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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. Five of seven patients receiving anti-CD19 chimeric antigen receptor (CAR) T cells obtained complete remissions. Four of the five CRs had long-term durability with durations of remissions ranging from 38 to 56 months. Remissions persisted despite recovery of normal B cells in three of the four patients with long-term remissions.

BackgroundAdoptive T-cell therapy has demonstrated clinical activity in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors experience primary or secondary resistance to this treatment modality. Target antigen loss resulting either from non-uniform antigen expression or defects in antigen processing and presentation machinery is one well-characterized resistance mechanism. Constitutively expressed membrane-anchored interleukin-12 (caIL-12) has demonstrated enhanced antitumor activity and low systemic exposure in multiple preclinical adoptive T-cell treatment models with homogeneous tumor antigen expression. In this study, we assess the therapeutic impact of caIL-12 on target antigen-negative variants in syngeneic mouse models.MethodsTarget antigen-positive tumors were generated by transducing B16F10 melanoma cells (B16) or Lewis Lung Carcinoma cells (LLC) with a construct expressing the OVA antigen, SIINFEKL, tagged to ubiquitin (B16-U-OVA, LLC-U-OVA), while B16 or LLC tumors served as antigen-negative variants. C57BL/6J mice were subcutaneously injected with heterogeneous tumors composed of 80% B16-U-OVA and 20% B16. Bilateral tumors were established by injecting the left flank with B16-U-OVA or LLC-U-OVA tumors and the right flank injected with B16 or LLC tumors. The tumor-bearing mice then underwent 5.5 Gy total body irradiation, followed by adoptive transfer of OT-I TCR-T cells engineered with or without caIL-12.ResultsTCR-T cells (OT-I) delivered caIL-12 to the B16-U-OVA tumor sites and induced robust tumor control and survival benefits in mice bearing a heterogeneous tumor with OVA-negative variants. caIL-12 exerted its effect on OVA-negative B16 variants primarily by priming and activating endogenous antitumor CD8 T cells via antigen spreading. In addition, antigen spreading induced by OT-I-caIL-12 resulted in controlling OVA-negative tumors implanted at distant sites. This therapeutic effect required antigen-specific TCR-T cells and caIL-12 to colocalize at the tumor site, along with endogenous CD8 T cells capable of recognizing shared tumor antigens.ConclusionExpression of caIL-12 by tumor-targeting T cells demonstrated therapeutic effect against target-antigen-negative tumor variants, primarily through the induction of antigen spreading. These findings highlight the potential of caIL-12 to address challenges of antigen escape and tumor heterogeneity that may limit the efficacy of T-cell therapy against solid tumors.

Understanding regional distribution of HLA frequencies is crucial for optimizing enrollment in HLA-restricted clinical trials and to promote trial diversity per the Food and Drug Administration’s 2020 mandate. Using US HLA frequency data and census demographics we developed a method to create high-resolution HLA class 1 genotypic frequency maps. Analyzing HLA-A*11:01 and HLA-B*58:01 as alleles of interest, we found significant US regional variations. HLA-A*11:01, which presents KRAS neoantigen mutations targeted by TCR T-cell therapies, showed 10–15% genotypic frequency (national average 11.2%), with western US states 1.5 times higher than average and local variations within California (10–19%). These insights can be used to guide clinical trial site selection, for example, in National Cancer Institute (NCI) cancer center catchment areas. For HLA-B*58:01, which reacts pharmacogenetically with allopurinol and results in severe cutaneous adverse reactions, Mississippi had a high frequency among US states, which could be used to guide potential public safety campaigns. This method can identify regions with high HLA type representation, aiding efficient patient identification and enrollment for HLA-specific clinical trials and health-awareness efforts.

BackgroundTriple-negative breast cancer (TNBC) represents a subtype of breast cancer with poorest prognosis due to limited effective targeted therapies. Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating hematological cancers, but its application in TNBC requires further development. One major obstacle is the lack of suitable tumor-specific target in TNBC. Inspired by recent success of trophoblast cell-surface antigen 2 (TROP2) antibody-drug conjugate in TNBC, we developed a second-generation CAR that specifically targets TROP2 and formally evaluated its antitumor activity and safety profile using in vitro and in vivo models.MethodsA CAR molecule targeting TROP2 was constructed based on the clinically-validated humanized antibody Sacituzumab and expressed in primary human T cells using a retroviral vector. Tumor cytotoxicity, cytokine production and T-cell proliferation of TROP2 CAR-T cells were tested against multiple TNBC cell lines in vitro. Antitumor efficacy was evaluated using orthotopic and metastatic models of cell line-derived xenograft in NSG mice and in patient-derived xenograft (PDX) model. The safety profile of TROP2 CAR-T cells was assessed using TROP2-humanized immunocompetent mice and an “AND”-logic gated SynNotch CAR targeting B7-H3 and TROP2 was engineered to minimize off-tumor, on-target toxicity of TROP2 CAR-T cells.ResultsHuman TROP2 CAR-T cells demonstrated robust antitumor activity in vitro and in orthotopic/metastatic/PDX xenograft mouse models. TROP2 CAR-T cells caused lethal on-target, off-tumor toxicity in TROP2-humanized immunocompetent mice, causing severe tissue damage in lungs and systemic inflammation. The B7-H3/TROP2 “AND”-logic gated SynNotch CAR-T cells showed comparable antitumor efficacy without causing apparent adverse effects as in TROP2 CAR-T cells.ConclusionsThese data indicate that while CAR-T therapy targeting TROP2 possesses potent antitumor activity against TNBC cell lines and PDX, its potential side effects could be lethal due to TROP2 expression in vital organs such as the lung. Using an “AND”-logic gated CAR is a viable solution to overcome its in vivo toxicity. Our study lays the groundwork for future development of TROP2 CAR-T cell therapy for TNBC.

Age-associated defects in T cell-mediated immunity can increase the risk of cancers, but how aging influences adoptive T-cell therapy (ACT) for cancers remains unclear. Here, using a mouse model of melanoma, we demonstrate that aging diminishes anti-tumor activity of engineered CD8 T cells expressing a tumor-specific T cell receptor (CD8 TCR-T cells) in ACT for solid tumors. Aged CD8 TCR-T cells cannot control tumor growth in either young or aged mice. Aged CD8 TCR-T cells are unable to accumulate efficiently in tumors and have higher tendency to become terminally exhausted T cells with lower expression of endothelial PAS domain-containing protein 1 (Epas1) compared to young cells. Crispr-mediated ablation of Epas1 promotes terminal exhaustion of young CD8 T cells in tumors, diminishing their anti-tumor activity in young mice. Conversely, retroviral expression of Epas1 enhances anti-tumor activity of aged CD8 TCR-T cells. These findings suggest that aging-induced reduction of Epas1 expression impairs anti-tumor activity of CD8 T cells in ACT against solid tumors, which can be therapeutically improved by expression of exogenous Epas1.

BackgroundPersonalized immunotherapy of pancreatic ductal adenocarcinoma (PDAC) through T-cell mediated targeting of tumor-specific, mutanome-encoded neoepitopes may offer new opportunities to combat this disease, in particular by countering recurrence after primary tumor resection. However, the sensitive and accurate calling of somatic mutations in PDAC tissue samples is compromised by the low tumor cell content. Moreover, the repertoire of immunogenic neoepitopes in PDAC is limited due to the low mutational load of the majority of these tumors.MethodsWe developed a workflow involving the combined analysis of next-generation DNA and RNA sequencing data from matched pairs of primary tumor samples and patient-derived xenograft models towards the enhanced detection of driver mutations as well as single nucleotide variants encoding potentially immunogenic T-cell neoepitopes. Subsequently, we immunized HLA/human T-cell receptor (TCR) locus-transgenic mice with synthetic peptides representing candidate neoepitopes, and molecularly cloned the genes encoding TCRs targeting these epitopes.ResultApplication of our pipeline resulted in the identification of greater numbers of non-synonymous mutations encoding candidate neoepitopes with increased confidence. Furthermore, we provide proof of concept for the successful isolation of HLA-restricted TCRs from humanized mice immunized with different neoepitopes, several of which would not have been selected based on mutanome analysis of PDAC tissue samples alone. These TCRs mediate specific T-cell reactivity against the tumor cells in which the corresponding mutations were identified.ConclusionEnhanced mutanome analysis and candidate neoepitope selection increase the likelihood of identifying therapeutically relevant neoepitopes, and thereby support the optimization of personalized immunotherapy for PDAC and other poorly immunogenic cancers.

