Targeting Leukemias by CD123 Specific Chimeric Antigen Receptor

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1908-1908
Author(s):  
Radhika Thokala ◽  
Harjeet Singh ◽  
Simon Olivares ◽  
Richard Champlin ◽  
Laurence J N Cooper
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Lymphoblastic Leukemia ◽  
Antigen Receptor ◽  
Leukemic Cell ◽  
Sleeping Beauty ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Abstract 1908 Chimeric antigen receptors (CARs) are employed to genetically modify T cells to redirect their specificity to target antigens on tumor cells. Typically a second generation CAR is derived by fusing an extracellular domain derived from the scFv of monoclonal antibody (CAR) specific to targeted antigen with CD3 zeta, and CD28 endodomains. CD123 (IL3RA) is expressed on 45% to 95%of acute myelogenous leukemia (AML) and B-cell lineage acute lymphoblastic leukemia (B-ALL). Expression of CD123 is high in the leukemic stem cell (LSC) population, but not in normal hematopoietic stem cells. Thus, CD123 appears to be potential target for immunotherapy in leukemias through chimeric antigen receptor (CAR). We hypothesized that the generation of CD123 specific CAR can redirect the specificity of T cells to CD123 and this was tested by cloning the scFv of CD123 mAb in our CAR construct. The sleeping beauty system was used to express the CAR and DNA plasmids were electroporated into peripheral blood mononuclear cells and cells were numerically expanded on artificial antigen presenting cells genetically modified to express co stimulatory molecules CD86, 4-1BBL, membrane-bound IL-15, and CD123 antigen in presence of IL-21 and 1L–2. CAR+ T numerically expanded to clinically relevant numbers and showed antigen specific cytotoxicity in leukemic celllines. CAR+ T cells expressed both effector and memory markers showing the potential for in vivo persistence after T cell infusion. The bonemarrow homing receptor CXCR4 was expressed by CAR T cells shows the potential to target LSC that reside in BM niches. The preliminary data suggests that mirroring an approach we are using to manufacture clinical grade CD19 specific CAR+ T cells.Figure 1:(A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.Figure 1:. (A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.CD3CD3 Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Development of a qPCR Assay for the Evaluation of the Dendritic Cell Chimeric Antigen Receptor Transcriptome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5895-5895
Author(s):  
Yan Leyfman ◽  
Anthony Daniyan ◽  
Renier J. Brentjens
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Qpcr Assay ◽  
Tumoricidal Activity ◽  
Intracellular Domain ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Chronic lymphocytic leukemia (CLL) is a hematological malignancy of B cells that accounts for a third of all new leukemia cases, but is incurable with standard first-line chemotherapies. Hematopoietic stem cell transplants offer the only curative treatment but are restricted to those with a matching donor. The recent advent of adoptive transfer therapy utilizing Chimeric Antigen Receptor (CAR) T cells, which are synthetic receptors that combine the target-specificity of antibodies to the effector capabilities of T cells, have emerged as a promising anticancer therapy due to its targeted tumoricidal activity. Clinical trials utilizing CD19-targeted CAR T cells have demonstrated strong efficacy against acute B-cell leukemia, but those performed in CLL have not been as successful for unclear reasons. This may be due to the dysfunctional tumor microenvironment which renders key immune effectors ineffective. Dendritic cells (DCs), a potent antigen presenting cell that functions as a bridge between the innate and adaptive immune systems, are rendered functionally and quantitatively deranged with an impaired ability to present antigen and thereby stimulate immunogenic responses. These limitations can potentially be rectified through an effective recruitment of an endogenous antitumor immune response and thus we hypothesize that genetically modified DCs should be capable of repolarizing the tolerogenic microenvironment induced by CLL toward one of immunogenicity to promote tumor eradication. In this study, we developed a qPCR assay to assess the DC CAR transcriptome. Because our CD19-targeted DC CARs with a CD40 intracellular domain signal via NF-κB to activate downstream elements, we needed to first assess the fidelity of our assay. Using the immortalized mouse DC cell line JAWS II, which lacks CD40, we used a potent TLR4 agonist, MPL, to stimulate our DCs through MyD88, an alternative pathway that also activates NF-κB. We saw elevated expression of downstream NF-κB elements, including IL-6, IL-10, IRF3, and IRF7. Establishing the sensitivity of our assay, we next evaluated the transcriptome of DC CARs by coculturing our CD19-targeted DC CARs containing a CD40 intracellular domain with HEK 293 cells, a human embryonic kidney cell line, retrovirally transduced with a human CD19 antigen to activate our CAR DCs and assess downstream gene expression. We observed expected elevation of downstream NF-κB elements including IL-6, IL-10, NOS2, IRF3, IFN-γ, TNFα, IL12p40, and TGF-β, indicating the viability of this assay. These positive results confirm the multidisciplinary potential of our assay as a promising technology that will allow us to better augment future CARs. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals The Impact of the Immunophenotyping Characteristics of Patients' Peripheral Blood on the Manufacturing and Clinical Outcome of CD7-Targeted Chimeric Antigen Receptor T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3830-3830
Author(s):  
Mingzhi Zhang ◽  
Xiaorui Fu ◽  
Huimin Meng ◽  
Min Wang ◽  
Yu Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Significant Difference ◽  
Car T ◽  
The Impact

