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CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFN γ R/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo ; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells. CAR-T therapy is a promising treatment modality for B-cell malignancies, yet many patients relapse. Using an in vivo genomewide screen in a model of B cell leukemia, we identify an unexpected mechanism of CAR-T resistance in which interferon gamma from the in vivo tumor microenvironment induces an adaptive T-cell resistance program in tumor cells.

Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8 + T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5 -mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses. UCHL5 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains. Here, the authors discover through in-vivo CRISPR-Cas9 screens that Uchl5 is involved in immune evasion and modulation of extracellular matrix deposition in head and neck squamous cell carcinoma.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.Here, the authors use genome-scale in vivo CRISPR screens to look at immune evasion mechanisms across cancer models, showing that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints facilitate immune escape.

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

BackgroundThe tyrosine phosphatases PTPN2 and PTPN1 negatively regulate several signaling pathways in immune and tumor cells. We previously demonstrated that oral administration of our recently discovered active site PTPN2/N1 small molecule inhibitor ABBV-CLS-484 (AC-484) promotes anti-tumor immunity in several syngeneic mouse tumor models. AC-484 improves T cell activation and function upon TCR stimulation and enhances dendritic cell and macrophage activity in vitro consistent with prior findings in PTPN2 or PTPN1 genetically deficient T cells and myeloid cells. However, a role for PTPN2 or PTPN1 in NK cells has not been previously described. NK cells are essential for eliminating tumors that typically evade the adaptive T cell response and are critically important to control metastasis formation. Given the role of inhibitory signaling pathways, we hypothesized that PTPN2 and PTPN1 may also negatively regulate NK activity and therefore AC-484 should enhance NK cell function and NK-mediated anti-tumor immunity.MethodsTo understand the impact of AC-484 on NK cells, we employed cytotoxicity assays in vitro and utilized immunophenotyping and single cell RNA sequencing of tumor-infiltrating immune cells isolated from mouse syngeneic tumor models. We also assessed the contribution of NK cells to AC-484-mediated efficacy in subcutaneous primary tumor and spontaneous lung metastasis formation models.ResultsAC-484 treatment enhanced NK cell function and NK-mediated tumor cell killing in vitro. Consistent with these findings, immunophenotyping and single-cell RNAseq analyses demonstrated that in vivo AC-484 therapy increased NK cell abundance and activation in mouse tumor models with varying responsiveness to immune checkpoint blockade. Further, in tumor models that do not rely on T cells for tumor control such as those with MHCI or Jak1 deficiency, AC-484 therapy improved NK-mediated efficacy. In addition to controlling primary tumors, AC-484 also potently prevented lung metastasis formation in the B16F10 intravenous and the 4T1 orthotopic breast cancer models in an NK cell-dependent manner.ConclusionsHere, we describe for the first time a role for PTPN2 and PTPN1 in NK cells. Our findings suggest that AC-484 can both control primary tumors and prevent tumor metastasis in an NK cell-dependent manner. We further show that AC-484 treatment overcomes various common immune evasion mechanisms developed by tumors, including those acquired via mutations in Beta-2-microglobulin, HLA, and JAK1/2. These findings, along with our previous reports, underscore how AC-484 significantly promotes anti-tumor efficacy through a multifaceted mechanism by sensitizing tumor cells to inflammation and enhancing the activity of a variety of immune subsets.Ethics ApprovalHumanHuman blood samples were acquired through the internal AbbVie Inc’s blood donation program in accordance with AbbVie’s Occupational Safety and Health Administration protocols or healthy donors from Stanford University.AnimalsAll in vivo experiments conducted at AbbVie were in compliance with the NIH Guide for Care and Use of Laboratory Animals guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). All in vivo studies conducted at the Broad Institute or Calico Life Sciences were approved by the respective IACUC committees.DisclosuresC.H.P., I.S., Y.L., M.N.P., and J.D.P. are employees of Calico Life Sciences LLC. K.A.M., K.L.K., J.D.A, K.H., J.T.K., A.W.S., K.M.H, J.M.F, P.R.K, and C.K.B. are employees of AbbVie Inc. H.E., A.J.M., P.T., O.A, K.C., K.O., K.B.Y., and R.T.M. are employees of the Broad Institute. The laboratory of R.T.M. at the Broad Institute receives research funding from Calico Life Sciences LLC. R.T.M. has served as a consultant for Bristol Myers Squibb and receives research funding from Calico Life Sciences LLC.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance 1 , 2 . The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity 3 – 6 . However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8 + T cell function by enhancing JAK–STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes. An orally bioavailable small-molecule active-site inhibitor of the phosphatases PTPN2 and PTPN1, ABBV-CLS-484, demonstrates immunotherapeutic efficacy in mouse models of cancer resistant to PD-1 blockade.

