Sequential CD7 CAR T-Cell Therapy and Allogeneic HSCT without GVHD Prophylaxis.

BACKGROUND: Patients with relapsed or refractory hematologic cancers have a poor prognosis. Chimeric antigen receptor (CAR) T-cell therapy as a bridge to allogeneic hematopoietic stem-cell transplantation (HSCT) has the potential for long-term tumor elimination. However, pre-HSCT myeloablation and graft-versus-host disease (GVHD) prophylaxis agents have toxic effects and could eradicate residual CAR T cells and compromise antitumor effects. Whether the integration of CAR T-cell therapy and allogeneic HSCT can preserve CAR T-cell function and improve tumor control is unclear. METHODS: We tested a novel "all-in-one" strategy consisting of sequential CD7 CAR T-cell therapy and haploidentical HSCT in 10 patients with relapsed or refractory CD7-positive leukemia or lymphoma. After CAR T-cell therapy led to complete remission with incomplete hematologic recovery, patients received haploidentical HSCT without pharmacologic myeloablation or GVHD prophylaxis drugs. Toxic effects and efficacy were closely monitored. RESULTS: After CAR T-cell therapy, all 10 patients had complete remission with incomplete hematologic recovery and grade 4 pancytopenia. After haploidentical HSCT, 1 patient died on day 13 of septic shock and encephalitis, 8 patients had full donor chimerism, and 1 patient had autologous hematopoiesis. Three patients had grade 2 HSCT-associated acute GVHD. The median follow-up was 15.1 months (range, 3.1 to 24.0) after CAR T-cell therapy. Six patients remained in minimal residual disease-negative complete remission, 2 had a relapse of CD7-negative leukemia, and 1 died of septic shock at 3.7 months. The estimated 1-year overall survival was 68% (95% confidence interval [CI], 43 to 100), and the estimated 1-year disease-free survival was 54% (95% CI, 29 to 100). CONCLUSIONS: Our findings suggest that sequential CD7 CAR T-cell therapy and haploidentical HSCT is safe and effective, with remission and serious but reversible adverse events. This strategy offers a feasible approach for patients with CD7-positive tumors who are ineligible for conventional allogeneic HSCT. (Funded by the National Natural Science Foundation of China and the Key Project of Science and Technology Department of Zhejiang Province; ClinicalTrials.gov numbers, NCT04599556 and NCT04538599.).

The Chimeric Chaoyang-Zika Vaccine Candidate Is Safe and Protective in Mice.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital syndromes including microcephaly and fetal demise in pregnant women. No commercial vaccines against ZIKV are currently available. We previously generated a chimeric ZIKV (ChinZIKV) based on the Chaoyang virus (CYV) by replacing the prME protein of CYV with that of a contemporary ZIKV strain GZ01. Herein, we evaluated this vaccine candidate in a mouse model and showed that ChinZIKV was totally safe in both adult and suckling immunodeficient mice. No viral RNA was detected in the serum of mice inoculated with ChinZIKV. All of the mice inoculated with ChinZIKV survived, while mice inoculated with ZIKV succumbed to infection in 8 days. A single dose of ChinZIKV partially protected mice against lethal ZIKV challenge. In contrast, all the control PBS-immunized mice succumbed to infection after ZIKV challenge. Our results warrant further development of ChinZIKV as a vaccine candidate in clinical trials.

Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism.

Chimeric antigen receptor (CAR)-T therapy has shown superior efficacy against hematopoietic malignancies. However, many patients failed to achieve sustainable tumor control partially due to CAR-T cell exhaustion and limited persistence. In this study, by performing single-cell multi-omics data analysis on patient-derived CAR-T cells, we identify CD38 as a potential hallmark of exhausted CAR-T cells, which is positively correlated with exhaustion-related transcription factors and further confirmed with in vitro exhaustion models. Moreover, inhibiting CD38 activity reverses tonic signaling- or tumor antigen-induced exhaustion independent of single-chain variable fragment design or costimulatory domain, resulting in improved CAR-T cell cytotoxicity and antitumor response. Mechanistically, CD38 inhibition synergizes the downregulation of CD38-cADPR -Ca2+ signaling and activation of the CD38-NAD+-SIRT1 axis to suppress glycolysis. Collectively, our findings shed light on the role of CD38 in CAR-T cell exhaustion and suggest potential clinical applications of CD38 inhibition in enhancing the efficacy and persistence of CAR-T cell therapy.

