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 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.

scholarly journals The roles of T cell competition and stochastic extinction events in chimeric antigen receptor T cell therapy

2021 ◽  
Vol 288 (1947) ◽  
Author(s):  
Gregory J. Kimmel ◽  
Frederick L. Locke ◽  
Philipp M. Altrock
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy is a remarkably effective immunotherapy that relies on in vivo expansion of engineered CAR T cells, after lymphodepletion (LD) by chemotherapy. The quantitative laws underlying this expansion and subsequent tumour eradication remain unknown. We develop a mathematical model of T cell–tumour cell interactions and demonstrate that expansion can be explained by immune reconstitution dynamics after LD and competition among T cells. CAR T cells rapidly grow and engage tumour cells but experience an emerging growth rate disadvantage compared to normal T cells. Since tumour eradication is deterministically unstable in our model, we define cure as a stochastic event, which, even when likely, can occur at variable times. However, we show that variability in timing is largely determined by patient variability. While cure events impacted by these fluctuations occur early and are narrowly distributed, progression events occur late and are more widely distributed in time. We parameterized our model using population-level CAR T cell and tumour data over time and compare our predictions with progression-free survival rates. We find that therapy could be improved by optimizing the tumour-killing rate and the CAR T cells' ability to adapt, as quantified by their carrying capacity. Our tumour extinction model can be leveraged to examine why therapy works in some patients but not others, and to better understand the interplay of deterministic and stochastic effects on outcomes. For example, our model implies that LD before a second CAR T injection is necessary.

scholarly journals A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2020 ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Semih U. Tareen ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and HIV sanctuary sites. Here, we describe a CAR RNA FISH histocytometry platform combined with a dnnRRS image analysis algorithm to quantitate spatial distribution and in vivo functional ability of a CAR T cell population at a single cell resolution. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions engineered cells in situ with their target cells in tissues with high statistical power and thus, as an important tool for preclinical and potentially clinical assessment of CAR T cell effectiveness.One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in human and mouse tissue.Impact statementWe developed an imaging platform and analysis pipeline to study large populations of engineered cells on a single cell level in situ.

scholarly journals JAK/STAT-Dependent Chimeric Antigen Receptor (CAR) Expression: A Design Benefiting From a Dual AND/OR Gate Aiming to Increase Specificity, Reduce Tumor Escape and Affect Tumor Microenvironment

2021 ◽  
Vol 12 ◽  
Author(s):  
Javad Khanali ◽  
Mohammadreza Azangou-Khyavy ◽  
Melika Boroomand-Saboor ◽  
Mobina Ghasemi ◽  
Hassan Niknejad
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Chimeric Antigen Receptor ◽  
Logic Gate ◽  
Antigen Receptor ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Recent advances in cancer immunotherapy have attracted great interest due to the natural capacity of the immune system to fight cancer. This field has been revolutionized by the advent of chimeric antigen receptor (CAR) T cell therapy that is utilizing an antigen recognition domain to redirect patients’ T cells to selectively attack cancer cells. CAR T cells are designed with antigen-binding moieties fused to signaling and co-stimulatory intracellular domains. Despite significant success in hematologic malignancies, CAR T cells encounter many obstacles for treating solid tumors due to tumor heterogeneity, treatment-associated toxicities, and immunosuppressive tumor microenvironment. Although the current strategies for enhancing CAR T cell efficacy and specificity are promising, they have their own limitations, making it necessary to develop new genetic engineering strategies. In this article, we have proposed a novel logic gate for recognizing tumor-associated antigens by employing intracellular JAK/STAT signaling pathway to enhance CAR T Cells potency and specificity. Moreover, this new-generation CAR T cell is empowered to secrete bispecific T cell engagers (BiTEs) against cancer-associated fibroblasts (CAFs) to diminish tumor metastasis and angiogenesis and increase T cell infiltration.

scholarly journals A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Michael Claus Jensen ◽  
Jia Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.

Results from Melanoma Antigen Redirected Vaccine Stimulated Autologous Lymphocytes (MARVSmALo): A pilot study.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15026-e15026
Author(s):  
Joy M. Fulbright ◽  
Gary C. Doolittle ◽  
Cliona M. Rooney ◽  
Siddhartha Ganguly ◽  
Sunil H. Abhyankar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Pilot Study ◽  
Metastatic Melanoma ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

