Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models.

OBJECTIVES: Autologous chimeric antigen receptor (CAR) αß T-cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T-cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood-derived CAR+ Vδ1 γδ T cells expressing a second-generation CAR targeting the B-cell-restricted CD20 antigen. METHODS: Donor-derived Vδ1 γδ T cells from peripheral blood were ex vivo-activated, expanded and engineered to express a novel anti-CD20 CAR. In vitro and in vivo assays were used to evaluate CAR-dependent and CAR-independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B-cell tumors. RESULTS: Anti-CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B-cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft-versus-host disease in immunodeficient mice. CONCLUSION: These preclinical data support the clinical evaluation of ADI-001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B-cell malignancies (NCT04735471).

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers.

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

R723, a selective JAK2 inhibitor, effectively treats JAK2V617F-induced murine myeloproliferative neoplasm.

The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.