Relapsed/Refractory Peripheral T-Cell Lymphoma-Associated Hemophagocytic Lymphohistiocytosis With UNC13D and CD27 Germline Mutations.

Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory disease characterized by familial and acquired forms. Here, we present the case of a 26-year-old male patient with relapsed/refractory peripheral T-cell lymphoma and concurrent HLH. Whole-exon sequencing revealed germline mutations associated with HLH, including those in critical genes such as CD27 and UNC13D and other germline heterozygous variants (NOTCH2, NOTCH3, IL2RA, TYK2, AGL, CFD, and F13A1). CD107a analyses consistently demonstrated impaired degranulation of cytotoxic T-lymphocytes and natural killer (NK) cells. Examination of the patient's family pedigree revealed that his father and mother harbored UNC13D and CD27 mutations, respectively; his brother carried the same CD27 heterozygous mutation. However, none of them manifested the disease. Despite the missense mutation of CD27 (c.779C>T; p.Pro260Leu) lacking previous documentation in databases, comprehensive analysis suggested non-pathogenic mutations in the CD27 variant, indicating minimal impact on T- and NK-cell functions. These results ultimately supported the option of hematopoietic stem cell transplantation (HSCT) as a successful curative therapeutic approach. As of this report, the patient has remained free of lymphoma and quiescent HLH 15.2 months post-HSCT. This study underscores the efficacy of genetic tests in identifying significant mutations and confirming their etiologies, providing an early basis for treatment decisions and the selection of suitable transplant donors.

HLA Fully-Mismatched Sibling-Derived CD7 CAR-T Therapy Bridging to Haploidentical Hematopoietic Stem Cell Transplantation for Hepatosplenic γδ T-cell Lymphoma.

While chimeric antigen receptor (CAR)-T-cell therapy has demonstrated remarkable effectiveness in the treatment of B-cell lymphomas and leukemias, research on T-cell malignancies is still limited. Here, we reported a patient with hepatosplenic γδ T-cell lymphoma refractory to multiple lines of chemotherapy, who eventually achieved first complete remission with flow cytometry-confirmed minimal residual disease negativity after human leukocyte antigen (HLA) fully-mismatched sibling-derived CD7 CAR-T therapy. However, given the allogeneic nature, CAR-T cells dropped rapidly after a peak of 83.4% of circulating T-cells. Cytokine release syndrome, cytopenia, and infections occurred but were manageable after treatments. After the consolidative haploidentical hematopoietic stem cell transplantation (HSCT), the patient remained in remission at the end of the follow-up (13 months post-CAR-T infusion). This is the first case of relapsed/refractory hepatosplenic γδ T-cell lymphoma who achieved lasting CR after HLA fully-mismatched sibling-derived CD7 CAR-T therapy bridging to haploidentical HSCT.

A distinct cytokine network distinguishes chimeric antigen receptor T cell (CAR-T)-associated hemophagocytic lymphohistiocytosis-like toxicity (carHLH) from severe cytokine release syndrome following CAR-T therapy.