e15026 Background: Despite improvements in treatment with targeted agents and immunotherapeutics metastatic melanoma still has a guarded prognosis. Many melanoma cells upregulate the disialoganglioside GD2. Early GD2 chimeric antigen receptor (CAR) T-cell studies in neuroblastoma demonstrated no toxicity but limited ability to expand in vivo. Strategies to expand these GD2.CAR T cells might improve efficacy while retaining safety. This pilot study aimed to evaluate the safety and efficacy of first generation 14g2a.zeta chimeric antigen receptor ( GD2.CAR ) transduced, activated T cells enriched for vaccine specific cytotoxic T-Lymphocytes (tvs-CTL). Methods: Patients with metastatic melanoma in which standard therapy had failed were eligible if they had recovered from effects of prior therapy, did not have rapidly progressive disease, were free of melanoma involving the CNS and did not have a contraindication to receiving Hepatitis B, Polio or DTAP vaccine. Patients received each of these vaccines prior to cell harvest, 4 days before and 28 days after autologous T cell infusion. Patient 1 was treated with 2 x 108 cell dose and patients 2 and 3 were treated with 4x108 cell dose. We used QPCR to measure transgene copy number in patients before and after infusion. Interferon-gamma enzyme linked immunospot (ELISPOT) assay was used to measure the frequencies of tetanus, pertussis, diphtheria, poliovirus and tumor antigens-specific T cells in peripheral blood. Results: GD2.CAR-tvs-CTL were manufactured and infused in 3 patients. Overall the infusions were safe. Seven low grade adverse events possibly related to study participation were reported. The first 2 patients did not demonstrate robust in vivo expansion of GD2.CAR-tvs-CTLs by QPCR and had rapid disease progression. In patient 3 a significant expansion of GD2.CAR-tvs-CTLs, i.e. 18,250 copies/ug genomic DNA was observed on day 7 and cells persisted at 159 copies/ug DNA for up to 12 months (latest measured time point). High pertussis-specific responses were also observed by INF-gamma ELISPOT in this patient starting from day 14 after the vaccination through month 12. Conclusions: We have demonstrated that GD2.CAR T cells expanded and persisted in melanoma patient for up to 12 months. The use of vaccination before blood procurement for T cell manufacture and boosting virus-specific T cell after CAR T cell infusion is a safe strategy and may have helped induced higher transgene levels in one of three patients. Clinical trial information: NCT02482532 .

scholarly journals Distribution Measurements of Chimeric Antigen Receptor-Modified T Cells Against CD19

2020 ◽  
Author(s):  
Zhitao Ying ◽  
Ting He ◽  
Xiaopei Wang ◽  
Wen Zheng ◽  
Ningjing Lin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Target Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Backgroud: The unprecedented efficacy of chimeric antigen receptor (CAR) T-cell immunotherapy of CD19+ B-cell malignancies has opened a new and useful way for the treatment of malignant tumor. Nonetheless, there are still formidable challenges in the field of CAR-T cell therapy, such as the biodistribution of CAR-T cells in vivo.Methods: We demonstrated the distribution of CAR-T cells in the absence of target cells or with target cells in the mice and the dynamic changes in the patient blood over time after infusion were deteced by qPCR and FACS. Results: CAR-T cells still proliferated in the mice without target cells and peaked at 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at 6 weeks. In the clinical trial, we found that CAR-T cells peaked at 7-21days after infusion and can last for as long as 510 days in the peripheral blood of patients. Simultaneously, mild side-effects were noted which can be effectively controlled within two months in these patients.Conclusions: CAR-T cells can expand themselves with or without target cells in mice. CAR-T cells can persistence for a long time in patients.

Chimeric Antigen Receptor T Cell Immunotherapy for Tumor: A Review of Patent Literatures

2019 ◽  
Vol 14 (1) ◽  
pp. 60-69
Author(s):  
Manxue Fu ◽  
Liling Tang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Limiting Factors ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Background: Chimeric Antigen Receptor (CAR) T cell immunotherapy, as an innovative method for tumor immunotherapy, acquires unprecedented clinical outcomes. Genetic modification not only provides T cells with the antigen-binding function but also endows T cells with better immunological functions both in solid and hematological cancer. However, the CAR T cell therapy is not perfect because of several reasons, such as tumor immune microenvironment, and autologous limiting factors of CAR T cells. Moreover, the safety of CAR T cells should be improved.Objective:Recently many patents and publications have reported the importance of CAR T cell immunotherapy. Based on the patents about CAR T cell immunotherapy, we conclude some methods for designing the CAR which can provide information to readers.Methods:In this review, we collect recent patents and publications, summarize some specific antigens for oncotherapy from patents and enumerate some approaches to conquering immunosuppression and reinforcing the immune response of CAR T cells. We also sum up some strategies for improving the safety of CAR T cell immunotherapy.Results:CAR T cell immunotherapy as a neotype cellular immunotherapy has been proved effective in oncotherapy and authorized by FDA. Improvements in CAR designing enhance functions of CAR T cells.Conclusion:This review, summarizing antigens and approaches to overcome defects of CAR T cell immunotherapy from patents and publications, might contribute to a broad readership.

scholarly journals Chimeric Antigen Receptor T Cell Exhaustion during Treatment for Hematological Malignancies

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Chunyi Shen ◽  
Zhen Zhang ◽  
Yi Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Hematological Malignancies ◽  
Antigen Receptor ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Immunotherapy, especially based on chimeric antigen receptor (CAR) T cells, has achieved prominent success in the treatment of hematological malignancies. However, approximately 30-50% of patients will have disease relapse following remission after receiving CD19-targeting CAR-T cells, with failure of maintaining a long-term effect. Mechanisms underlying CAR-T therapy inefficiency consist of loss or modulation of target antigen and CAR-T cell poor persistence which mostly results from T cell exhaustion. The unique features and restoration strategies of exhausted T cells (Tex) have been well described in solid tumors. However, the overview associated with CAR-T cell exhaustion is relatively rare in hematological malignancies. In this review, we summarize the characteristics, cellular, and molecular mechanisms of Tex cells as well as approaches to reverse CAR-T cell exhaustion in hematological malignancies, providing novel strategies for immunotherapies.

scholarly journals HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Matthew T. Ollerton ◽  
Edward A. Berger ◽  
Elizabeth Connick ◽  
Gregory F. Burton
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Follicular Dendritic

ABSTRACT The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4+ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought. IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4+ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4+ T cells and the FDC reservoir in vitro. Although CAR-T cells eliminated CD4+ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

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