Targeting IDH2R140Q and other neoantigens in acute myeloid leukemia.

ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, ∼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.

Antitumor efficacy and safety of unedited autologous CD5.CAR T cells in relapsed/refractory mature T-cell lymphomas.

ABSTRACT: Despite newer targeted therapies, patients with primary refractory or relapsed (r/r) T-cell lymphoma have a poor prognosis. The development of chimeric antigen receptor (CAR) T-cell platforms to treat T-cell malignancies often requires additional gene modifications to overcome fratricide because of shared T-cell antigens on normal and malignant T cells. We developed a CD5-directed CAR that produces minimal fratricide by downmodulating CD5 protein levels in transduced T cells while retaining strong cytotoxicity against CD5+ malignant cells. In our first-in-human phase 1 study (NCT0308190), second-generation autologous CD5.CAR T cells were manufactured from patients with r/r T-cell malignancies. Here, we report safety and efficacy data from a cohort of patients with mature T-cell lymphoma (TCL). Among the 17 patients with TCL enrolled, CD5 CAR T cells were successfully manufactured for 13 out of 14 attempted lines (93%) and administered to 9 (69%) patients. The overall response rate (complete remission or partial response) was 44%, with complete responses observed in 2 patients. The most common grade 3 or higher adverse events were cytopenias. No grade 3 or higher cytokine release syndrome or neurologic events occurred. Two patients died during the immediate toxicity evaluation period due to rapidly progressive disease. These results demonstrated that CD5.CAR T cells are safe and can induce clinical responses in patients with r/r CD5-expressing TCLs without eliminating endogenous T cells or increasing infectious complications. More patients and longer follow-up are needed for validation. This trial was registered at www.clinicaltrials.gov as #NCT0308190.

Engineering T cells to suppress acute GVHD and leukemia relapse after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) limits the therapeutic benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and requires immunosuppressive prophylaxis that compromises antitumor and antipathogen immunity. OX40 is a costimulatory receptor upregulated on circulating T cells in aGVHD and plays a central role in driving the expansion of alloreactive T cells. Here, we show that OX40 is also upregulated on T cells infiltrating GVHD target organs in a rhesus macaque model, supporting the hypothesis that targeted ablation of OX40+ T cells will mitigate GVHD pathogenesis. We thus created an OX40-specific cytotoxic receptor that, when expressed on human T cells, enables selective elimination of OX40+ T cells. Because OX40 is primarily upregulated on CD4+ T cells upon activation, engineered OX40-specific T cells mediated potent cytotoxicity against activated CD4+ T cells and suppressed alloreactive T-cell expansion in a mixed lymphocyte reaction model. OX40 targeting did not inhibit antiviral activity of memory T cells specific to Epstein-Barr virus, cytomegalovirus, and adenoviral antigens. Systemic administration of OX40-targeting T cells fully protected mice from fatal xenogeneic GVHD mediated by human peripheral blood mononuclear cells. Furthermore, combining OX40 targeting with a leukemia-specific chimeric antigen receptor in a single T cell product provides simultaneous protection against leukemia and aGVHD in a mouse xenograft model of residual disease posttransplant. These results underscore the central role of OX40+ T cells in mediating aGVHD pathogenesis and support the feasibility of a bifunctional engineered T-cell product derived from the stem cell donor to suppress both disease relapse and aGVHD following allo-HSCT.

Retraction Note: A homing system targets therapeutic T cells to brain cancer.

This Article has been retracted; see accompanying Retraction.

Engineering naturally occurring CD7- T cells for the immunotherapy of hematological malignancies.

Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.

Long-term follow-up for the development of subsequent malignancies in patients treated with genetically modified IECs.

Subsequent malignancies are well-documented complications in long-term follow-up of cancer patients. Recently, genetically modified immune effector (IE) cells have shown benefit in hematologic malignancies and are being evaluated in clinical trials for solid tumors. Although the short-term complications of IE cells are well described, there is limited literature summarizing long-term follow-up, including subsequent malignancies. We retrospectively reviewed data from 340 patients treated across 27 investigator-initiated pediatric and adult clinical trials at our center. All patients received IE cells genetically modified with γ-retroviral vectors to treat relapsed and/or refractory hematologic or solid malignancies. In a cumulative 1027 years of long-term follow-up, 13 patients (3.8%) developed another cancer with a total of 16 events (4 hematologic malignancies and 12 solid tumors). The 5-year cumulative incidence of a first subsequent malignancy in the recipients of genetically modified IE cells was 3.6% (95% confidence interval, 1.8% to 6.4%). For 11 of the 16 subsequent tumors, biopsies were available, and no sample was transgene positive by polymerase chain reaction. Replication-competent retrovirus testing of peripheral blood mononuclear cells was negative in the 13 patients with subsequent malignancies tested. Rates of subsequent malignancy were low and comparable to standard chemotherapy. These results suggest that the administration of IE cells genetically modified with γ retroviral vectors does not increase the risk for subsequent malignancy.