BACKGROUND AIMS: With the increasing application of chimeric antigen receptor (CAR)-T cell therapy in various malignancies, an extra toxicity profile has been revealed, including a severe complication resembling hemophagocytic lymphohistiocytosis (HLH), which is usually disguised by severe cytokine release syndrome (CRS). METHODS: In a clinical trial in whom 99 patients received B-cell maturation antigen CAR-T cells, we identified 20 (20.20%) cases of CAR-T cell-associated HLH (carHLH), most of whom possessed a background of severe CRS (grade ≥3). The overlapping features of carHLH and severe CRS attracted us to further explore the differences between them. RESULTS: We showed that carHLH can be distinguished by extreme elevation of interferon-γ, granzyme B, interleukin-1RA and interleukin-10, which can be informative in developing prevention and management strategies of this toxicity. Moreover, we developed a predictive model of carHLH with a mean area under the curve of 0.81 ± 0.07, incorporating serum lactate dehydrogenase at day 6 post-CRS and serum fibrinogen at day 3 post-CRS. CONCLUSIONS: The incidence of carHLH in CAR-T recipients might be relatively higher than we previously thought. relatively higher than we previously. A cytokine network distinguished from CRS is responsible for carHLH. And corresponding cytokine-directed therapies, especially targeting IL-10, are worth trying.

Rapid Generation of Recombinant Flaviviruses Using Circular Polymerase Extension Reaction.

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

Safety and efficacy of CRISPR-based non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells in patients with relapsed or refractory Non-Hodgkin's lymphoma: a first-in-human phase I study.

Background: Thus far, all approved chimeric antigen receptor (CAR)-T products are manufactured using modified viruses, which increases the risk of tumorigenesis, costs and production time. We aimed to evaluate the safety and efficacy of a kind of virus-free CAR-T cells (PD1-19bbz), in which an anti-CD19 CAR sequence is specifically integrated at the PD1 locus using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, in adults with relapsed/refractory (r/r) B cell non-Hodgkin's lymphoma (B-NHL). Methods: This single-arm phase I dose-escalation clinical trial evaluated PD1-19bbz in adult patients with r/r B-NHL from May 3rd 2020 to August 10th 2021. The patients were recruited and treated at the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Patients underwent leukapheresis and lymphodepleting chemotherapy before PD1-19bbz infusion. After the dose-escalation phase including three cohorts: 2 × 106/kg, 4 × 106/kg, 6 × 106/kg with three patients at each dose level, the optimal biological dose was determined to be 2 × 106/kg, which was then applied to an extended cohort of nine patients. The primary endpoint was the incidence of dose-limiting toxicities (DLT). The secondary endpoint was the response and survival. This trial was registered at www.clinicaltrials.gov as #NCT04213469. Findings: Twenty-one patients received PD1-19bbz infusion. Among all treated patients, 19 (90%) patients were diagnosed with stage III or IV disease. Meanwhile, 19 (90%) were stratified as intermediate risk or worse. Of note, four participants had >50% programmed death ligand-1 (PD-L1) expression in pre-treatment tumour sample, including two with extremely high levels (∼80%). There was no DLT identified. Fourteen patients had low-grade (1-2) cytokine release syndrome and two patients received tocilizumab. Four patients experienced immune effector cell-associated neurotoxicity syndrome of grade 1-2. The most common adverse events were hematologic toxicities, including anaemia (n = 6), lymphocyte count decreased (n = 19), neutrophil count decreased (n = 17), white blood cell count decreased (n = 10), and platelet count decreased (n = 2). All patients had objective response and 18 patients reached complete response. At a median follow-up of 19.2 months, nine patients remained in remission, and the estimated median progression-free survival duration was 19.5 months (95% confidence interval: 9.9-infinity), with the median overall survival not reached. Interpretation: In this first-in-human study of non-viral specifically integrated CAR-T products, PD1-19bbz exhibited promising efficacy with a manageable toxicity profile. A phase I/II trial of PD1-19bbz in a larger patient cohort is underway. Funding: National Key R&D Program of China, National Natural Science Foundation of China, Key Project of Science and Technology Department of Zhejiang Province, Shanghai Zhangjiang National Independent Innovation Demonstration Area, Key Projects of Special Development Funds.

Incidence, clinical characteristics and prognosis of tumor lysis syndrome following B-cell maturation antigen-targeted chimeric antigen receptor-T cell therapy in relapsed/refractory multiple myeloma.

