CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T ...cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.
We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance.
One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients.
CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).
Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed ...by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.
Many viruses alter expression of proteins on the surface of infected cells including molecules important for immune recognition, such as the major histocompatibility complex (MHC) class I and II ...molecules. Virus-induced downregulation of surface proteins has been observed to occur by a variety of mechanisms including impaired transcription, blocks to synthesis, and increased turnover. Viral infection or transient expression of the Ebola virus (EBOV) glycoprotein (GP) was previously shown to result in loss of staining of various host cell surface proteins including MHC1 and β1 integrin; however, the mechanism responsible for this effect has not been delineated. In the present study we demonstrate that EBOV GP does not decrease surface levels of β1 integrin or MHC1, but rather impedes recognition by steric occlusion of these proteins on the cell surface. Furthermore, steric occlusion also occurs for epitopes on the EBOV glycoprotein itself. The occluded epitopes in host proteins and EBOV GP can be revealed by removal of the surface subunit of GP or by removal of surface N- and O- linked glycans, resulting in increased surface staining by flow cytometry. Importantly, expression of EBOV GP impairs CD8 T-cell recognition of MHC1 on antigen presenting cells. Glycan-mediated steric shielding of host cell surface proteins by EBOV GP represents a novel mechanism for a virus to affect host cell function, thereby escaping immune detection.
The advent of engineered T cells as a form of immunotherapy marks the beginning of a new era in medicine, providing a transformative way to combat complex diseases such as cancer. Following FDA ...approval of CAR T cells directed against the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma, CAR T cells are poised to enter mainstream oncology. Despite this success, a number of patients are unable to receive this therapy due to inadequate T cell numbers or rapid disease progression. Furthermore, lack of response to CAR T cell treatment is due in some cases to intrinsic autologous T cell defects and/or the inability of these cells to function optimally in a strongly immunosuppressive tumor microenvironment. We describe recent efforts to overcome these limitations using CRISPR/Cas9 technology, with the goal of enhancing potency and increasing the availability of CAR-based therapies. We further discuss issues related to the efficiency/scalability of CRISPR/Cas9-mediated genome editing in CAR T cells and safety considerations. By combining the tools of synthetic biology such as CARs and CRISPR/Cas9, we have an unprecedented opportunity to optimally program T cells and improve adoptive immunotherapy for most, if not all future patients.
The use of lentiviral vectors in cell and gene therapy is steadily increasing, both in commercial and investigational therapies. Although existing data increasingly support the usefulness and safety ...of clinical-grade lentiviral vectors used in cell manufacturing, comprehensive studies specifically addressing their long-term stability are currently lacking. This is significant considering the high cost of producing and testing GMP-grade vectors, the limited number of production facilities, and lengthy queue for production slots. Therefore, an extended shelf life is a critical attribute to justify the investment in large vector lots for investigational cell therapies. This study offers a thorough examination of essential stability attributes, including vector titer, transduction efficiency, and potency for a series of clinical-grade vector lots, each assessed at a minimum of 36 months following their date of manufacture. The 13 vector lots included in this study were used for cell product manufacturing in 16 different clinical trials, and at the time of the analysis had a maximum storage time at −80°C of up to 8 years. The results emphasize the long-term durability and efficacy of GMP-grade lentiviral vectors for use in ex vivo cell therapy manufacturing.
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As the use of GMP-grade lentiviral vectors for clinical cell therapy manufacturing increases, the high cost, limited production facilities, and long queues necessitate that vector lots remain stable in long-term storage for extended use. This comprehensive analysis demonstrates long-term stability by titer, product transduction efficiency, and potency.
BackgroundPreclinical combinations of CAR-T therapies with oncolytic viruses have shown promise,1 2 but thus far no clinical data are available. Here we report interim results for three patients with ...epithelial ovarian- and pancreatic cancer treated with an intravenous infusion of VCN-01, an investigational PH20 hyaluronidase-armed oncolytic virus3 followed by autologous T cells transduced to express a chimeric antigen receptor directed against mesothelin (huCART-meso) from an ongoing Phase I study.MethodsVCN-01 is a genetically modified, oncolytic adenovirus with tumor tropism enhanced by an integrin-binding motif and selective replication in pRB-defective cells.4 It encodes, PH20 hyaluronidase to degrade tumor hyaluronan and might thereby increase tumor penetrance of coadministered therapies.4–6 HuCART-meso are autologous T cells transduced with a fully humanized chimeric antigen receptor composed of anti-mesothelin ‘M5’ ScFv fused to 4–1BB and TCRzeta signaling domains.7 This is a phase I trial to establish safety and feasibility of the combination of VCN-01 and huCART-meso (table 1).ResultsThree patients (table 2) received combination therapy at their assigned dose and no dose limiting toxicities observed. One subject experienced cytokine release syndrome (CRS), grade 2 in severity and lasting 2 days. No neurotoxicity was observed. Transient grade 3 anemia and lymphopenia were observed, and AST elevation, as expected from VCN-01. There was no grade 4 or higher adverse events. All three patients were retreated with huCART-meso cells via IV infusion post-Month 2. VCN-01 and huCART-meso were detected in the peripheral blood for extended periods (figure 1). The two patients with measurable disease (both with ovarian cancer) showed SD up to 150 and 300 days with the biggest decrease of disease volume 15% and 29.1% respectively by RECIST criteria (figure 2 and table 3).ConclusionsA regimen of IV delivery of 3.3 x 1012 VP VCN-01 followed 14 d later by 5 x 107 huCART-meso T cells is feasible and appears safe. Repeat dosing with huCART-meso post-Month 2 is also feasible. VCN-01 persistence suggests tumor infection and active replication. The peak and duration of huCART-meso T cells in the peripheral blood as well as duration of stable disease in evaluable patients show encouraging trends. The study will test higher doses of VCN-01 and will interrogate tumor biopsies to gain further insights. The results will inform and guide optimization of the combination of CAR T cells with oncolytic virus.AcknowledgementsWe wish to acknowledge the clinical trial patients and their families, Theriva Biologics for providing VCN-01, and the members of the Clinical Trials Unit at the Abramson Cancer Center. Funding – UPenn private funds.Trial RegistrationNCT05057715 (trial in progress)ReferencesGuedan S, Alemany R. CAR-T Cells and Oncolytic Viruses: Joining Forces to Overcome the Solid Tumor Challenge. Front Immunol. 2018;9:2460.McGrath K, Dotti G. Combining Oncolytic Viruses with Chimeric Antigen Receptor T Cell Therapy. Hum Gene Ther. 2021;32(3–4):150–157.Garcia-Carbonero R, Bazan-Peregrino M, Gil-Martin M, et al. Phase I, multicenter, open-label study of intravenous VCN-01 oncolytic adenovirus with or without nab-paclitaxel plus gemcitabine in patients with advanced solid tumors. J Immunother Cancer. 2022;10(3).Rodriguez-Garcia A, Gimenez-Alejandre M, Rojas JJ, et al. Safety and efficacy of VCN-01, an oncolytic adenovirus combining fiber HSG-binding domain replacement with RGD and hyaluronidase expression. Clin Cancer Res. 2015;21(6):1406–1418.Bazan-Peregrino M, Garcia-Carbonero R, Laquente B, et al. VCN-01 disrupts pancreatic cancer stroma and exerts antitumor effects. J Immunother Cancer. 2021;9(11).Farrera-Sal M, Moreno R, Mato-Berciano A, Maliandi MV, Bazan-Peregrino M, Alemany R. Hyaluronidase expression within tumors increases virotherapy efficacy and T cell accumulation. Mol Ther Oncolytics. 2021;22:27–35.Haas AR, Golden RJ, Litzky LA, et al. Two Cases of Severe Pulmonary Toxicity from Highly Active Mesothelin-Directed CAR T Cells. Mol Ther. 2023.Ethics ApprovalUniversity of Pennsylvania, Institutional Review Board, Federalwide Assurance: 00004028; Confirmation #: dgjegcid; Protocol Number: 849937\28-September 2022ConsentPC4b-03821-VCN-01=M5 Main IFC V3.12.03.2021Abstract 671 Figure 1Pharmacokinetics of VCN-01 and huCART-meso in the peripheral blood.Abstract 671 Figure 2Response to Combination Therapy. Change in tumor volume from baseline (%) is shown for the two patients with disease measurable by RECIST. Timepoints for infusion of VCN-01, huCART-meso (first dose) and huCART-meso retreatment are shown.Abstract 671 Table 1Study DesignAbstract 671 Table 2Patient Demographics and Disease CharacteristicsAbstract 671 Table 3Response to Combination Therapy
Chimeric antigen receptor (CAR) T cells are a promising therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a rational target in multiple myeloma (MM).
We conducted a phase I ...study of autologous T cells lentivirally-transduced with a fully-human, BCMA-specific CAR containing CD3ζ and 4-1BB signaling domains (CART-BCMA), in subjects with relapsed/refractory MM. Twenty-five subjects were treated in 3 cohorts: 1) 1-5 x 108 CART-BCMA cells alone; 2) Cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART-BCMA cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART-BCMA cells. No pre-specified BCMA expression level was required.
CART-BCMA cells were manufactured and expanded in all subjects. Toxicities included cytokine release syndrome and neurotoxicity, which were grade 3-4 in 8 (32%) and 3 (12%) subjects, respectively, and reversible. One subject died at day 24 from candidemia and progressive myeloma, following treatment for severe CRS and encephalopathy. Responses (based on treated subjects) were seen in 4/9 (44%) in cohort 1, 1/5 (20%) in cohort 2, and 7/11 (64%) in cohort 3, including 5 partial, 5 very good partial, and 2 complete responses, 3 of which were ongoing at 11, 14, and 32 months. Decreased BCMA expression on residual MM cells was noted in responders; expression increased at progression in most. Responses and CART-BCMA expansion were associated with CD4:CD8 T cell ratio and frequency of CD45RO-CD27+CD8+ T cells in the pre-manufacturing leukapheresis product.
CART-BCMA infusions with or without lymphodepleting chemotherapy are clinically active in heavily-pretreated MM patients.
NCT02546167.
University of Pennsylvania-Novartis Alliance and NIH.
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