scholarly journals The Application of CAR-T Cells in Haematological Malignancies

2020 ◽  
Vol 68 (6) ◽  
Author(s):  
Katarzyna Skorka ◽  
Katarzyna Ostapinska ◽  
Aneta Malesa ◽  
Krzysztof Giannopoulos
Keyword(s):  
T Cells ◽  
Mechanism Of Action ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
Haematological Malignancies ◽  
Treatment Regimens ◽  
Car T Cells ◽  
Car T

AbstractChimeric antigen receptor (CAR)-T cells (CART) remain one of the most advanced and promising forms of adoptive T-cell immunotherapy. CART represent autologous, genetically engineered T lymphocytes expressing CAR, i.e. fusion proteins that combine components and features of T cells as well as antibodies providing their more effective and direct anti-tumour effect. The technology of CART construction is highly advanced in vitro and every element of their structure influence their mechanism of action in vivo. Patients with haematological malignancies are faced with the possibility of disease relapse after the implementation of conventional chemo-immunotherapy. Since the most preferable result of therapy is a partial or complete remission, cancer treatment regimens are constantly being improved and customized to individual patients. This individualization could be ensured by CART therapy. This paper characterized CART strategy in details in terms of their structure, generations, mechanism of action and published the results of clinical trials in haematological malignancies including acute lymphoblastic leukaemia, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia and multiple myeloma.

scholarly journals Automated Generation and Phenotypic Characterization of Clinical Grade CD19 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1675-1675
Author(s):  
Ashish Sharma ◽  
Anne Roe ◽  
Filipa Blasco Lopes ◽  
Ruifu Liu ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Central Memory ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown enormous promise in the treatment of certain B cell malignancies. Access to treatment is still limited due to a variety of issues, including pricing and centralized manufacturing models. Generation of CAR-T cells using an automated platform, followed by rigorous functional phenotyping, may contribute to the development of a robust long-lasting therapy. METHODS: Here, we used the Miltenyi Prodigy (Miltenyi Biotech, Bergisch Gladbach, Germany) to automate the process of manufacturing genetically manipulated T cells in a closed system. The system obviates the need for clean room infrastructure. We tested the feasibility of utilizing the Miltenyi Prodigy to manufacture CAR-T cells using a CD19 scFV vector with the 4-1BB co-stimulatory domain. (Lentigen Technology, Inc, Gaithersburg, MD). The purity, differentiation capacity and effector function of the enriched CAR-T cells was studied using high-dimensional flow cytometry. Finally, the functional potential of these cells was tested in vitro and by treating NOD-SCID-gamma (NSG) mice infused with B cell lymphoma cells (Raji B cell), with the CAR-T cells. RESULTS: Starting with 1 x 108 CD4 and CD8 cells from donor apheresis products, CAR-T cells were expanded for 12 days in culture media containing with TransAct (Miltenyi Biotech), IL7 and IL15. The mean fold-expansion at day 12 was 44 ± 5.6, range 39-50 (n=3). The mean transduction efficiency of CAR-T vector was 20%, range 10-25% (n=3), which is similar to other reported methods. The CD19 CAR-T product was enriched in both the CD4 and CD8 T cells subsets, and showed high-level of cytotoxicity against CD19+ cell lines in vitro and in vivo (Figure 1: Mice treated with the CD19-CAR T demonstrated a marked reduction in disease burden as compared to T cell control as measured by bioluminescence imaging and flow cytometric analysis). The CAR-T product was enriched in cell subsets with both effector (CD27-CCR7-; ~20% of total cells) and central memory phenotypes (CD27+CCR7+; ~30% of total T cells). The effector CD4 and CD8 T cells showed increased expression of major functional T cell differentiation transcription factors (i.e. T-bet and GATA3) critical for the development of anti-tumor responses. Whereas, the central CD4 and CD8 T cells were enriched for the expression of TCF7 (a stemness related member of the WNT signaling known to increase longevity of these cells). The frequencies and phenotypes of these cells were maintained in peripheral blood of NSG mice infused with B cell lymphoma cells (Raji B cells), 1 week after treatment. A significant expansion of CD8+ effector T cells and a dramatic reduction in tumor burden was observed over the next 4 weeks in all major organs. Interestingly, we observed that smaller proportion of central-memory like cells (with higher TCF7 levels) continued to persist 6 weeks post-treatment, potentially contributing to a long-lived recallable response. Based on these data we have initiated a phase 1 clinical trial to test the therapeutic potential of the CAR-T product in patients with advanced/refractory B cell lymphoma. The first clinical grade manufacturing run resulted in a CD19 + cell yield of 1.4 x109. CONCLUSION: Our data highlight that the automated CAR-T generation platform (i.e. Miltenyi Prodigy) is effective at the generating purified functionally competent CAR-T cells. Further, findings from our phenotyping analyses show that the CAR-T product is enriched in both effector and central memory subsets and is effective at tumor clearance in vivo. Thus far, we have treated one patient with CD19 CAR-T manufactured on this platform and 2 more have been enrolled. Though this initial study is based on CD19 CAR-T cells, the approach described here could easily be utilized to genetically engineer T cells with gene constructs that are more relevant for specific cancers and infectious diseases. Disclosures No relevant conflicts of interest to declare.

