scholarly journals Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma

2020 ◽  
Author(s):  
Liqing Kang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
Wei Qi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

Abstract Background: Treatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells. Method: The scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells. Results: The in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting. Conclusion: We report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo . The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.

scholarly journals Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma

2020 ◽  
Author(s):  
Liqing Kang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
Wei Qi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

Abstract Background: Chimeric antigen receptor (CAR) engineered T cells directed B cell maturation antigen (BCMA) showed transient recovery to multiple myeloma (MM). However, the expression of CD19 on immature plasma cell may escape the recognition by BCMA-CAR T, which restrict the efficacy and facilitate to relapse. The purpose of this study is to characterize a novel CAR structure with a tandem orientation of scFv-BCMA and scFv-CD19, tandem CAR (tan-CAR), to provide an effective solution for killing both BCMA and/or CD19 expression MM cells.Method: Single-chain variable fragment (scFv) sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T cell signaling domains to achieve tan-CAR construct. The therapeutic specificity and efficiency were analyzed for tan-CAR T cells activation, proliferation, cytokine release and cytolytic toxicity in vitro. Also, in vivo efficacy evaluation conducted in xenograft mouse models with the combination of two corresponding target tumor cells, in comparison with conventional CAR.Results: The in vitro studies demonstrated specific activation of tan-CAR T cells to the K562 tumor cell overexpressing CD19, BCMA, or both. Besides, it also elicits the comparable immunoreactivities, in terms of proliferation, cytokine release and cytolytic activity compared to single scFv modified CAR T cells. Importantly, the in vivo studies of tan-CAR-transduced T cells results specific inhibition of tumor growth in xenograft model that express combined tumor antigen i.e. CD19 and BCMA. Moreover, systemic administration of tan-CAR resulted in complete tumor remission, whilst neither BCMA-CAR T nor CD19-CAR-T could. Conclusion: A novel tan-CAR T was successfully designed and showed the significant antitumor efficacy for combined antigen-positive tumor cells in vitro and in vivo. However, the single CAR T cells with targeting one antigen didn’t achieve similar potency. The data from this study suggest a novel strategy to help reduce relapse due to existing CD19-expressing multiple myeloma cells or downregulation of the BCMA antigen after CAR-based treatment of multiple myeloma.

scholarly journals Characterization of the Therapeutic Effects of Novel Chimeric Antigen Receptor T Cells Targeting CD38 on Multiple Myeloma

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaorui Li ◽  
Yaru Feng ◽  
Fengqin Shang ◽  
Zhuoying Yu ◽  
Tieshan Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Second Generation ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Ifn Γ

Multiple myeloma (MM) is a tumor type characterized by the unregulated proliferation of clonal plasma cells in the bone marrow. Immunotherapy based on chimeric antigen receptor T cell (CAR-T) therapy has achieved exciting success in the treatment of hematological malignant tumors. CD38 is highly and evenly expressed in MM and is an attractive target for MM treatment. Here, we successfully constructed two novel second-generation CAR-T cells targeting CD38 by retroviral vector transduction. CD38 CAR-T cells could be activated effectively after stimulation with CD38-positive tumor cells and secrete cytokines such as IFN-γ and TNF-α to promote tumor cell apoptosis in in vitro experiments. Real-time fluorescence monitoring experiments, luciferase detection experiments and flow cytometry experiments revealed the efficient and specific killing abilities of CD38 CAR-T cells against CD38-positive tumor cells. The proliferation ability of CD38 CAR-T cells in vitro was higher than that of untransduced T cells. Further antitumor experiments in vivo showed that CD38 CAR-T cells could be quickly activated to secrete IFN-γ and eliminate tumors. Thus, novel CD38-targeted second-generation CAR-T cells have efficient and specific antitumor activity and may become a novel therapy for the clinical treatment of MM.