Background aims: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor-T cell (CAR-T) therapy is used for refractory or relapsed multiple myeloma (r/r MM). However, CAR-T-related tumor lysis syndrome (TLS) has been observed. We aimed to elucidate the incidence, clinical and laboratory characteristics, and prognosis of CAR-T cell-related TLS. Methods: Patients (n=105) with r/r MM treated with BCMA-targeted CAR-T cell therapy were included. Patient characteristics, laboratory parameters, and clinical outcomes were assessed. Results: Eighteen (17.1%) patients developed TLS after BCMA-targeted CAR-T cell therapy. The median time till TLS onset was 8 days. Patients with TLS had steep rise in uric acid (UA), creatinine, and lactate dehydrogenase (LDH) within 6 days following CAR-T cell infusion and presented earlier and persistent escalation of cytokines (C-reactive protein [CRP], interleukin-6 [IL-6], interferon-γ [IFN-γ], and ferritin levels). All 18 patients had cytokine release syndrome (CRS), of which 13 (72.2%) developed grade 3-4 CRS. Three of 18 patients (16.7%) developed immune effector cell-associated neurotoxicity syndrome (ICANS): two patients with grade 1 ICANS and one with grade 2 ICANS. TLS development had a negative effect on the objective response rate (77.8% in the TLS group vs. 95.4% in the non-TLS group, p<0.01). During the median follow-up of 15.1 months, the median PFS was poorer of patients with TLS (median: 3.4 months in the TLS group vs. 14.7 months in the non-TLS group, p<0.001, hazard ratio [HR]=3.5 [95% confidence interval [CI] 1.5-8.5]). Also, TLS development exhibited significant effects on OS (median: 5.0 months in the TLS group vs. 39.8 months in the non-TLS group, p<0.001, hazard ratio [HR]=3.7 [95% CI 1.3-10.3]). TLS was associated with a higher tumor burden, elevated baseline creatinine and UA levels, severe CRS, pronounced CAR-T cell expansion, and corticosteroid use. Conclusion: TLS is a frequently observed CAR-T therapy complication and negatively influences clinical response and prognosis. Close monitoring for TLS should be implemented during CAR-T cell therapy, especially for those at high TLS risk.

HBV reactivation in patients with chronic or resolved HBV infection following BCMA-targeted CAR-T cell therapy.

B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor-T cell (CAR-T) therapy is used for refractory or relapsed multiple myeloma (r/r MM). Concern of the safety and efficacy of CAR-T cell therapy in patients with chronic or resolved HBV infection is raised. In this study, we retrospectively reviewed 99 patients with r/r MM treated with BCMA-targeted CAR-T cell therapy, of which 7 (7.1%) patients had chronic HBV infection, 43 (43.4%) with resolved HBV infection, and the remaining 49 (49.49%) HBV-uninfected. Patients' characteristics before CAR-T cell administration were comparable in different status of HBV infection. Patients' liver function, cytokine levels, CAR-T cell expansion and cytokine release syndrome (CRS) grade after CAR-T cell therapy did not differ in different HBV serologic status. Furthermore, chronic HBV infection or resolved HBV infection did not affect clinical response, progress-free survival (PFS), or overall survival (OS). Four (4.04%) patients experienced HBV reactivation, 3 (6.98%) with resolved HBV infection, and 1 (14.29%) chronic HBV infection. Of 4 patients with HBV reactivation, 2 cases (50%) of severe hepatitis were noted and reported. Drops of serum IgG and elevation of alanine aminotransferase (ALT), alanine aminotransferase (AST), total bilirubin (TB) were observed in all four patients around the date of HBV reactivation.