Human platelet lysate enhances in vivo activity of CAR-Vδ2 T cells by reducing cellular senescence and apoptosis.

BACKGROUND AIMS: Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. METHODS: Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. RESULTS: We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. CONCLUSIONS: This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement.

Taking T-Cell Oncotherapy Off-the-Shelf.

Banked allogeneic or 'off-the-shelf' (OTS) T cells from healthy human donors are being developed to address the limitations of autologous cell therapies. Potential challenges of OTS T cell therapies are associated with their allogeneic origin and the possibility of graft-versus-host disease (GvHD) and host-versus-graft immune reactions. While the risk of GvHD from OTS T cells has been proved to be manageable in clinical studies, approaches to prevent immune rejection of OTS cells are at an earlier stage of development. We provide an overview of strategies to generate OTS cell therapies and mitigate alloreactivity-associated adverse events, with a focus on recent advances for preventing immune rejection.

A homing system targets therapeutic T cells to brain cancer.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies.

Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7-, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering.

Combinatorial antigen targeting strategies for acute leukemia: application in myeloid malignancy.

BACKGROUND AIMS: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse. METHODS: For co-transduction with the authors' previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens. RESULTS: The in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors. CONCLUSIONS: Overall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.

Transcription factor ELF4 controls the proliferation and homing of CD8+ T cells via the Krüppel-like factors KLF4 and KLF2.

Transcription factors that regulate the quiescence, proliferation and homing of lymphocytes are critical for effective immune system function. Here we demonstrate that the transcription factor ELF4 directly activated the tumor suppressor KLF4 'downstream' of T cell antigen receptor signaling to induce cell cycle arrest in naive CD8(+) T cells. Elf4- and Klf4-deficient mice accumulated CD8(+)CD44(hi) T cells during steady-state conditions and generated more memory T cells after immunization. The homeostatic population expansion of CD8(+)CD44(hi) T cells in Elf4-null mice resulted in a redistribution of cells to nonlymphoid tissue because of lower expression of the transcription factor KLF2 and the surface proteins CCR7 and CD62L. Our work describes the combinatorial effect of lymphocyte-intrinsic factors on the homeostasis, activation and homing of T cells.

CD7 CAR T Cells for the Therapy of Acute Myeloid Leukemia.

Chimeric antigen receptor (CAR) T cell therapy for the treatment of acute myeloid leukemia (AML) has the risk of toxicity to normal myeloid cells. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients but is absent on normal myeloid and erythroid cells. Since CD7 expression by malignant blasts is also linked with chemoresistance and poor outcomes, targeting this antigen may be beneficial for this subset of AML patients. Here, we show that expression of a CD7-directed CAR in CD7 gene-edited (CD7KO) T cells effectively eliminates CD7+ AML cell lines, primary CD7+ AML, and colony-forming cells but spares myeloid and erythroid progenitor cells and their progeny. In a xenograft model, CD7 CAR T cells protect mice against systemic leukemia, prolonging survival. Our results support the feasibility of using CD7KO CD7 CAR T cells for the non-myeloablative treatment of CD7+ AML.

CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies.

Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7+) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO+CCR7- effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells.

Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lectin-like Molecule 1.

The successful immunotherapy of acute myeloid leukemia (AML) has been hampered because most potential antigenic targets are shared with normal hematopoietic stem cells (HSCs), increasing the risk of sustained and severe hematopoietic toxicity following treatment. C-type lectin-like molecule 1 (CLL-1) is a membrane glycoprotein expressed by >80% of AML but is absent on normal HSCs. Here we describe the development and evaluation of CLL-1-specific chimeric antigen receptor T cells (CLL-1.CAR-Ts) and we demonstrate their specific activity against CLL-1+ AML cell lines as well as primary AML patient samples in vitro. CLL-1.CAR-Ts selectively reduced leukemic colony formation in primary AML patient peripheral blood mononuclear cells compared to control T cells. In a human xenograft mouse model, CLL-1.CAR-Ts mediated anti-leukemic activity against disseminated AML and significantly extended survival. By contrast, the colony formation of normal progenitor cells remained intact following CLL-1.CAR-T treatment. Although CLL-1.CAR-Ts are cytotoxic to mature normal myeloid cells, the selective sparing of normal hematopoietic progenitor cells should allow full myeloid recovery once CLL-1.CAR-T activity terminates. To enable elective ablation of the CAR-T, we therefore introduced the inducible caspase-9 suicide gene system and we show that exposure to the activating drug rapidly induced a controlled decrease of unwanted CLL-1.CAR-T activity against mature normal myeloid cells.

A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.

Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms.