Multiple myeloma is a usually incurable malignancy of plasma cells. New therapies are urgently needed for multiple myeloma. Adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is ...a promising new therapy for hematologic malignancies, but an ideal target antigen for CAR-expressing T-cell therapies for multiple myeloma has not been identified. B-cell maturation antigen (BCMA) is a protein that has been reported to be selectively expressed by B-lineage cells including multiple myeloma cells. Our goal was to determine if BCMA is a suitable target for CAR-expressing T cells.
We conducted an assessment of BCMA expression in normal human tissues and multiple myeloma cells by flow cytometry, quantitative PCR, and immunohistochemistry. We designed and tested novel anti-BCMA CARs.
BCMA had a restricted RNA expression pattern. Except for expression in plasma cells, BCMA protein was not detected in normal human tissues. BCMA was not detected on primary human CD34(+) hematopoietic cells. We detected uniform BCMA cell-surface expression on primary multiple myeloma cells from five of five patients. We designed the first anti-BCMA CARs to be reported and we transduced T cells with lentiviral vectors encoding these CARs. The CARs gave T cells the ability to specifically recognize BCMA. The anti-BCMA-CAR-transduced T cells exhibited BCMA-specific functions including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. Importantly, anti-BCMA-CAR-transduced T cells recognized and killed primary multiple myeloma cells.
BCMA is a suitable target for CAR-expressing T cells, and adoptive transfer of anti-BCMA-CAR-expressing T cells is a promising new strategy for treating multiple myeloma.
Adoptive transfer of genetically modified T cells is an attractive approach for generating antitumor immune responses. We treated a patient with advanced follicular lymphoma by administering a ...preparative chemotherapy regimen followed by autologous T cells genetically engineered to express a chimeric antigen receptor (CAR) that recognized the B-cell antigen CD19. The patient's lymphoma underwent a dramatic regression, and B-cell precursors were selectively eliminated from the patient's bone marrow after infusion of anti–CD19-CAR-transduced T cells. Blood B cells were absent for at least 39 weeks after anti–CD19-CAR-transduced T-cell infusion despite prompt recovery of other blood cell counts. Consistent with eradication of B-lineage cells, serum immunoglobulins decreased to very low levels after treatment. The prolonged and selective elimination of B-lineage cells could not be attributed to the chemotherapy that the patient received and indicated antigen-specific eradication of B-lineage cells. Adoptive transfer of anti–CD19-CAR-expressing T cells is a promising new approach for treating B-cell malignancies. This study is registered at www.clinicaltrials.gov as #NCT00924326.
Acute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an ...opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4+ CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.
•Donor-derived allogeneic CAR T cells are functional and eradicate ALL.•Allogeneic CD4+ CAR T cells can mediate acute GVHD but only when CD19+ leukemia is present.
Abstract
B-cell–depleting therapies may lead to prolonged disease and viral shedding in individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and this viral ...persistence raises concern for viral evolution. We report sequencing of early and late samples from a 335-day infection in an immunocompromised patient. The virus accumulated a unique deletion in the amino‐terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts.
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify ...actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Prolonged myelosuppression after chimeric antigen receptor (CAR) T-cell therapy is common and poorly understood. A retrospective analysis of 43 patients was conducted to investigate factors ...contributing to CAR T-cell-related cytopenias. Thirty-five patients were evaluable for analysis of delayed cytopenias occurring after initial hematologic recovery. Time to hematologic recovery (TTHR) was defined as number of days after CAR T-cell infusion for recovery to hemoglobin ≥8.0 g/dL, platelets ≥50.0 k/µL, and neutrophil count ≥1.0 k/µL without transfusions or growth factors for 7 days. Baseline percent bone marrow (BM) malignancy involvement correlated with TTHR (p = .0047). Patients with grades 3-4 cytokine-release syndrome (CRS) had longer TTHR than those with grades 0-2 CRS (p = .0479). Patients who developed prolonged or delayed cytopenias after anti-BCMA CAR T cells had a higher percentage of BM aspirate CAR
+
cells at 2 months (n = 10; p = .0159).
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T cell signaling domains. CD30 is a cell surface protein expressed on Hodgkin's ...lymphoma, some T cell lymphomas, and some B cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared three different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8α, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z, except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity, including degranulation, cytokine release, and proliferation, when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain deficient (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30
T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30
T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all
and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.
Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly ...used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.
We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes.
We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses.
We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.
As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical ...necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells PBMNCs) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.
Panch et al. demonstrate that CART cryopreservation during manufacture minimally affects final product characteristics, in vivo levels, persistence, and clinical response. However, increases in early apoptotic markers and cell damage pathways in cryo-thawed CARTs require careful consideration to better formulate dosing strategies.
New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically ...modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD. This trial was registered at www.clinicaltrials.gov as #NCT01087294.
•Donor-derived anti-CD19-CAR T cells cause regressions of refractory malignancies after allogeneic transplantation.
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