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A major limitation to developing chimeric antigen receptor (CAR)-T cell therapies for solid tumors is identifying surface proteins highly expressed in tumors but not in normal tissues. Here, we identify Tyrosinase Related Protein 1 (TYRP1) as a CAR-T cell therapy target to treat patients with cutaneous and rare melanoma subtypes unresponsive to immune checkpoint blockade. TYRP1 is primarily located intracellularly in the melanosomes, with a small fraction being trafficked to the cell surface via vesicular transport. We develop a highly sensitive CAR-T cell therapy that detects surface TYRP1 in tumor cells with high TYRP1 overexpression and presents antitumor activity in vitro and in vivo in murine and patient-derived cutaneous, acral and uveal melanoma models. Furthermore, no systemic or off-tumor severe toxicities are observed in an immunocompetent murine model. The efficacy and safety profile of the TYRP1 CAR-T cell therapy supports the ongoing preparation of a phase I clinical trial. A main challenge for the use of CAR-T in solid tumours is the identification of surface proteins as feasible targets. Here, the authors show TYRP1 as a target for CAR-T cell therapy in preclinical models of cutaneous, acral and uveal melanoma.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells 1 – 14 . The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies 15 – 17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen–HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR–Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8 + T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time. Effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8 + T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells. Adoptive cell transfer (ACT) of T cells for tumor treatment often requires IL-2 administration. Here, the authors show that a modified IL-2 cytokine (NKTR-214) can outperform IL-2 in a melanoma mouse model.

Background Interferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway. Methods We performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death. Results We show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins. Conclusions Our results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance.

Whole-exome sequencing was performed on four patients' tumors before exposure to pembrolizumab and after disease progression following a response to treatment. Acquired mutations involving antigen presentation and interferon response were noted. Durable responses in metastatic cancers have been achieved with a variety of immunotherapies such as interleukin-2, adoptive cell transfer of tumor-infiltrating lymphocytes, antibodies that block cytotoxic T-lymphocyte–associated antigen 4 (CTLA4), 1 – 5 and antibodies that block programmed death 1 (PD-1). 6 – 10 However, in a recent study, approximately 25% of patients with melanoma who had had an objective response to PD-1 blockade therapy had disease progression at a median follow-up of 21 months. 11 The mechanisms of immune-resistant cancer progression are mostly unknown. Previous studies involving humans examined the loss of beta-2-microglobulin as a mechanism of acquired resistance to several forms of cancer . . .

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes 1 , 2 using recombinant viral vectors, which do not target transgenes to specific genomic sites 3 , 4 . The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair 5 , 6 . Here we developed a CRISPR–Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor ( TCR ) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells. A non-viral strategy to introduce large DNA sequences into T cells enables the correction of a pathogenic mutation that causes autoimmunity, and the replacement of an endogenous T-cell receptor with an engineered receptor that can recognize cancer antigens.

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

The Millennium Drought lasted more than a decade and is notable for causing persistent shifts in the relationship between rainfall and runoff in many southeastern Australian catchments. Research to date has successfully characterised where and when shifts occurred and explored relationships with potential drivers, but a convincing physical explanation for observed changes in catchment behaviour is still lacking. Originating from a large multi-disciplinary workshop, this paper presents and evaluates a range of hypothesised process explanations of flow response to the Millennium Drought. The hypotheses consider climatic forcing, vegetation, soil moisture dynamics, groundwater, and anthropogenic influence. The hypotheses are assessed against evidence both temporally (e.g. why was the Millennium Drought different to previous droughts?) and spatially (e.g. why did rainfall–runoff relationships shift in some catchments but not in others?). Thus, the strength of this work is a large-scale assessment of hydrologic changes and potential drivers. Of 24 hypotheses, 3 are considered plausible, 10 are considered inconsistent with evidence, and 11 are in a category in between, whereby they are plausible yet with reservations (e.g. applicable in some catchments but not others). The results point to the unprecedented length of the drought as the primary climatic driver, paired with interrelated groundwater processes, including declines in groundwater storage, altered recharge associated with vadose zone expansion, and reduced connection between subsurface and surface water processes. Other causes include increased evaporative demand and harvesting of runoff by small private dams. Finally, we discuss the need for long-term field monitoring, particularly targeting internal catchment processes and subsurface dynamics. We recommend continued investment in the understanding of hydrological shifts, particularly given their relevance to water planning under climate variability and change.