BackgroundImmune checkpoint blockade is effective for a subset of patients across many cancers, but most patients are refractory to current immunotherapies and new approaches are needed to overcome resistance.1 2 The protein tyrosine phosphatase PTPN2 and the closely related PTPN1 are central regulators of inflammation, and their genetic deletion in either tumor cells or host immune cells promotes anti-tumor immunity.3–6 However, phosphatases are challenging drug targets and in particular, the active site has been considered undruggable. Here, we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2/N1 active site inhibitor.MethodsIn this study, we characterize AC484 and evaluate its effects in vitro and in vivo. We conduct in vitro experiments to investigate the interferon response and the activation and function of various immune cell subsets in response to AC484. We employ murine cancer models resistant to PD-1 blockade and assess the anti-tumor efficacy of AC484 monotherapy in these models. Additionally, through single-cell transcriptional profiling of tumor-infiltrating immune cells, we examine the transcriptional and functional effects of AC484 treatment, with a focus on CD8+ T cells.ResultsAC484 treatment demonstrates the ability to amplify the response to interferon and enhance the activation and function of multiple immune cell subsets in vitro. In murine cancer models resistant to PD-1 blockade, monotherapy AC484 treatment generates robust anti-tumor immunity. Transcriptomic and functional analyses of tumor-infiltrating immune cells reveal that AC484 treatment elicits broad effects on myeloid and lymphoid compartments, particularly influencing CD8+ T cells. Surprisingly, we find that AC484 treatment induces a unique transcriptional state in CD8+ T cells mediated by enhanced JAK-STAT signaling, whereby T cells display a highly cytotoxic effector profile, increased memory signatures, and reduced exhaustion and dysfunction.ConclusionsOur results demonstrate that oral administration of small molecule inhibitors of PTPN2/N1 can induce potent anti-tumor immunity. PTPN2/N1 inhibitors offer a promising new strategy for cancer immunotherapy and are currently being evaluated clinically in patients with advanced solid tumors (NCT04777994). More broadly, our study shows that small molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics targeting this important class of enzymes.ReferencesHugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell. 2017;168:542.Fares CM, Van Allen EM, Drake CG, Allison JP, Hu-Lieskovan S. Mechanisms of resistance to immune checkpoint blockade: why does checkpoint inhibitor immunotherapy not work for all patients? Am Soc Clin Oncol Educ Book. 2019;39:147–164.Manguso RT, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017;547:413–418.Wiede F, et al. PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours. EMBO J. 2020;39:e103637.LaFleur MW, et al. PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nat. Immunol. 2019;20:1335–1347.Flosbach M, et al. PTPN2 deficiency enhances programmed T cell expansion and survival capacity of activated T cells. Cell Rep. 2020;32:107957.Ethics ApprovalThe protocol, under which human blood samples were acquired, was approved by and is reviewed on an annual basis by WCG IRB (Puyallup, Washington). WCG IRB is in full compliance with the Good Clinical Practices as defined under the U.S. Food and Drug Administration (FDA) Regulations, U.S. Department of Health and Human Services (HHS) regulations and the International Conference on Harmonisation (ICH) Guidelines. All human research participants signed informed consent forms. All animal studies at AbbVie, were reviewed and approved by AbbVie’s Institutional Animal Care and Use Committee and in compliance with the NIH Guide for Care and Use of Laboratory Animals guidelines. Animal studies were conducted in an AAALAC accredited program where veterinary care and oversight was provided to ensure appropriate animal care. All in vivo studies conducted at the Broad Institute were approved by the Broad Institute IACUC committee and mice were housed in a specific-pathogen free facility. All in vivo studies at Calico were conducted according to protocols approved by the Calico Institutional Animal Care and Use Committee.

Despite striking efficacy against hematologic malignancies, the cost and complexity of CAR T manufacturing present significant barriers to broader patient access. Beyond manufacturing challenges, ex vivo expansion of T cells may be detrimental for their function and persistence. Thus, delivery of CARs to reprogram host cells in vivo would represent a significant advance toward 'off-the-shelf' therapy but has been limited by low efficiency, low specificity, and immunogenicity of viral vectors. Here we describe the design of pseudotyped lentiviral vectors (LV) with superior functionality and high target specificity. We show that LV pseudotyped with chimeric envelope glycoproteins from dolphin morbillivirus (DMV) can be engineered to selectively infect human T cells and evade neutralizing antibody responses in measles-vaccinated human serum. We further demonstrate that camelid-derived nanobodies are a superior retargeting domain, overcoming limitations inherent to the use of single chain variable fragment antibodies. Using a chimeric DMV-pseudotyped virus targeting the CD7 receptor, we demonstrate efficient and highly specific infection of T cells both in vitro and in vivo, generating functional CAR T cells and inducing therapeutic efficacy in a preclinical B cell lymphoma model.