Preclinical Evaluation of Allogeneic Anti-Bcma Chimeric Antigen Receptor T Cells with Safety Switch Domains and Lymphodepletion Resistance for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 381-381 ◽  
Author(s):  
Bijan Boldajipour ◽  
Roman Galetto ◽  
Cesar Sommer ◽  
Thomas Pertel ◽  
Julien Valton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Preclinical Evaluation ◽  
Car T Cells ◽  
Car T

Abstract Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool. Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule. Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis' know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab. Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity. Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. Disclosures Boldajipour: Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

scholarly journals CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

2021 ◽  
Author(s):  
Katherine Mueller ◽  
Nicole Piscopo ◽  
Matthew Forsberg ◽  
Louise Saraspe ◽  
Amritava Das ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tumor Regression ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cells traditionally harbor viral vectors that encode the CAR transgene in the genome. However, viral vector manufacturing typically is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. Here, CAR T cells were generated within nine days using recombinant SpCas9 protein and nucleic acids, without any viral vectors or animal components. In comparison to traditional retroviral CAR T cells, nonviral CRISPR CAR T cells exhibit TRAC-targeted genomic integration of the CAR transgene, higher frequency of gene expression signatures associated with a memory phenotype, low receptor signaling prior to infusion, and potent cytotoxicity against GD2+ neuroblastoma in vitro and in vivo. This proof-of-principle study eliminating viral vectors and animal components during CAR gene transfer could enable more flexible and scalable manufacturing of clinically-relevant, high-quality CAR T cells to treat cancers, including solid tumors.

scholarly journals High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

2021 ◽  
Vol 11 ◽  
Author(s):  
Radhika Thokala ◽  
Zev A. Binder ◽  
Yibo Yin ◽  
Logan Zhang ◽  
Jiasi Vicky Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood–brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

scholarly journals CAR T Cells Equipped With a Fully Human scFv Targeting Trop2 Can Be Used to Treat Pancreatic Cancer

2021 ◽  
Author(s):  
Hong Jia Zhu ◽  
Yujie Jia ◽  
Jingwen Tan ◽  
Xiaoyan Fang ◽  
Jing Ye ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
Cancer Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Pancreatic Cancer Cells ◽  
Car T

Abstract Purpose: Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in treating haematologic malignancies but has not been effective against solid tumours thus far. Trop2 is a tumour-related antigen broadly overexpressed on a variety of tumours and has been reported as a promising target for pancreatic cancers. Our study aimed to determine whether CAR T cells designed with a fully human Trop2-specific single-chain fragment variable (scFv) can be used in the treatment of Trop2-positive pancreatic tumours.Methods: We designed Trop2-targeted chimeric antigen receptor engineered T cells with a novel human anti-Trop2 scFv (2F11) and then investigated the cytotoxicity, degranulation, and cytokine secretion profiles of the anti-Trop2 CAR T cells when they were exposed to Trop2+ cancer cells in vitro. We also studied the antitumour efficacy and toxicity of Trop2-specific CAR T cells in vivo using a BxPC-3 pancreatic xenograft model.Results: Trop2-targeted CAR T cells designed with 2F11 effectively killed Trop2-positive pancreatic cancer cells and produced high levels of cytotoxic cytokines in vitro. In addition, Trop2-targeted CAR T cells, which persistently circulate in vivo and efficiently infiltrate into tumour tissues, significantly blocked and even eliminated BxPC-3 pancreatic xenograft tumour growth without obvious deleterious effects observed after intravenous injection into NSG mice. Moreover, disease-free survival was efficiently prolonged.Conclusion: These results show that Trop2-targeted CAR T cells equipped with a fully human anti-Trop2 scFv could be a potential treatment strategy for pancreatic cancer and could be useful for clinical evaluation.

scholarly journals Engineering T cells with hypoxia-inducible chimeric antigen receptor (HiCAR) for selective tumor killing

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Qibin Liao ◽  
Huan He ◽  
Yunyu Mao ◽  
Xiangqing Ding ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Normal Tissues ◽  
Car T Cells ◽  
Car T ◽  
Tumor Killing

Abstract Chimeric antigen receptor-modified T cells (CAR-T cells) have shown good effects in the treatment of hematologic cancers; however, they may cause on-target off-tumor toxicity because of minimal expression of tumor-associated antigens (TAAs) on normal tissues, particularly in the context of treating solid tumors. Hypoxia is a common hallmark of solid tumors because of the Warburg effect. To minimize side effects, we designed a hypoxia-inducible CAR (HiCAR), which is driven by a hypoxia response element (HRE), and consists of a conventional CAR and an oxygen-dependent degradation domain (ODD) that is actively degraded under normoxia but stabilized under hypoxia. HiCAR-T cells showed enhanced cytotoxicity against tumor cells under hypoxia compared to normoxia in vitro and antitumor efficacy comparable to that of conventional CAR-T cells in vivo. Overall, our study demonstrates the potential of the HiCAR for improving the safety of CAR-T cells to promote the clinical application of CAR-T immunotherapy.