GPRC5D CAR T cells (OriCAR-017) in patients with relapsed or refractory multiple myeloma (POLARIS): a first-in-human, single-centre, single-arm, phase 1 trial.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy targeting B-cell maturation antigen (BCMA) has shown activity in treating relapsed or refractory multiple myeloma; however, relapse is still common, and new targets are needed. We aimed to assess the activity and safety profile of G protein-coupled receptor class C group 5 member D (GPRC5D)-targeted CAR T cells (OriCAR-017) in patients with relapsed or refractory multiple myeloma. METHODS: POLARIS was a first-in-human, single-centre, single-arm, phase 1 trial of GPRC5D-targeted CAR T cells (OriCAR-017) done at the First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China. Eligible patients were adults aged 18-75 years with a diagnosis of relapsed or refractory multiple myeloma and an ECOG performance status of 0-2, had GPRC5D expression in bone marrow plasma cells greater than 20% or were positive for GPRC5D by immunohistochemistry, and had received at least three previous lines of treatment including proteasome inhibitors, immunomodulatory drugs, and chemotherapy. Patients were consecutively assigned to receive a single dose of intravenous OriCAR-017 at 1 × 106 CAR T cells per kg, 3 × 106 CAR T cells per kg, or 6 × 106 CAR T cells per kg in the dose-escalation phase. In the expansion phase, patients received the recommended phase 2 dose. Recruitment to the expansion phase terminated early due to the COVID-19 pandemic on May 1, 2022. The primary endpoints were safety, the maximum tolerated dose and the recommended phase 2 dose. Safety and activity analyses included all patients who received OriCAR-017. This trial is registered with ClinicalTrials.gov, NCT05016778. This trial has been completed and is entering long-term follow-up. FINDINGS: Between June 9, 2021, and Feb 28, 2022, we recruited 13 patients for inclusion into the study. One patient was excluded because of GPRC5D negativity and two patients discontinued after apheresis because of rapid progression. Nine patients were assigned to the dose escalation phase (three received 1 × 106 CAR T cells per kg, three received 3 × 106 CAR T cells per kg, and three received 6 × 106 CAR T cells per kg). The maximum tolerated dose was not identified, because no dose-limiting toxic effects were observed. On the basis of safety and preliminary activity, the recommended phase 2 dose was set at 3 × 106 CAR T cells per kg, which was received by one additional patient in the dose expansion phase. Five patients (50%) were female, five (50%) were male, and all were Chinese. Five patients (50%) were previously treated with BCMA-targeted CAR T-cell therapy. Median follow-up was 238 days (IQR 182-307). There were no serious adverse events and no treatment-related deaths. The most common grade 3 or worse adverse events were haematological, including neutropenia (ten [100%] of ten patients), thrombocytopenia (nine [90%]), leukopenia (nine [90%]), and anaemia (seven [70%]). All patients had cytokine release syndrome (nine [90%] grade 1 and one [10%] grade 2). No neurological toxic effects were reported. Ten (100%) of ten patients had an overall response, of whom six (60%) had a stringent complete response and four (40%) had very good partial response. Two patients discontinued due to disease progression (one GPRC5D-positive patient in the middle-dose group and one GPRC5D-negative patient in the low-dose group). INTERPRETATION: The results of this study suggest that GPRC5D is an active target for immunotherapy in multiple myeloma. GPRC5D-targeted CAR T-cell therapy is a promising treatment modality for patients with relapsed or refractory multiple myeloma and deserves further testing. FUNDING: OriCell Therapeutics.

Preparation and characterization of a neutralizing murine monoclonal antibody against tetanus toxin.

