scholarly journals In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Béatrice Clémenceau ◽  
Sandrine Valsesia-Wittmann ◽  
Anne-Catherine Jallas ◽  
Régine Vivien ◽  
Raphaël Rousseau ◽  
...  
Keyword(s):  
Chimeric Antigen Receptor ◽  
Tumor Regression ◽  
Critical Role ◽  
Antigen Receptor ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Her2 Positive ◽  
Positive Target

The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ(NK-92CD16) or a trastuzumab-based scFv-FcεRIγchimeric receptor (NK-92CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition byNK-92CD16was always inferior to that observed after direct recognition byNK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer ofNK-92CD16+ trastuzumab but not ofNK-92CARinduced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of theNK-92CARin the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

scholarly journals In Vitro Comparison of ADCC and CAR Sensitivity of Adult HER-2+ B-ALL Using the NK-92 Human Cell Line Transduced with a Human CD16 (ADCC) or an Anti-HER2 Chimeric Antigen Receptor (CAR)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5193-5193
Author(s):  
Beatrice Clemenceau ◽  
Thierry Guillaume ◽  
Nelly Robillard ◽  
Regine Vivien ◽  
Pierre Peterlin ◽  
...  
Keyword(s):  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Her2 Expression ◽  
51Cr Release ◽  
Her2 Positive ◽  
Car T ◽  
Anti Cd20

Abstract Introduction: Prognosis of acute lymphoblastic leukemia (ALL) in adults remains dismal. Recent advances have consisted of targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T lymphocytes (anti-CD19 CAR T cells), showing very encouraging results. However, relapse still occur after this new therapeutics and increasing the armamentarium for ALL is still required. Her2 is expressed in above one third of adult B-ALL (Chevallier,Haematologica 2009). The anti-HER2 antibodytrastuzumab has been tested prospectively in such patients, showing a low efficacy (Chevallier, Blood 2012).The present work was designed to compare, in vitro, thelysis sensibility of B-ALL HER2+ mediated by two mechanisms of HER2 recognition based on antibody (Ab) specificity: firstly, when the anti-HER2 monoclonalAbtrastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (antibodydependant cellular cytotoxicity, ADCC) andsecondly, when HER2-positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (anti-HER2 CAR). Methods: Frozen samples from 7 HER2+ B-ALL adults were available, including onesample expressing also CD20. Characteristics of Patients and controls are presented in Table 1. Three human breast-cancer cell lines (BCCL) expressing different validated (HercepTest, Dako) levels of HER2 (BT474 (HER2 3+), MCF-7 (HER2 0-/1+) and MDA-MB-231 (HER2 0-/1+) were used as positive control. Leukemic HER2-negative B-ALL cells from two patients were used as negative control, including one patient with CD20+ expression. The level of HER2 expression by HER2+ B-ALL was determined by fluorescence-activated cell sorter (FACS) and compared to the HER2 expressing BCCL.Cytotoxic activity was assessed at a 30-to-1 effector-to-target ratio in a 4 hours 51Cr-release assay.We used the same cytotoxic effector lymphocytes (the human NK cell line NK-92), armed with eitheraFcγRIIIa/FcεRIγ receptor (referred to as NK-92CD16) or with an anti-HER2/FcεRIγ CAR receptor (referred to as NK-92CAR) as previously described (Clémenceau, JImmunol Research 2015). For ADCC/CAR assays, target cells were pre-incubated withtrastuzumab and /or rituximab at 10µg/ml each. The study was approved by our local review board and twoalivepatients gave informed consent. Results: The level of HER2 expression by HER2+ B-ALL (n=7) was highly variable with a mean relative fluorescence intensity ratio (RFI) of 13 (range 5 to 30). According to the HercepTestranking used for BCCL lines (see above) all B-ALL would be classified as HER2 0-/1+. In vitro HER2+ BCCL were efficiently lysed by anti-HER2 NK-92CAR in this 4 hours 51Cr-release assay and this direct pathway of killing by CAR was always more efficient than the indirect pathway mediated by ADCC. In addition, thelysis level was correlated to HER2 level expression. Trastuzumabalone has no effect on the spontaneous 51Cr-release of B-ALL cells (not shown). Anti-HER2 CAR mediatedlysis was observed for 5 out of 6 HER2+ B-ALL cells (83%, 1 case (#5) non interpretable). Although lysis levels were low, they werespecificsince the CAR mediatedlysis was inhibited when the B-ALL were pre-incubated with 10 µg/mltrastuzumab (not shown). In contrast, for these 5 HER2+ B-ALL, no anti-HER2 ADCClysis was observed. These results confirmed that in these experimental conditions, HER2 targeting by CAR is more efficient than by ADCC. For the two HER2- B-ALL, no ADCC or CARlysis were observed. Finally, for the two B-ALL expressing CD20, anti-CD20 ADCClysiscan be observed demonstrating that B-ALL cells are not intrinsically resistant to ADCCcytoxicity. Regarding the unique case of B-ALL expressing both CD20/HER2 antigens, only anti-CD20 ADCClysiswas observed. Results are presented in Table 2. Conclusion: Together, these preliminary results suggest that for the CD20-negative and HER2-positive B-ALLs, one cannot rely on ADCC as a mechanism of target celllysis, while the interest of an anti-HER2-CAR approach deserve further studies to better correlate the level of HER2 expression and the sensitivity tolysisby HER2-specific CAR. Disclosures No relevant conflicts of interest to declare.