scholarly journals Chlorotoxin Redirects Chimeric Antigen Receptor T Cells for Specific and Effective Targeting of Glioblastoma

2020 ◽  
Author(s):  
Dongrui Wang ◽  
Renate Starr ◽  
Wen-Chung Chang ◽  
Brenda Aguilar ◽  
Darya Alizadeh ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Matrix Metalloproteinase 2 ◽  
Cell Surface Expression ◽  
Binding Potential ◽  
Car T Cells ◽  
Car T

AbstractWhile chimeric antigen receptor (CAR) T cells have demonstrated antitumor activity against glioblastoma (GBM), tumor heterogeneity remains a critical challenge. To more effectively target heterogeneous GBMs, we report the development of a novel peptide-based CAR exploiting the GBM-binding potential of chlorotoxin (CLTX). CLTX bound a greater proportion of tumor cells than GBM-associated antigens EGFR, HER2 and IL13Rα2. CAR T cells bearing CLTX as the targeting domain (CLTX-CAR), mediated potent in vitro and in vivo anti-GBM activity, and efficiently targeted tumors lacking expression of other GBM-associated antigens. Importantly, CLTX-CAR T cells exhibited no observable off-target effector activity against normal cells, or when adoptively transferred into mice. Effective targeting by CLTX-CAR T cells required cell surface expression of matrix metalloproteinase-2 (MMP-2). Our results are the first demonstration of a peptide toxin utilized as a CAR targeting domain, expanding the repertoire of tumor-selective CAR T cells with the potential to reduce antigen escape.One Sentence SummaryChimeric antigen receptors incorporating chlorotoxin as the tumor targeting domain recognize and kill glioblastoma with high specificity and potency.

scholarly journals Split chimeric antigen receptor-modified T cells targeting glypican-3 suppress hepatocellular carcinoma growth with reduced cytokine release

2020 ◽  
Vol 12 ◽  
pp. 175883592091034 ◽  
Author(s):  
Xuan Liu ◽  
Jianyun Wen ◽  
Honglei Yi ◽  
Xiaorui Hou ◽  
Yue Yin ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Tumor Growth ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Lower Amount ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

Background: Human glypican-3 (hGPC3) is a protein highly expressed in hepatocellular carcinoma (HCC) but limited in normal tissues, making it an ideal target for immunotherapy. The adoptive transfer of hGPC3-specific chimeric antigen receptor T (CAR-T) cells for HCC treatment has been conducted in clinical trials. Due to the rigid construction, conventional CAR-T cells have some intrinsic limitations, like uncontrollable overactivation and inducing severe cytokine release syndrome. Methods: We redesigned the hGPC3-specific CAR by splitting the traditional CAR into two parts. By using coculturing assays and a xenograft mouse model, the in vitro and in vivo cytotoxicity and cytokine release of the split anti-hGPC3 CAR-T cells were evaluated against various HCC cell lines and compared with conventional CAR-T cells. Results: In vitro data demonstrated that split anti-hGPC3 CAR-T cells could recognize and lyse hGPC3+ HepG2 and Huh7 cells in a dose-dependent manner. Impressively, split anti-hGPC3 CAR-T cells produced and released a significantly lower amount of proinflammatory cytokines, including IFN-γ, TNF-α, IL-6, and GM-CSF, than conventional CAR-T cells. When injected into immunodeficient mice inoculated subcutaneously with HepG2 cells, our split anti-hGPC3 CAR-T cells could suppress HCC tumor growth, but released significantly lower levels of cytokines than conventional CAR-T cells. Conclusions: We describe here for the first time the use of split anti-hGPC3 CAR-T cells to treat HCC; split anti-hGPC3 CAR-T cells could suppress tumor growth and reduce cytokine release, and represent a more versatile and safer alternative to conventional CAR-T cells treatment.

scholarly journals High Affinity Chimeric Antigen Receptor with Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

2021 ◽  
Author(s):  
Radhika Thokala ◽  
Zev A. Binder ◽  
Yibo Yin ◽  
Logan Zhang ◽  
Jiasi Vicky Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against Epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

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