using-crisprcas9-to-knock-out-gm-csf-in-car-t-cells

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

dynamic-imaging-chimeric-antigen-receptor-t-cells-with

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [<sup>18</sup>F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Mayo Clinic

Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell-associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF-deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSF CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts.

Management of cytokine release syndrome: an update on emerging antigen-specific T cell engaging immunotherapies.

Although Pneumocystis pneumonia (PCP), a life-threatening infection, has been reported in patients with non-Hodgkin B-cell lymphoma (BNHL) who were treated with rituximab-containing chemotherapies (R-CTX), the PCP prophylaxis regimen awaits establishment to date. This study reports a retrospective analysis of the efficacy and safety of a low-dose trimethoprim/sulfamethoxazole (TMP/SMX) in patients with BNHL receiving R-CTX. We retrospectively analyzed 156 patients newly diagnosed with BNHL who received R-CTX at our institute from 2010 to 2015. We collected patients' clinical and laboratory data, including lymphocytes count, IgG level, PCP prophylaxis regimens, and adverse events (AEs). Patients were categorized into the following two groups based on the TMP/SMX regimen: group A (33 patients; 80 mg/400 mg×3/week) or group B (65 patients; 160 mg/800 mg×2/week). Both lymphocytes count and IgG level declined during R-CTX. No patient developed PCP. Patients in group B exhibited a significantly higher incidence of AEs (18.2% vs. 63.1%; p<0.05) and increased AST (6.1% vs. 26.6%; p<0.05), compared with those in group A. Thus, TMP/SMX (80 mg/400 mg×3/week) effectively prevents PCP and is preferable because of the lower rates of AEs.

[Pneumocystis pneumonia prophylaxis with low-dose trimethoprim/sulfamethoxazole during rituximab-containing chemotherapy].

Baseline immune dysregulation in autologous stem cell transplant recipients is associated with a 'graft versus host'-like syndrome and poor outcomes.

To assess the efficacy and safety of lenzilumab in patients with severe coronavirus disease 2019 (COVID-19) pneumonia.

GM-CSF Neutralization With Lenzilumab in Severe COVID-19 Pneumonia: A Case-Cohort Study.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1 CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor T (CART) cell immunotherapy has yielded significant clinical success in treating certain hematological malignancies. However, despite high initial response rates, most patients eventually relapse. Resistance to CART cell therapy can stem from tumor cell mutations, T cell defects, and tumor microenvironment (TME) immunosuppression. Tumor cells can downregulate target antigen expression to evade CART cell detection or mutate death receptor pathways to resist CART cell cytotoxicity. Patient T cells can be intrinsically defective, and CART cells often undergo exhaustion. The TME is abundant with immunosuppressive cells and factors which contribute to suboptimal CART cell activity. Collectively, issues originating in tumor cells, T cells, and the TME present significant hurdles to long-term remission after CART cell therapy. Various strategies to combat CART cell resistance have shown promise in preclinical studies and early clinical trials and are crucial to achieving durable responses.

Resistance to CART cell therapy: lessons learned from the treatment of hematological malignancies.

Chimeric antigen receptor T (CART) cells are a promising immunotherapy that has induced dramatic anti-tumor responses in certain B cell malignancies. However, CART cell expansion and trafficking are often insufficient to yield long-term remissions, and serious toxicities can arise after CART cell administration. Visualizing CART cell expansion and trafficking in patients can detect an inadequate CART cell response or serve as an early warning for toxicity development, allowing CART cell treatment to be tailored accordingly to maximize therapeutic benefits. To this end, various imaging platforms are being developed to track CART cells , including nonspecific strategies to image activated T cells and reporter systems to specifically detect engineered T cells. Many of these platforms are clinically applicable and hold promise to provide valuable information and guide improved CART cell treatment.

 CART cell imaging: Paving the way for success in CART cell therapy.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor  expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NISCART19 and NISBCMA-CART, respectively) and tested the sensitivity of F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NISCART19 and NISBCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive I uptake . F-TFB-PET detected NISCART cells  to a sensitivity level of 40,000 cells. F-TFB-PET confirmed NISBCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, F-TFB-PET revealed significant systemic uptake, correlating with CART-cell  expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. F-TFB-PET detected CART-cell trafficking to tumor sites and  expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions .

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Pivotal clinical trials of B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent FDA approval. Despite their success, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART-cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We demonstrated that CAFs inhibit CART-cell anti-tumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and SLAMF7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, we generated CART cells targeting both MM cells and CAFs. Our dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

ArticleTotal : 2	Year
Publication title Cited by 34	2019
Publication title Cited by 8	2022

Article	Year
GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts. Blood\| PubMed ID: 30463995	2019
Management of cytokine release syndrome: an update on emerging antigen-specific T cell engaging immunotherapies. Immunotherapy\| PubMed ID: 31161844	2019
[Pneumocystis pneumonia prophylaxis with low-dose trimethoprim/sulfamethoxazole during rituximab-containing chemotherapy]. [Rinsho ketsueki] The Japanese journal of clinical hematology\| PubMed ID: 31167996	2019
Baseline immune dysregulation in autologous stem cell transplant recipients is associated with a 'graft versus host'-like syndrome and poor outcomes. Bone marrow transplantation\| PubMed ID: 32161319	2020
GM-CSF Neutralization With Lenzilumab in Severe COVID-19 Pneumonia: A Case-Cohort Study. Mayo Clinic proceedings\| PubMed ID: 33153629	2020
Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 33388419	2021
Resistance to CART cell therapy: lessons learned from the treatment of hematological malignancies. Leukemia & lymphoma\| PubMed ID: 33682608	2021
CART cell imaging: Paving the way for success in CART cell therapy. Molecular therapy oncolytics\| PubMed ID: 33816781	2021
Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity. Cancer immunology research\| PubMed ID: 34244299	2021
Targeting Cancer-Associated Fibroblasts in the Bone Marrow Prevents Resistance to CART-Cell Therapy in Multiple Myeloma. Blood\| PubMed ID: 35090171	2022

Reona Sakemura

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