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 Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

2021 ◽  
Author(s):  
Yannick D. Muller ◽  
Leonardo M.R. Ferreira ◽  
Emilie Ronin ◽  
Patrick Ho ◽  
Vinh Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Chimeric Antigen Receptor ◽  
Mononuclear Cells ◽  
Antigen Receptor ◽  
Single Chain ◽  
Transplant Tolerance ◽  
Peripheral Blood Mononuclear

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-zeta signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

scholarly journals CD137 Co-Stimulation Improves The Antitumor Effect Of LMP1-Specific Chimeric Antigen Receptor T Cells In Vitro And In Vivo

2019 ◽  
Vol Volume 12 ◽  
pp. 9341-9350 ◽  
Author(s):  
Xiaojun Tang ◽  
Qi Tang ◽  
Yuan Mao ◽  
Xiaochen Huang ◽  
Lizhou Jia ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antitumor Effect ◽  
Antigen Receptor

scholarly journals Functional effects of chimeric antigen receptor co-receptor signaling domains in human Tregs

2019 ◽  
Author(s):  
Nicholas A.J. Dawson ◽  
Isaac Rosado-Sánchez ◽  
German E. Novakovsky ◽  
Vivian C.W. Fung ◽  
Qing Huang ◽  
...  
Keyword(s):  
Chimeric Antigen Receptor ◽  
Cellular Therapy ◽  
Antigen Receptor ◽  
In Vitro Assays ◽  
Receptor Signaling ◽  
Graft Versus Host ◽  
Signaling Domain ◽  
Signaling Domains

SummaryAntigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptor (CARs) are a potent immunosuppressive cellular therapy in multiple disease models. To date the majority of CAR Treg studies employed second generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs. Using an HLA-A2-specific CAR platform and human Tregs, we compared ten CARs with different co-receptor signaling domains and systematically tested their function. Tregs expressing a CAR encoding wild-type CD28 were markedly superior to all other CARs tested in anin vivomodel of graft-versus-host disease. In vitro assays revealed stable expression of Helios and ability to suppress CD80 expression on DCs as keyin vitropredictors ofin vivofunction. This comprehensive study of CAR signaling-domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.

Development of human NKG2D-CD3ε chimeric antigen receptor (CAR) for T-cell-mediated cancer immunotherapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 150-150
Author(s):  
Sergei Kusmartsev ◽  
Johaness Vieweg ◽  
Victor Prima
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Adaptor Protein ◽  
T Cell Subsets ◽  
Human T Cells