Abstract Background T cell acute lymphoblastic leukemia and T cell acute lymphoblastic lymphoma (T-ALL/LBL) is a highly aggressive hematological tumor characterized by immature lymphoblasts invading the bone marrow. Treatment options for patients with T-ALL/ LBL are currently limited. Chimeric antigen receptor (CAR)- T cell therapy has opened an era in the treatment of B-cell malignancies. However, the development of CAR-T therapy for T-ALL/LBL faces many challenges. One of them is that therapeutic targets are usually expressed on both tumor and normal T cells, which causes the potential risk of "cell fratricide". Therefore, the difficulty of manufacturing CAR T cells for T-ALL/LBL is dramatically increased. CD7, is 40kD membrane-bound glycoprotein majorly expressed on peripheral T-cells and NK cells and their precursors. CD7 is highly expressed in almost all T-ALL/LBL and considered to be one of the most promising targets for T-ALL/LBL treatment. Patients and Methods This study is based on a phase I clinical trial (NCT04004637) for patients with relapse/refractory CD7 + NK/T cell lymphoma and T-ALL/LBL. To manufacture CAR-T cells, the peripheral blood mononuclear cells (PBMC) were collected from the patients who met the enrollment criteria. The proportion of viability and the ratio of the T cell markers were analyzed. Subsequently, the isolated T cells were co-transduced with CD7 protein expression blocker (PEBL) and CD7-CAR lentiviruses to obtain CD7-CAR-T cells, which can avoid the fratricide of CD7-CAR-T cells. Before the infusion, the phenotypic characteristics and cytotoxicity of CD7-CAR-T products were analyzed. Then peripheral blood (PB) of patients was collected regularly after receiving treatment to analyze the immunophenotyping of T cells. Results From August 2019 to June 2021, 24 leukopaks from patients with CD7-positive T-ALL/LBL were collected, and a total of 32 batches of CD7-CAR T cells were manufactured, with a 78.13% (25/32) successful rate. Among the 7 batches of failure cases, one patient had undergone blood collection twice and CAR-T preparation for three times, but all of three attempts failed (brown icon). Another four patients failed to prepare once. Eight patents were recruited for CD7-CAR-T treatment and 87.5% of complete remission (CR) rate was achieved (7/8), of which a patient (P4, blue icon) has been maintaining CR for more than 15 months. Two other patients, P7 (red icon) and P8 (light red icon), had CD7 - relapse at the time of 6th month and 3rd month after CR, respectively. We divided all samples into successful preparation group (GS), infusion group (GI) and preparation failure group (GF). As shown in Fig. 1A, all three groups exhibited good viability of PBMC. There was no significant difference between GS and GF, but GI was higher than that of GF. The proportion of CD3 + cells in PBMC of GS was significantly lower than that of GF, and GI also showed this feature. Meanwhile, GS and GI both have a higher CD4 +/CD8 + ratio compared with GF. The immunophenotyping results showed CD7-CAR-T products had a majority of the central memory subsets (T CM; 69.41 ± 10.71%) and effect memory subsets (T EM; 28.56 ± 10.19%), with limited number of effector T cell (T E) and naive T cells (T N) (Fig. 1B). The percentage of CAR +CD8 +CD27 + and CD4 +CD25 +CD127 - subsets associated with T cells activation and proliferation, as well as CD223 + and CD279 + subsets related to T cells suppression and exhaustion were lower, except for CD366 + subgroup that also indicated depletion signal (Fig. 1B). In addition, CD7-CAR-T cells showed strong cytotoxicity against CEM (CD7 +) tumor cells accompanied by the release of cytokines, in which the level of IL-2 is extremely low (Fig. 1C). Subsequently, we performed statistics on the proportion of CD3 + and CD4 +/CD8 + cells in the PB of patients after infusion. The proportion of CD3 + cells in the PB of the P4 has been maintained at a high level, and the ratio of CD4 +/CD8 + keeps low (Fig. 1D). P7 showed a significant decrease in the amount of T cells on the 60th day after CAR T infusion, while the ratio of CD4 +/CD8 + showed an upward trend. Conclusion The results indicate that the success rate of CD7-CAR-T manufacturing is positively correlated with higher viability, lower CD3 + and higher CD4 + of PBMC. There was no significant difference among P4 (CR more than 15 months), P7 (CD7 - relapse at 6 th month after CR) and P8 (CD7 - relapse at 3rd month after CR). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas

