for hematological malignancies. METHODS: Patients received standard of care or up to three escalating doses of ADTs generated through CD25+/CD71+ immunomagnetic depletion. The primary endpoint of the study was circulating CD3+ T-cell count at 4 months post-SCT. Twenty-one patients were treated, 13 in the ADT arm and eight in the control arm. RESULTS: The authors observed a trend toward improved CD3+ T-cell count at 4 months in the ADT arm versus the control arm (230/µL versus 145/µL, P = 0.18), and three ADT patients achieved normal CD3+ T-cell count at 4 months (>700/µL). The rates of significant graft-versus-host disease (GVHD) were comparable in both cohorts, with grade ≥2 acute GVHD in seven of 13 and four of eight patients and chronic GVHD in three of 13 and three of eight patients in the ADT and control arms, respectively. CONCLUSIONS: These data suggest that adoptive transfer of ADTs is safe, but that in the MUD setting the benefit in terms of T-cell reconstitution is limited. This approach may be of more use in the context of more rigorous T-cell depletion

Dual targeting of CD19 and CD22 with Bicistronic CAR-T cells in Patients with Relapsed/Refractory Large B Cell Lymphoma

Relapse following CD19-directed chimeric antigen receptor T-cells (CAR-T) for relapsed/refractory large B-cell lymphoma (r/r LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multi-antigen targeting and PD-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in r/r LBCL as inpatient or outpatient therapy (NCT03289455, https://clinicaltrials.gov/ct2/show/NCT03289455). Endpoints include toxicity (primary) and response rates (secondary). AUTO3 was manufactured for 62 patients using autologous leukapheresis, modified with a bicistronic transgene. 52 patients received AUTO3 (7/52,50x106; 45/52,150-450x106) and 48/52 received pembrolizumab. Median age was 59 years (range,27-83) and 46/52 had stage III/IV disease. Median follow-up was 21.6 months (range,15.1-51.3) at last data cut (Feb 28, 2022). AUTO3 was safe: grade 1-2 and grade 3 CRS affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), HLH affected 2 patients, and no Pembrolizumab-associated autoimmune sequalae were observed. On this basis, outpatient administration was tested in 20 patients, saving a median of 14 hospital days/patient. AUTO3 was effective: overall response rates were 66% (48.9%, CR; 17%, PR). For patients with CR, median DOR was not reached, with 54.4% (CI: 32.8, 71.7) projected to remain progression-free beyond 12 months after onset of remission. DOR for all responding patients was 8.3 months (95% CI: 3.0, NE) with 42.6% projected to remain progression-free beyond 12 months after onset of remission. Overall, AUTO3 +/- pembrolizumab for r/r LBCL was safe, lending itself to outpatient administration, and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion/persistence in vivo, and selection of CAR binders active at low antigen densities

Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity