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The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor β (FRβ) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRβ + TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8 + T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRβ + TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens. Several strategies have been attempted to target immune suppressive populations in the tumor microenvironment. Here the authors show that folate receptor β-targeted CAR-T cells eliminate immunosuppressive tumor associated macrophages, promoting endogenous antitumor immune responses and adoptive T-cell therapy in pre-clinical models.

‘T cell exhaustion’ is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1– and the self-renewing TCF1+ population from which they derive. These TCF1+ cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer 1 – 3 , but dysfunction due to T cell exhaustion is an important barrier to progress 4 – 6 . To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion 6 . Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells 7 – 10 . Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents. Chimeric antigen receptor (CAR) T cells engineered to overexpress the canonical AP-1 transcription factor c-Jun are resistant to T cell exhaustion, and provide enhanced therapeutic benefit in mouse tumour models.

Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

Background The small non-coding microRNAs play an important role in development by regulating protein translation, but their involvement in axon guidance is unknown. Here, we investigated the role of microRNA-134 (miR-134) in chemotropic guidance of nerve growth cones. Results We found that miR-134 is highly expressed in the neural tube of Xenopus embryos. Fluorescent in situ hybridization also showed that miR-134 is enriched in the growth cones of Xenopus spinal neurons in culture. Importantly, overexpression of miR-134 mimics or antisense inhibitors blocked protein synthesis (PS)-dependent attractive responses of Xenopus growth cones to a gradient of brain-derived neurotrophic factor (BDNF). However, miR-134 mimics or inhibitors had no effect on PS-independent bidirectional responses of Xenopus growth cones to bone morphogenic protein 7 (BMP7). Our data further showed that Xenopus LIM kinase 1 (Xlimk1) mRNA is a potential target of miR-134 regulation. Conclusions These findings demonstrate a role for miR-134 in translation-dependent guidance of nerve growth cones. Different guidance cues may act through distinct signaling pathways to elicit PS-dependent and -independent mechanisms to steer growth cones in response to a wide array of spatiotemporal cues during development.

Background Redirection of T lymphocytes against tumor antigens can induce dramatic regression of advanced stage malignancy. The use of bispecific antibodies (BsAbs) that bind both the T-cell receptor (TCR) and a target antigen is one promising approach to T-cell redirection. However, BsAbs indiscriminately bind all CD3+ T-cells and trigger TCR activation in the absence of parallel costimulatory signals required to overcome T-cell unresponsiveness or anergy. Methods To address these limitations, a combination platform was designed wherein a unique BsAb referred to as frBsAb exclusively engages T-cells engineered to express a novel chimeric receptor comprised of extracellular folate receptor fused to intracellular TCR and CD28 costimulatory signaling domains in tandem; a BsAb-binding immune receptor (BsAb-IR). As a surrogate TCR, the BsAb-IR allows for concomitant TCR and costimulatory signaling exclusively in transduced T-cells upon engagement with specific frBsAbs, and can therefore redirect T-cells on command to desired antigen. Human primary T-cells were transduced with lentiviral vector and expanded for 14–18 days. BsAb-IRs were harvested and armed with frBsAbs to test for redirected cytotoxicity against CD20 positive cancer cell lines. Results Using frBsAbs specific for CD20 or HER2, the lytic activity of primary human T-cells expressing the BsAb-IR was specifically redirected against CD20+ leukemic cells or HER2+ epithelial cancer cells, respectively, while non-engineered T-cells were not activated. Notably, elimination of the CD28 costimulatory domain from the BsAb-IR construct significantly reduced frBsAb-redirected antitumor responses, confirming that frBsAbs are capable of delivering simultaneous TCR activation and costimulatory signals to BsAb-IR T-cells. Conclusion In summary, our results establish the proof of concept that the combination of BsAbs with optimized gene-engineered T-cells provides the opportunity to specify and augment tumor antigen-specific T-cell activation and may improve upon the early success of conventional BsAbs in cancer immunotherapy.

Since previous work using a nonreplicating adenovirus-expressing mouse interferon-β (Ad.mIFNβ) showed promising preclinical activity, we postulated that a vector-expressing IFNβ at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNβ (VV.mIFNβ) was tested. VV.mIFNβ-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNβ had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNβ vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.