After Clostridium tetani infects the human body, it propagates under anaerobic conditions and produces tetanus neurotoxin (TeNT). TeNT can affect the central nervous system, inhibit the release of neurotransmitters, and result in respiratory failure, which are the root causes of death in tetanus patients. Identifying monoclonal antibodies (mAbs) targeting TeNT with neutralizing activity is urgently needed for the prevention and treatment of tetanus infection. In this study, through immunizing BALB/c mice with tetanus toxoid (TT), we obtained six positive hybridoma cell lines (1A7, 2C7, 3A7, 3H4, 4C1, and 4E12). Antibody isotyping showed that the antibodies are all of the IgG1/κ subclass. Ascites fluid was prepared by allogeneic ascites induction and the antibodies were purified through protein G affinity chromatography columns. Purities of the produced murine mAbs were all greater than 95%. All six antibodies bound to linear epitopes, among which 3A7 bound to the TeNT/L domain and the other five antibodies bound to the TeNT/Hc domain. Moreover, the affinity constants of these six antibodies against the antigen were all in the nanomolar range, and the affinity of 4E12 antibody reached the picomolar range. Results from toxin-neutralization assays in mice showed that 2C7 antibody delayed animal death, while 1A7, 3A7, 3H4, and 4E12 antibodies conferred partial protection. Additionally, 4C1 antibody offered complete protection, as 200 µg of 4C1 antibody fully protected against toxin challenge with 10 LD50 of TeNT and had a window period of 1 h. Antibody epitope grouping results revealed that the binding epitopes of 4C1 antibody were different from those of the other five antibodies. When 4C1 antibody was used in combination with another antibody, the neutralizing activities of antibodies were all evidently enhanced. Specifically, 4C1 combined with 3A7 antibody led to the greatest improvement in neutralizing activities, and 20 µg antibodies total (10 + 10 µg) fully protected against toxin challenge with 10 LD50. When 4E12, 3A7, and 4C1 antibodies were used in combination, 18 µg antibodies total (6 + 6 + 6 µg) completely neutralized 10 LD50 toxin. The present study derived murine mAbs with neutralizing activities and laid the foundation for follow-up therapeutic drug development for TeNT poisoning as well as establishment of TeNT detection methods.

Evaluation of Zika virus DNA vaccines based on NS1 and domain III of E.

BACKGROUND: A large-scale outbreak of Zika virus (ZIKV) has occurred in Brazil and other South American countries, and has rapidly spread to 60 countries and regions worldwide since 2015, but no approved anti-ZIKV vaccines are available as of 2021. METHODS: We developed four types of anti-ZIKV DNA vaccine candidates: VPC-NS1, VPC-prME, VPC-prME-NS1, and VPC-EIII-NS1. They were developed against the structural proteins prM and E, and non-structural protein 1 (NS1) of ZIKV using the mammalian cell expression vector pcDNA3.1(+) as the backbone. For immunization, we intramuscularly injected mice with each vaccine candidate (n = 12 to 15 per group) on day 0 and day 14, with mice injected with phosphate-buffered saline (PBS) and pcDNA3.1(+) backbone vector as controls. On day 7, 21, and 35 after initial immunization, the effect of DNA vaccines was evaluated by ZIKV-specific humoral immunity determined by enzyme-linked immunosorbent assay (ELISA), ZIKV-specific T cell immunity determined by intracellular cytokine staining by flow cytometry and serum neutralization capacity determined by plaque reduction neutralization test (PRNT50) assay. RESULTS: The sequencing results showed that DNA vaccine vectors were successfully constructed. Western blotting and immunofluorescence results demonstrated the successful expression of immunogens carried by the DNA vaccines. On day 21 and 35 after the initial immunization, the levels of serum total immunoglobulin (Ig)G in all vaccine-given groups were slightly higher (approximately 1.5- to 2-fold) than those in the control groups. By contrast, ZIKV-specific IgG levels of all vaccine-given groups were significantly higher (approximately 10- to 1000- fold) than those of the control groups. The PRNT50 assay showed that the average serum dilution factors for neutralizing half ZIKV virions from vaccine-given groups were at least 32-fold (highest, 93-fold), while the sera from control group showed no protection. For cellular immunity, the proportions of CD11b+ myeloid cells, CD19+ B lymphocytes and CD3+ T lymphocytes in the mouse spleens as well as the percentages of CD4+ and CD8+ subsets of T cell were not changed 35 days after initial immunization. By contrast, the proportions of ZIKV-specific CD4+T cell and CD8+T cell in all vaccine-given groups were 2- to 10-folds and 2- to 30-fold than those in the control groups, respectively. CONCLUSION: All four DNA vaccines designed for the ZIKV induced neutralizing IgGs and cellular immune responses against ZIKV. Particularly, VPC-EIII-NS1 induced high level of humoral response comparable to the vaccine candidate containing prM, E and NS1 polyprotein, suggesting a potent reduced ADE effect and reserved neutralizing activity. Our findings may provide guidance for improving safety of anti-ZIKV vaccines in the future.

Genetically modified CD7-targeting allogeneic CAR-T cell therapy with enhanced efficacy for relapsed/refractory CD7-positive hematological malignancies: a phase I clinical study.

Chimeric antigen receptor (CAR)-T cell therapy against T cell malignancies faces major challenges including fratricide between CAR-T cells and product contamination from the blasts. Allogeneic CAR-T cells, generated from healthy donor T cells, can provide ready-to-use, blast-free therapeutic products, but their application could be complicated by graft-versus-host disease (GvHD) and host rejection. Here we developed healthy donor-derived, CD7-targeting CAR-T cells (RD13-01) with genetic modifications to resist fratricide, GvHD and allogeneic rejection, as well as to potentiate antitumor function. A phase I clinical trial (NCT04538599) was conducted with twelve patients recruited (eleven with T cell leukemia/lymphoma, and one with CD7-expressing acute myeloid leukemia). All patients achieved pre-set end points and eleven proceeded to efficacy evaluation. No dose-limiting toxicity, GvHD, immune effector cell-associated neurotoxicity or severe cytokine release syndrome (grade ≥ 3) were observed. 28 days post infusion, 81.8% of patients (9/11) showed objective responses and the complete response rate was 63.6% (7/11, including the patient with AML). 3 of the responding patients were bridged to allogeneic hematopoietic stem cell transplantation. With a median follow-up of 10.5 months, 4 patients remained in complete remission. Cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) reactivation was observed in several patients, and one died from EBV-associated diffuse large B-cell lymphoma (DLBCL). Expansion of CD7-negative normal T cells was detected post infusion. In summary, we present the first report of a Phase I clinical trial using healthy donor-derived CD7-targeting allogeneic CAR-T cells to treat CD7+ hematological malignancies. Our results demonstrated the encouraging safety and efficacy profiles of the RD13-01 allogeneic CAR-T cells for CD7+ tumors.

Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL.

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1-7. However, CAR-T cell therapy currently has several limitations8-12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti-Leukemia Efficacy of CAR-T Cells via SOCE-Calcineurin-NFAT and Glycolysis Pathways.

Chimeric antigen receptor (CAR) T cells are potent agents for recognizing and eliminating tumors, and have achieved remarkable success in the treatment of patients with refractory leukemia and lymphoma. However, dysfunction of T cells, including exhaustion, is an inevitable obstacle for persistent curative effects. Here, the authors initially found that calcium signaling is hyperactivated via sustained tonic signaling in CAR-T cells. Next, it is revealed that the store-operated calcium entry (SOCE) inhibitor BTP-2, but not the calcium chelator BAPTA-AM, markedly diminishes CAR-T cell exhaustion and terminal differentiation of CAR-T cells in both tonic signaling and tumor antigen exposure models. Furthermore, BTP-2 pretreated CAR-T cells show improved antitumor potency and prolonged survival in vivo. Mechanistically, transcriptome and metabolite analyses reveal that treatment with BTP-2 significantly downregulate SOCE-calcineurin-nuclear factor of activated T-cells (NFAT) and glycolysis pathways. Together, the results indicate that modulating the SOCE-calcineurin-NFAT pathway in CAR-T cells renders them resistant to exhaustion, thereby yielding CAR products with enhanced antitumor potency.

BCMA CAR-T Therapy Is Safe and Effective for Refractory/Relapsed Multiple Myeloma With Central Nervous System Involvement.