BackgroundThe tumor microenvironment is frequently hypoxic and characterized by a scarcity of nutritional resources including a shortage of glucose. As effector T cells have high energy demands, tumor metabolism can contribute to T-cell dysfunction and exhaustion.MethodsIn this study, we determined hypoxia in spleen and tumor tissue from tumor-bearing C57BL/6J mice using reverse transcription polymerase chain reaction (RT-PCR), histology and flow cytometry. Next, CD8+ T cells isolated from C57BL6J mice or P14+ mice were transduced with Thy1.1 (Control) or Thy1.1-Myoglobin (Mb) packaged retrovirus. Expression of Mb was confirmed with RT-PCR and western blot. Cellular metabolism was determined by flow cytometry, transmission electron microscopy, focused ion beam scanning electron microscopy, Seahorse, metabolomics and luminescence assays. Mb expressing or control P14+ or OT-I+ T cells were transferred in B16F10-gp33 or MC38-ova tumor-bearing mice respectively and analyzed using flow cytometry and histology. B16F10-gp33 tumor-bearing mice were additionally treated with anti-programmed cell death protein-1 (PD-1) checkpoint inhibitor.ResultsHere we demonstrate that expression of the oxygen-binding protein myoglobin in T cells can boost their mitochondrial and glycolytic metabolic functions. Metabolites and tricarboxylic acid compounds were highly increased in the presence of myoglobin (Mb), which was associated with increased ATP levels. Mb-expressing T cells exhibited low expression of hypoxia-inducible factor-1α after activation and during infiltration into the tumor microenvironment (TME). Accordingly, Mb expression increased effector T-cell function against tumor cells in vitro with concomitant reductions in superoxide levels. Following adoptive transfer into tumor-bearing mice, Mb expression facilitated increased infiltration into the TME. Although T cells expressing Mb exhibited increased expression of effector cytokines, PD-1 was still detected and targetable by anti-PD-1 monoclonal antibodies, which in combination with transfer of Mb-expressing T cells demonstrated maximal efficacy in delaying tumor growth.ConclusionTaken together, we show that expression of Mb in T cells can increase their metabolism, infiltration into the tumor tissue, and effector function against cancer cells.

BackgroundTumor-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) facilitates tumor progression, but the role of immune cell-intrinsic PCSK9 in tumor control remains unclear.MethodsOrthotopic models of pancreatic cancer and melanoma in Pcsk9-deficient mice were established and tumor-infiltrating immune cells were analyzed using single-cell RNA sequencing and flow cytometry. The effect of genetic disruptions of PCSK9 on murine CD8+ T cells and human chimeric antigen receptor (CAR)-T cells was evaluated both in vitro and in vivo.ResultsAblation of host Pcsk9 remarkably suppressed tumor growth and prolonged the survival of tumor-bearing mice, while tumor cells still express PCSK9. The enhanced tumor suppression in Pcsk9-deficient mice depended on CD8+ T cells. Notably, PCSK9 expression was induced in CD8+ tumor-infiltrating lymphocytes (TILs). Consequently, Pcsk9 ablation potentiated the antitumor capacity of CD8+ T cells, showing increased intratumoral infiltration and improved cytotoxic function, along with higher proportions of both effector-memory precursor exhausted (TPEX) and terminally exhausted (TTEX) CD8+ TILs. Additionally, disruption of PCSK9 in both murine CD8+ T cells and human CAR-T cells, synergistic with PD-1 blockade, promoted tumor suppression.ConclusionThese findings indicate that PCSK9 inhibits the antitumor function of CD8+ T cells, suggesting it may be a promising target for enhancing T-cell-based cancer immunotherapy.

Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases. Wang and colleagues show that a novel design of CAR based on adnectin, a class of scaffold molecules derived from the tenth type III domain of human fibronectin, can target EGFR-positive tumor cells and exhibit a similar functional profile as compared with the conventional scFv-derived CAR.

Purpose of ReviewIn this paper, we review the current state and modalities of adoptive cell therapies (ACT) in non-small cell lung carcinoma (NSCLC). We also discuss the challenges hampering the use of ACT and the approaches to overcome these barriers.Recent FindingsSeveral trials are ongoing investigating the three main modalities of T cell-based ACT: tumor-infiltrating lymphocytes (TILs), genetically engineered T-cell receptors (TCRs), and chimeric antigen receptor (CAR) T cells. The latter, in particular, has revolutionized the treatment of hematologic malignancies. However, the efficacy against solid tumor is still sparse. Major limitations include the following: severe toxicities, restricted infiltration and activation within the tumors, antigen escape and heterogeneity, and manufacturing issues.SummaryACT is a promising tool to improve the outcome of metastatic NSCLC, but significant translational and clinical research is needed to improve its application and expand the use in NSCLC.