Immunomodulatory Agent Lenalidomide Enhances Antitumor Functions of Chimeric Receptor-Modified T Cells in Vitro and in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 805-805 ◽  
Author(s):  
Otáhal Pavel ◽  
Dana Prukova ◽  
Vlastimil Král ◽  
Radek Jaksa ◽  
Lucie Lateckova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Chimeric Receptor ◽  
Car T Cells ◽  
Car T

Abstract Tumor immunotherapy based on the use of Chimeric Receptor Modified T cells (CAR T cells) is a promising approach for the treatment of a refractory hematological cancer. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable. In order to enhance the effectiveness of CAR-based immunotherapy we tested the immunoadjuvant properities of lenalidomide in combination with CAR19 T cells in a mouse model of B cell lymphoma. CAR19 construct which was used is composed of anti-CD19scFv joined with signaling domain of 4-1BB and TCR zeta and was delivered into T cell via lentiviral transduction. Lenalidomide is an immunomodulatory drug used for the treatment of multiple myeloma and selected B-cell malignancies, e.g. mantle cell lymphoma (MCL) or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). However, the precise mechanism of action is not very well understood and it is believed that is mediated by a modulation of activity of E3 ubiquitin ligase cereblon which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors resulting in changes of expression of various receptors on the surface of tumor cells. To test our hypothesis, immunodeficient NSG mice (NOD-SCID-gamma chain null mice) were s.c. transplanted with various human B cells lymphoma cells (MCL or ABC-DLBCL) followed by i.v treatment with CAR19 T cells with or without daily i.p. lenalidomide. First, when we measured the growth of tumors following treatment with CAR19 T cells plus lenalidomide we found that this combination more effectively suppressed growth of s.c. B-NHL tumors than treatment with only CAR19 T cells or only lenalidomide (Figure 1, 1x10e7 Nemo tumor cell s.c., followed with 2 doses of 1x10(7) CAR19 T cells + Lenalidomide daily, tumor weight was measured 14 days after treatment). Additionally, in this experiment lenalidomide significantly enhanced infiltration of residual tumors by CD8+CAR19 T cells (not shown). Next, we tested the response of CAR19 T cells in vitro to B-NHL cells in the presence or, absence of lenalidomide to determine the costimulatory effect of lenalidomide on signaling via CAR, our data show that lenalidomide significantly enhanced functional response of CAR19 T cells following recognition of B cells in vitro which is demonstrated by enhanced production of IFN-gamma and by increased expression of CD69 by CAR19 T cells, interestingly, this effect was seen only if CAR19 T cells but not B-NHL cells were pre-treated with lenalidomide or, when we activated CAR19 T cell with antibody to CAR but not with antibody to CD3. Thus, our data indicate that lenalidomide might work through direct effects on T cells and specifically enhance signaling via CAR. The biochemical events underlying this costimulatory effect of lenalidomide on signaling by CAR are currently being investigated. In summary, our data support the use of lenalidomide for augmentation CAR-based immunotherapy in clinical settings. Figure 1 Figure 1. Disclosures Klener: Cellgene: Research Funding.

scholarly journals TGF-β/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma

2021 ◽  
Vol 22 (16) ◽  
pp. 8706
Author(s):  
Kyung-Eun Noh ◽  
Jun-Ho Lee ◽  
So-Yeon Choi ◽  
Nam-Chul Jung ◽  
Ji-Hee Nam ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Single Chain ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-β1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-β-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals 114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A126-A126
Author(s):  
John Goulding ◽  
Mochtar Pribadi ◽  
Robert Blum ◽  
Wen-I Yeh ◽  
Yijia Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Tumor Immunity ◽  
Immune Cell ◽  
Nk Cell ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2205-2205 ◽  
Author(s):  
Elisa De Togni ◽  
Miriam Y Kim ◽  
Matt L Cooper ◽  
Julie Ritchey ◽  
Julie O'Neal ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Cytotoxic Effects ◽  
Car T Cells ◽  
Human T Cells ◽  
Car T ◽  
Acute Myeloid

Abstract Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML. C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML. We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B). We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells. In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership.

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