Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery.

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRß chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

A Phase 1 Clinical Trial of NKTR-255 with CD19-22 CAR-T Cell Therapy for Refractory B-cell Acute Lymphoblastic Leukemia.

While chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies, many patients relapse and therefore strategies to improve antitumor immunity are needed. We previously designed a novel autologous bispecific CAR targeting CD19 and CD22 (CAR19-22), which was well tolerated and associated with high response rates but relapse was common. Interleukin-15 (IL15) induces proliferation of diverse immune cells and can augment lymphocyte trafficking. Here, we report the results of a phase 1 clinical trial of the first combination of a novel recombinant polymer-conjugated IL15 receptor agonist (NKTR-255), with CAR19-22, in adults with relapsed / refractory B-cell acute lymphoblastic leukemia. Eleven patients were enrolled, nine of whom successfully received CAR19-22 followed by NKTR-255. There were no dose limiting toxicities, with transient fever and myelosuppression as the most common possibly related toxicities. We observed favorable efficacy with eight out of nine patients (89%) achieving measurable residual disease negative remission. At 12 months, progression-free survival for NKTR-255 was double that of historical controls (67% vs 38%). We performed correlative analyses to investigate the effects of IL15 receptor agonism. Cytokine profiling showed significant increases in IL15 and the chemokines CXCL9 and CXCL10. The increase in chemokines was associated with decreases in absolute lymphocyte counts and CD8+ CAR T-cells in blood and ten-fold increases in CSF CAR-T cells, suggesting lymphocyte trafficking to tissue. Combining NKTR-255 with CAR19-22 was safe, feasible and associated with high rates of durable responses (NCT03233854).

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing.

BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

CD22-directed CAR T-cell therapy induces complete remissions in CD19-directed CAR-refractory large B-cell lymphoma.

The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Development of CAR T Cells Expressing a Suicide Gene Plus a Chimeric Antigen Receptor Targeting Signaling Lymphocytic-Activation Molecule F7.

Chimeric antigen receptors (CARs) are fusion proteins that contain antigen-recognition domains and T cell signaling domains. Signaling lymphocytic-activation molecule F7 (SLAMF7) is a promising target for CAR T cell therapies of the plasma cell malignancy multiple myeloma (MM) because SLAMF7 is expressed by MM but not normal nonhematopoietic cells. We designed CARs targeting SLAMF7. We transduced human T cells with anti-SLAMF7 CARs containing either CD28 or 4-1BB costimulatory domains. T cells expressing CD28-containing CARs or 4-1BB-containing CARs recognized SLAMF7 in vitro. SLAMF7-specific cytokine release was higher for T cells expressing CARs with CD28 versus 4-1BB domains. In murine solid tumor and disseminated tumor models, anti-tumor activity of T cells was superior with CD28-containing CARs versus 4-1BB-containing CARs. Because of SLAMF7 expression on some normal leukocytes, especially natural killer cells that control certain viral infections, the inclusion of a suicide gene with an anti-SLAMF7 CAR is prudent. We designed a construct with a CD28-containing anti-SLAMF7 CAR and a suicide gene. The suicide gene encoded a dimerization domain fused to a caspase-9 domain. T cells expressing the anti-SLAMF7 CAR plus suicide-gene construct specifically recognized SLAMF7 in vitro and eliminated tumors from mice. T cells expressing this construct were eliminated on demand by administering the dimerizing agent AP1903 (rimiducid).

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit+ population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue.

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

BACKGROUND: Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. METHODS: TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. RESULTS: The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. CONCLUSION: Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.

Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 10(6) CAR(+) T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient's MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967.

Long-Duration Complete Remissions of Diffuse Large B Cell Lymphoma after Anti-CD19 Chimeric Antigen Receptor T Cell Therapy.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) can induce complete remissions (CRs) of diffuse large B cell lymphoma (DLBCL). The long-term durability of these remissions is unknown. We administered anti-CD19 CAR T cells preceded by cyclophosphamide and fludarabine conditioning chemotherapy to patients with relapsed DLBCL. Five of the seven evaluable patients obtained CRs. Four of the five CRs had long-term durability with durations of remission of 56, 51, 44, and 38 months; to date, none of these four cases of lymphomas have relapsed. Importantly, CRs continued after recovery of non-malignant polyclonal B cells in three of four patients with long-term complete remissions. In these three patients, recovery of CD19+ polyclonal B cells took place 28, 38, and 28 months prior to the last follow-up, and each of these three patients remained in CR at the last follow-up. Non-malignant CD19+ B cell recovery with continuing CRs demonstrated that remissions of DLBCL can continue after the disappearance of functionally effective anti-CD19 CAR T cell populations. Patients had a low incidence of severe infections despite long periods of B cell depletion and hypogammaglobulinemia. Only one hospitalization for an infection occurred among the four patients with long-term CRs. Anti-CD19 CAR T cells caused long-term remissions of chemotherapy-refractory DLBCL without substantial chronic toxicities.

T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.

BACKGROUND: Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. METHODS: This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696. FINDINGS: Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients). INTERPRETATION: CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur. FUNDING: National Institutes of Health Intramural funds and St Baldrick's Foundation.

Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation.

New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD.

B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.

Inosine induces stemness features in CAR-T cells and enhances potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

CD22 CAR T cells demonstrate high response rates and safety in pediatric and adult B-ALL: Phase 1b results.

Chimeric antigen receptor (CAR) T cells targeting CD22 (CD22-CAR) provide a therapeutic option for patients with CD22+ malignancies with progression after CD19-directed therapies. Using on-site, automated, closed-loop manufacturing, we conducted parallel Phase 1b clinical trials investigating a humanized CD22-CAR with 41BB costimulatory domain in children and adults with heavily treated, relapsed/refractory (r/r) B-ALL. Of 19 patients enrolled, 18 had successful CD22-CAR manufacturing, and 16 patients were infused. High grade (3-4) cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS) each occurred in only one patient; however, three patients experienced immune-effector-cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). Twelve of 16 patients (75%) achieved CR with an overall 56% MRD-negative CR rate. Duration of response was overall limited (median 77 days), and CD22 expression was downregulated in 4/12 (33%) available samples at relapse. In summary, we demonstrate that closed-loop manufacturing of CD22-CAR T cells is feasible and is associated with a favorable safety profile and high CR rates in pediatric and adult r/r B-ALL, a cohort with limited CD22-CAR reporting.

A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer.

Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201-restricted peptides of MAGE-A3: 112-120 (KVAELVHFL) or MAGE-A3: 271-279 (FLWGPRALV), and T cell clones were generated. MAGE-A3-specific TCR α- and ß-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112-120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti-MAGE-A3 TCR.

Neoantigen-directed therapeutics in the clinic: where are we?

In the past decade, immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cell therapy have brought immunotherapy to the forefront of cancer treatment; however, only subsets of patients benefit from current approaches. Neoantigen-driven therapeutics specifically redirect the immune system of the patient to enable or reinduce its ability to recognize and eliminate cancer cells. The tumor specificity of this strategy spares healthy and normal cells from being attacked. Consistent with this concept, initial clinical trials have demonstrated the feasibility, safety, and immunogenicity of neoantigen-directed personalized vaccines. We review neoantigen-driven therapy strategies as well as their promise and clinical successes to date.

Challenges in neoantigen-directed therapeutics.

A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy.

Inosine Induces Stemness Features in CAR T cells and Enhances Potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing.