Recent progress in chimeric antigen receptor-modified T-cell (CAR-T cell) technology in cancer therapy is extremely promising, especially in the treatment of patients with B-cell acute lymphoblastic ...leukemia. In contrast, due to the hostile immunosuppressive microenvironment of a solid tumor, CAR T-cell accessibility and survival continue to pose a considerable challenge, which leads to their limited therapeutic efficacy. In this study, we constructed two anti-MUC1 CAR-T cell lines. One set of CAR-T cells contained SM3 single chain variable fragment (scFv) sequence specifically targeting the MUC1 antigen and co-expressing interleukin (IL) 12 (named SM3-CAR). The other CAR-T cell line carried the SM3 scFv sequence modified to improve its binding to MUC1 antigen (named pSM3-CAR) but did not co-express IL-12. When those two types of CAR-T cells were injected intratumorally into two independent metastatic lesions of the same MUC1
+
seminal vesicle cancer patient as part of an interventional treatment strategy, the initial results indicated no side-effects of the MUC1 targeting CAR-T cell approach, and patient serum cytokines responses were positive. Further evaluation showed that pSM3-CAR effectively caused tumor necrosis, providing new options for improved CAR-T therapy in solid tumors.
Chimeric antigen receptor (CAR)-T cell therapy is a novel type of immunotherapy. However, the use of CAR-T cells to treat acute myeloid leukaemia (AML) has limitations. B7-H3 is expressed in several ...malignancies, including some types of AML cells. However, its expression in normal tissues is low. Therefore, B7-H3 is ideal for targeted AML therapy.
First, we constructed B7-H3 CAR that can target B7-H3, and then constructed B7-H3-CAR-T cells in vitro, which were co-incubated with six AML cell lines expressing different levels of B7-H3, respectively. The toxicity and cytokines were detected by flow cytometry. In vivo, AML model was established in B-NSG mice to study the toxicity of B7-H3-CAR T on AML cells.
In vitro functional tests showed that B7-H3-CAR-T cells were cytotoxic to B7-H3-positive AML tumor cells and had good scavenging effect on B7-H3-expressing AML cell lines, and the cytokine results were consistent. In vivo, B7-H3-CAR-T cells significantly inhibited tumor cell growth in a mouse model of AML, prolonging mouse survival compared with controls.
B7-H3-CAR-T cells may serve as a novel therapeutic method for the targeted treatment of AML.
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Background: CD7 represents a potential target for T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LBL). We developed CD7 nanobody derived chimeric antigen receptor T-cells ...(CD7-CART), and established a non-gene editing strategy by anchoring CD7 in the ER and/or Golgi to overcome the CART fratricide. Methods: This single-arm, open-label, phase I study is to investigate CD7-CART cell manufacturing feasibility without contamination of malignant T cells, and the safety and efficacy of the CART on patients with CD7 positive relapsed/refractory T-ALL/T-LBL. 3 subjects, identified as both CD4 and CD8 negative T-ALL or T-LBL were enrolled. CART cells were manufactured by using CD4+/CD8+ sorted T cells from leukapheresis. All patients (Pts) were pretreated with Flu/Cy prior to CART infusion. 1x10
6
/kg CART cells were given to case 2 and 3, while 1.5x10
6
/kg to case 1. Results: Case 1 was diagnosed as refractory ALL with myeloid differentiation, who had received intensive chemotherapy and allogeneic hematopoietic stem cell microtransplantation. Case 2 was diagnosed as ALL (T/B mixed type) but relapsed with CNS involvement, and received radiotherapy in addition to intensive chemotherapy. Prior to CART infusion, case 2 had no abnormal B cells but 17.69% of abnormal early T cellsfrom BM. Case 3 had stage VI of T-LBL, which recurred after multi-cycle chemotherapy of BFM-90 regimen and autologous SCT. After CART treatment, no neurotoxicity was observed in all pts. Case 1 had grade 3 CRSwhile case 2 and 3 had grade 1, although increased IL-6 was detected in all pts. Significant CART expansion and persistence were observed in case 2 and 3, and MRD negative CR was confirmed on day 28 in both pts. The number of generalized lymphadenopathy, lymph node size, and the degree of metabolism were all significantly reduced in case 3. Case 1 had only moderate CART expansion, but abnormal early T cells from BM decreased from 70.03% to 19.57% on day 30. After CART infusion, the number of peripheral abnormal T cells became either undetectable in case 2 and 3, or significantly decreased in case 1. Interestingly, CART had unsustained effect on normal T cells in all pts. As of Feb-10-2020, case 1 has 5 months of OS, including 3 months of PFS. Case 2 and 3 has reached 2 and 1 months of PFS and is still in remission. Conclusions: CD7-CART cells can be manufactured without contamination of malignant T cells. CD7-CART therapy is well-tolerated and has great therapeutic potential for relapsed/refractory CD7 positive T cell malignancies. Clinical trial information: NCT04004637 .
CD7 protein as a target is being used to treat CD7+ lymphoma; however, the role of CD7 in the hematopoietic system remains largely unknown. Therefore, we evaluated the effects of CD7 KO in mice. The ...differentiation of the hematopoietic system in the bone marrow and the number of various cell types in the thymus and spleen did not differ between CD7 KO and WT mice. After subcutaneous inoculation of B16–F10 melanoma cells, tumors from CD7 KO mice grew more rapidly, and the proportion of CD8+ T cells in the spleen and tumors decreased. In vitro, the infiltration and adhesion of CD8+ T cells from the spleen of CD7 KO mice were weakened. Blocking CD7 in normal T cells did not alter the migration and infiltration, but in Jurkat, CCRF-CEM, and KG-1a tumor cell lines, migration and invasion were significantly reduced after blocking CD7. Therefore, CD7 does not affect hematopoietic system development but plays a crucial role in T cell infiltration into tumors.
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B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. ...In the present study, the full-length cDNA of BAFF (designated bBAFF) from the bat (Vespertilio superans Thomas) was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of bBAFF consists of 986 bases including an 873bp open reading frame encoding 290 amino acids. Sequence comparison indicated that the amino acid of bBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the bsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that bBAFF mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO (Small Ubiquitin-like Modifier)-bsBAFF was efficiently expressed in Escherichia coliBL21 (DE3) and confirmed by SDS-PAGE and Western blotting analysis. Laser scanning confocal microscopy analysis showed that bsBAFF could bind to its receptors on B cells. In vitro, the MTT assays indicated that SUMO-bsBAFF was not only able to promote survival/proliferation of bat lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These findings indicate that bsBAFF plays an important role in the survival/proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.
► This is the first report of BAFF gene cloned from chiroptera mammals. ► The deduced amino acid sequence was analyzed using bioinformatics methods. ► Protein was expressed in E. coli and purified by Ni column. ► Indirect immunofluorescence staining was conducted to determine receptor-binding activity. ► MTT analysis tested the bioactivity of BAFF to B cells.
Limited efficacy of chimeric antigen receptor T (CAR-T) cells in treating solid tumors is largely due to the antigen heterogeneity and immunosuppressive tumor microenvironment (TME). B7-H3 is ...over-expressed in most kind of solid tumors, making it a promising target for cancer treatment. This study aims to explore the effect of B7-H3-CAR-T therapy combined with radiotherapy in treating solid tumor models.
Irradiated tumor cell lines were prepared and tested. A humanized B7-H3-CAR-T was constructed, and it was evaluated that B7-H3-CAR-T cytotoxicity against solid tumor models with preconditioning of radiotherapy in vitro and vivo.
Irradiation was found to increase expression level of B7-H3 in pancreatic cancer (PANC-1), colorectal cancer (HCT-15, SW620), acute myelocytic leukemia (AML-5), epidermoid carcinoma (KB) and glioma (U87-MG) human cell lines significantly. 6Gy irradiation was also found to up-regulate tumor-infiltration molecule like intracellular adhesion molecule-1 ICAM-1 or FAS in HCT-15 cells, supporting a possible synergistic enhancement effect of radiotherapy. In vitro and in vivo experiments demonstrated that irradiation indeed significantly enhanced the ability of B7-H3-CAR-T to infiltrate and kill tumors. Interestingly in dual-tumor mouse model study, not only tumor cells on irradiation side were eradicated completely, irradiation also enhanced CAR-T tumor-killing ability on non-irradiated side, confirming the abscopal effect of irradiation existed with CAR-T therapy.
Our results suggest that B7-H3-CAR-T therapy combined with radiotherapy may be a promising modality in treating solid tumors.
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CAR T cells have shown clinical efficacy for acute lymphoblastic leukemia, but this therapy has not been effective for acute myeloid leukemia (AML), and other treatment options are needed. ...Theoretically, CAR-NK cells have a more favorable toxicity profile compared to CAR T cells, especially in avoiding adverse effects such as cytokine release syndrome. However, the clinical evidence for this has not yet been reported. In the current study, we tested the safety of CD33-CAR NK cells in patients with relapsed and refractory AML. At doses up to 5 × 10
(5 billion) cells per patient, no significant adverse effects were observed. CAR NK-92 cells can be produced at much lower cost compared to CAR T cells, and we believe after being optimized, they will be widely accessible for the treatment of cancer.
Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies; however, designing CARs for T-cell based diseases remain a challenge since most ...target antigens are shared between normal and malignant cells, leading to CAR-T cell fratricide. CD7 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL), but it is not expressed in one small group of normal T lymphocytes. Here, we constructed monovalent CD7-CAR-NK-92MI and bivalent dCD7-CAR-NK-92MI cells using the CD7 nanobody VHH6 sequences from our laboratory. Both CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells consistently showed specific and potent anti-tumor activity against T-cell leukemia cell lines and primary tumor cells. We observed robust cytotoxicity of the bivalent mdCD7-CAR-NK-92MI monoclonal cells against primary T-ALL samples. In agreement with the enhanced cytotoxicity of mdCD7-CAR-NK-92MI cells, significant elevations in the secretion of Granzyme B and interferon γ (IFN-γ) were also found in mdCD7-CAR-NK-92MI cells in response to CD7-positive primary T-ALL cells compared with NK-92MI-mock cells. Furthermore, we also demonstrated that mdCD7-CAR-NK-92MI cells significantly inhibited disease progression in xenograft mouse models of T-ALL primary tumor cells. Our data suggest that CD7-CAR-NK-92MI cells can be used as a new method or a complementary therapy for treating T-cell acute lymphocytic leukemia.
Since CD7 may represent a potent target for T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) immunotherapy, this study aimed to investigate safety and efficacy of autologous CD7-chimeric antigen ...receptor (CAR) T cells in relapsed and refractory (R/R) T-ALL/LBL patients, as well as its manufacturing feasibility.
Preclinical phase was conducted in NPG{trade mark, serif} mice injected with Luc+ GFP+CCRF-CEM cells. Open label phase I clinical trial (NCT04004637) enrolled patients with R/R CD7-positive T-ALL/LBL who received autologous CD7-CAR T cells infusion. Primary endpoint was safety, secondary endpoints included efficacy, pharmacokinetic and pharmacodynamic parameters.
CD7 blockade strategy was developed using tandem CD7 nanobody VHH6 coupled with an ER/Golgi-retention motif peptide to intracellularly fasten CD7 molecules. In preclinical phase CD7 blockade CAR T-cells prevented fratricide and exerted potent cytolytic activity, significantly relieving leukemia progression and prolonged the median survival of mice. In clinical phase, the complete remission (CR) rate was 87.5% (7/8) three months after CAR T cells infusion; one leukemia patient achieved minimal residual disease negative CR and one lymphoma patient achieved CR for more than 12 months. Majority of patients (87.5%) only had grade 1 or 2 cytokine release syndrome with no T-cell hypoplasia or any neurological toxicities observed. The median maximum concentration of CAR T cells was 857.2 cells/µL at approximately 12 days and remained detectable up to 270 days.
Autologous nanobody-derived fratricide-resistant CD7-CAR T cells demonstrated a promising and durable antitumor response in R/R T-ALL/LBL with tolerable toxicity, warranting further studies in highly aggressive CD7-positive malignancies.
Objectives
Despite the great success of CD19 CAR‐T cell therapy, its clinical efficacy has been greatly hampered by the high relapse rate. In this study, we designed and compared four structures of ...CD19/CD22 bispecific CAR‐T cells with different linkers and different orders of the antibody sequences.
Methods
We detected the cytotoxicity, cytokine secretion levels, sustainable killing ability, differentiation, exhaustion of these four CAR‐T cells in vitro. The optimal Bis‐C CAR‐T cells were evaluated the efficacy using NSG mice.
Results
The two structures of CD19/CD22 bispecific CAR‐T cells using (EAAAK)3 as linker had more significant cytotoxicity and cytokine secretion levels. In the process of continuous killing, Bis‐C CAR‐T cells showed better sustained killing ability, memory phenotype differentiation, and exhaustion. In the in vivo experiment mimicking CD19‐negative relapse, Bis‐C CAR‐T was more able to control the tumor progression of mice in the CD19 low expression or no expression groups than CD19 CAR‐T.
Conclusions
This study has generated a novel bispecific CAR‐T cell that can simultaneously target CD19 or CD22 positive tumor cells, providing a new strategy to address the limitations of single‐targeted CAR‐T therapy in B‐cell tumors (limited response or relapse).
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