Blood ◽  
2018 ◽  
Vol 132 (14) ◽  
pp. 1495-1506 ◽  
Author(s):  
Irene Scarfò ◽  
Maria Ormhøj ◽  
Matthew J. Frigault ◽  
Ana P. Castano ◽  
Selena Lorrey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Lymphoblastic Leukemia ◽  
Antigen Receptor ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
T Cell Lymphomas ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A124-A124
Author(s):  
Letizia Giardino ◽  
Ryan Gilbreth ◽  
Cui Chen ◽  
Erin Sult ◽  
Noel Monks ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Single Chain ◽  
High Affinity ◽  
Car T Cells ◽  
Car T ◽  
Animal Care ◽  
Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

scholarly journals Monoclonal antibodies and chimeric antigen receptor (CAR) T cells in the treatment of colorectal cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ke-Tao Jin ◽  
Bo Chen ◽  
Yu-Yao Liu ◽  
H uan-Rong Lan ◽  
Jie-Ping Yan
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
Immune Responses ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Therapeutic Approaches ◽  
Great Ability ◽  
Car T Cells ◽  
Car T

AbstractColorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer deaths worldwide. Besides common therapeutic approaches, such as surgery, chemotherapy, and radiotherapy, novel therapeutic approaches, including immunotherapy, have been an advent in CRC treatment. The immunotherapy approaches try to elicit patients` immune responses against tumor cells to eradicate the tumor. Monoclonal antibodies (mAbs) and chimeric antigen receptor (CAR) T cells are two branches of cancer immunotherapy. MAbs demonstrate the great ability to completely recognize cancer cell-surface receptors and blockade proliferative or inhibitory pathways. On the other hand, T cell activation by genetically engineered CAR receptor via the TCR/CD3 and costimulatory domains can induce potent immune responses against specific tumor-associated antigens (TAAs). Both of these approaches have beneficial anti-tumor effects on CRC. Herein, we review the different mAbs against various pathways and their applications in clinical trials, the different types of CAR-T cells, various specific CAR-T cells against TAAs, and their clinical use in CRC treatment.

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