150 Background: NKG2D is a lectin-like type 2 transmembrane receptor that expressed by natural killer cells and some T cell subsets. Stimulation of NKG2D receptor with specific agonistic ligands produces activating signals through signaling adaptor protein DAP10 leading to the enhanced cytokine production, proliferation, and cytotoxicity against tumor cells. There is strong evidence that NKG2D ligands are expressed in many human tumors, including melanoma, leukemia, myeloma, glioma, and carcinomas of the prostate, breast, lung, and colon. Recent studies also demonstrated that T cells bearing chimeric antigen receptor (CAR) NKG2D linked to CD3ζ (zeta) chain produce marked in vitro and in vivo anti-tumor effects. The aim of current study was to determine whether human T cells bearing chimeric antigen receptor (CAR) NKGD2 linked to CD3ε (epsilon) chain could be activated by the NKG2D-specific stimulation and able to kill human cancer cells. Given the important role of CD3ε in activation and survival of T cells, we hypothesized that NKG2D-CDε-bearing T cells could exert strong in vitro and in vivo anti-tumor effects. Methods: NKG2D CAR was produced by linking human NKG2D to DAP10 and the cytoplasmic portion of the CD3ε chain. Original full-length human cDNA clones were obtained from NIH Mammalian Gene Collection (MGC). Functional domain analysis and oligonucleotide design in the in-Fusion system of DNA cloning (Clontech) was used to generate the retroviral expression constructs. Results: Human PBMC-derived T cells were retrovirally transduced with newly generated NKG2D-CD3ε CAR DNA construct. These NKG2D CAR-expressing human T cells responded to NKG2D-specific activation by producing IFN-γ and exhibited significant cellular cytotoxicity against human tumor cells in vitro. In vivo studies demonstrated that NKG2D-CD3ε-bearing cells are capable of inhibiting growth of DU-145 human prostate cancer in the immunodeficient mice. Conclusions: Collectively, our data indicate the feasibility of developing chimeric antigen receptor NKG2D-CD3ε for T cells and suggest that adoptive transfer of T cells bearing NKG2D-CD3ε CAR could be potentially effective for immunotherapy of cancer patients.

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.

Abstract 206: Stem Cell-derived Exosomes Induce Cardiac Repair via mRNA Modification

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Prabhu Mathiyalagan ◽  
Yaxuan Liang ◽  
Adriano S Martins ◽  
Douglas W Losordo ◽  
Roger J Hajjar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Myocardial Ischemia ◽  
Target Genes ◽  
Critical Role ◽  
Cell Communication ◽  
Target Cells ◽  
Mouse Heart ◽  
Loss Of Function

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34 + stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.

scholarly journals Selective Cytotoxicity of Single and Dual Anti-CD19 and Anti-CD138 Chimeric Antigen Receptor-Natural Killer Cells against Hematologic Malignancies

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Sudjit Luanpitpong ◽  
Jirarat Poohadsuan ◽  
Phatchanat Klaihmon ◽  
Surapol Issaragrisil
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Chimeric Antigen Receptor ◽  
Nk Cell ◽  
Antigen Receptor ◽  
Antigen Expression ◽  
In Vivo Studies ◽  
Selective Cytotoxicity

Natural killer (NK) cells are part of the first line of defense that rapidly respond to malignant transformed cells. Chimeric antigen receptor- (CAR-) engineered NK cells, although are still at the preliminary stage, have been shown to be alternative to CAR-T cells, mainly due to the absence of graft-versus-host disease and safer clinical profile. Allogeneic human NK cell line NK-92 cells, equipped by CAR, are being developed for clinical applications. Herein, we designed third-generation CARs, optimized the production protocol, and generated CAR-NK-92 cells, targeting CD19 and/or CD138 antigens that employ CD28, 4-1BB, and CD3ζ signaling, with >80% CAR expression, designated as CD19-NK-92, CD138-NK-92, and dual-NK-92 cells. The generated CAR-NK-92 cells displayed high and selective cytotoxicity toward various corresponding leukemia, lymphoma, and multiple myeloma cell lines in vitro. Multitargeting approach using a mixture of CD19-NK-92 and CD138-NK-92 cells was also evaluated at various ratios to test the idea of personalized formulation to match the patients’ antigen expression profile. Our data indicate that increasing the ratio of CD19-NK-92 to CD138-NK-92 could improve NK cytotoxicity in leukemia cells with a relatively higher expression of CD19 over CD138, supporting the personalized proof of concept. This information represents the basis for further in vivo studies and future progress to clinical trials.

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