BackgroundEffective solid tumor cell therapy requires new strategies to improve T-cell activation, persistence, and durable function. 1 2 We developed four complementary, stackable T-cell reprogramming technologies to enhance chimeric antigen receptor (CAR) T-cell therapy in solid tumors: 1) overexpression of the activator protein 1 (AP-1) family transcription factor c-Jun to delay T-cell exhaustion and improve functional activity 1 3 4 ; 2) nuclear receptor subfamily 4A member 3 (NR4A3) gene knock-out (KO) to further delay exhaustion and enhance functionality 5–7 ; 3) Epi-RTM manufacturing protocol to promote stem-like characteristics 1 8–10 ; and 4) Stim-RTM technology a synthetic biomimetic to improve T cell potency compared with conventional activating reagents.11 LYL119 is a ROR1-targeted CAR T-cell product candidate that combines these technologies to create potent CAR T cells with durable function.MethodsHealthy donor T cells were manufactured at research or clinical scale with the Epi-R protocol, activated with Stim-R technology or a standard reagent, and transduced with a tri-cistronic lentiviral vector encoding ROR1 CAR, c-Jun, and truncated EGFR tag. The NR4A3 gene or a safe-harbor control gene was edited. CAR T-cell cytotoxicity, cytokine production, and phenotype were evaluated in vitro after repeated antigen-stimulation assays designed to promote exhaustion. Finally, CAR T cells were evaluated in vivo using a ROR1-expressing H1975 human lung cancer xenograft model in mice.ResultsLYL119 products were successfully manufactured at research and clinical scale with comparable product phenotype. We achieved ~90% genomic editing efficiency at the NR4A3 target gene resulting in 13-fold reduction in protein expression compared to a non-edited control. CAR T cells manufactured with Stim-R technology displayed an activated phenotype, as indicated by elevated CD25 expression, as well as high TCF-7 and CD127 expression suggesting maintenance of stem-like populations.LYL119 demonstrated superior cytotoxicity and sustained cytokine production upon repeated antigen stimulation compared to various controls lacking one or more of the reprogramming technologies (figure 1). LYL119 also had reduced surface expression of inhibitory receptors and maintained higher expression of CD127 compared to non-edited CAR T cells manufactured using standard reagents, suggesting reduced T-cell exhaustion and improved maintenance of stem-like characteristics. Moreover, LYL119 showed robust antitumor efficacy in vivo across a 10-fold dose range, including a very low dose of 0.1x106 CAR T cells (figure 2).ConclusionsThese data suggest that LYL119, which combines c-Jun overexpression, NR4A3 KO, Epi-R protocol, and Stim-R technology, can limit exhaustion, maintain stem-like features, and has potential to provide effective and durable CAR T-cell antitumor activity in patients with ROR1+ solid tumors.References1. Park S, et al. AACR 2022 Poster. Abstract 2754.2. Krishna S, et al. Science. 2020;370:1328–1334.3. Lynn RC, et al. Nature. 2019;576:293–300.4. Park S, et al. ASGCT 2022 Poster. Abstract 661.5. Chen J, et al. Nature. 2019;567:530–534.6. Liu X, et al. Nature. 2019;567:525–529.7. Lam V, et al. SITC 2022 Poster. Abstract 243.8. Patel Y, et al. SITC 2022 Poster. Abstract 370.9. Harris BD, et al. SITC 2022 Poster. Abstract 340.10. Patel Y, et al. AACR Special Conference: Tumor Immunology and Immunotherapy 2022 Poster. Abstract A54.11. Li A, et al. SITC 2022 Poster. Abstract 252.Abstract 278 Figure 1Successive lysis of ROR1-expressing H1975-NucLight Red (NLR) target cells in one representative donor (n=4 donors) at an effector-to-target ratio of 1:10. Lysis of H1975-NLR target cells was quantified by measuring total NLR intensity. NLR intensity was normalized relative to the starting intensity after replating for each round of stimulation. LYL119 showed significant functional potency at the last timepoint of the fourth stimulation compared to other T cells tested. Asterisks represent p-value significance of each condition compared to LYL119 (unpaired t-test, ns — not significant, * p < 0.05, ** p < 0.005).Abstract 278 Figure 2Antitumor efficacy at three CAR T-cell doses in one representative experiment tested in an in vivo H1975 xenograft model (n=2 donors with 10 mice/group). LYL119 impaired tumor growth at a very low CAR T-cell dose (0.1x106). (* p < 0.05, ** p < 0.005, ***p < 0.0001, Tukey one-way ANOVA).

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.