Central nervous system (CNS) involvement is a rare complication of multiple myeloma (MM) that portends an extremely poor prognosis. Although chimeric antigen receptor (CAR)-T cell therapy is considered a promising strategy for patients with MM, the role of CAR-T cell therapy in MM involving the CNS has not been fully elucidated. In this study, we retrospectively analyzed 4 cases of B-cell maturation antigen CAR-T cell therapy for patients with relapsed/refractory MM involving the CNS. Patients received a range of 2-7 lines of prior therapy, including 1 autologous hematopoietic stem cell transplant. The most common adverse event was cytokine release syndrome, which was observed in all 4 patients, including 2 with grade 1 and 2 with grade 2. No patient was complicated with immune effector cell-associated neurotoxicity syndrome. Within the follow-up (median: 257 d, range: 116-392 d), 3 of 4 patients reached complete remission (CR), and 1 patient reached partial response. At the data cutoff, 1 patient continued to remain in CR at day 220, and the patient with partial response died at day 116. The other 2 patients relapsed at 317 and 111 days with CR durations of 287 and 81 days, respectively. Our results show promising effectiveness and acceptable safety of CAR-T cell therapy for heavily pretreated patients with CNS MM.

Risk Factors Associated with Durable Progression-Free Survival in Patients with Relapsed or Refractory Multiple Myeloma Treated with Anti-BCMA CAR T-cell Therapy.

PURPOSE: B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy results in high remission rates in patients with relapsed/refractory (R/R) multiple myeloma. However, the factors associated with prognosis following CAR T-cell therapy are unknown. PATIENTS AND METHODS: Between July 1, 2018 and July 31, 2020, 61 patients with R/R multiple myeloma received anti-BCMA CAR T-cell therapy (Chictr.org number, ChiCTR1800017404). Step-wise multivariate Cox regression and competing risk analyses were conducted to identify poor prognosis-associated risk factors. RESULTS: Sixty patients (98.4%) experienced cytokine release syndrome (CRS), including 33, 23, and 4 cases of CRS grades 1 to 2, 3, and 4, respectively. The objective response rate (ORR) was 98.3%, and the complete remission (CR) rate was 70.3%. With a median follow-up period of 21.1 months, the 1-year overall survival (OS) and progression-free survival (PFS) rates were 78.0% and 50.2%, respectively. The median PFS was 12.7 months. Cox modeling revealed that poor PFS was associated with extramedullary disease [HR = 2.59, 95% confidence interval (95% CI) = 1.29-5.21, P = 0.008], light chain multiple myeloma (HR = 2.53, 95% CI = 1.03-5.97, P = 0.035), high-risk cytogenetics (HR = 2.80, 95% CI = 1.27-6.14, P = 0.01), and prior treatment with more than 3 therapeutic lines (HR = 3.14, 95% CI = 1.34-7.34, P = 0.008). Among the 41 CR cases, competing risk analyses demonstrated higher relapse predispositions in those with extramedullary disease (HR = 4.51, 95% CI = 1.86-10.9, P = 0.001), light chain multiple myeloma (HR = 4.89, 95% CI = 1.52 - 15.7, P = 0.008), or high-risk cytogenetics (HR = 5.09, 95% CI = 1.63-15.9, P = 0.005). CONCLUSIONS: Anti-BCMA CAR T-cell therapy is safe and effective for R/R multiple myeloma. For patients with high-risk factors, improvements to extend remission and more specific individualized therapies are needed.

Dasatinib enhances anti-leukemia efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion.

Relapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cells persistence. Sustained tonic signaling or strong activation drives CART cell differentiation and exhaustion, which limit the therapeutic efficacy and persistence of CART cells. Here, we identified dasatinib as the optimal candidate to prevent or reverse both CD28/CART and 4-1BB/CART cell differentiation and exhaustion during ex vivo expansion, which profoundly enhanced the therapeutic efficacy and in vivo persistence. Moreover, strong activation-induced CART cells differentiation, exhaustion and apoptosis driven by CD3/CD28 stimulation or antigen exposure were dramatically prevented or reversed by dasatinib treatment. Mechanistically, dasatinib markedly reduced the phosphorylation of Src and Lck, and downregulated the expression of genes involved in CAR signaling pathways, which resulted in the optimization of cell differentiation, exhaustion and apoptosis-related gene expression. Our study proposes a promising pharmacological approach for optimizing CART cells manufacture, and provides an experimental basis for reinvigorating CART cells in clinical application.