scholarly journals Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line

2020 ◽  
Vol 21 (23) ◽  
pp. 9163
Author(s):  
Chung Hyo Kang ◽  
Yeongrin Kim ◽  
Heung Kyoung Lee ◽  
So Myoung Lee ◽  
Hye Gwang Jeong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Single Chain ◽  
Car T Cells ◽  
Car T ◽  
Nk T Cell

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3094-3094 ◽  
Author(s):  
Alena A. Chekmasova ◽  
Holly M. Horton ◽  
Tracy E. Garrett ◽  
John W. Evans ◽  
Johanna Griecci ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Drug Product ◽  
Single Chain ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Targeting T-Cell Receptor β-Constant Domain for Immunotherapy of T-Cell Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 811-811
Author(s):  
Paul Michael Maciocia ◽  
Patrycja Wawrzyniecka ◽  
Brian Philip ◽  
Ida Ricciardelli ◽  
Ayse U. Akarca ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Jurkat Cells ◽  
Car T Cells ◽  
T Cell Lymphomas ◽  
Car T ◽  
Equity Ownership ◽  
T Cell Malignancies

Abstract T-cell lymphomas and leukemias are aggressive, treatment-resistant cancers with poor prognosis. Immunotherapeutic approaches have been limited by a lack of target antigens discriminating malignant from healthy T-cells. While treatment of B-cell cancers has been enhanced by targeting pan B-cell antigens, an equivalent approach is not possible for T-cell malignancies since profound T-cell depletion, unlike B-cell depletion, would be prohibitively toxic. We propose an immunotherapeutic strategy for targeting a pan T-cell antigen without causing severe depletion of normal T-cells. The α/β T-cell receptor (TCR) is a pan T-cell antigen, expressed on >90% of T-cell lymphomas and all normal T-cells. An overlooked feature of the TCR is that the β-constant region comprises 2 functionally identical genes: TRBC1 and TRBC2. Each T-cell expresses only one of these. Hence, normal T-cells will be a mixture of individual cells expressing either TRBC1 or 2, while a clonal T-cell cancer will express TRBC1 or 2 in its entirety. Despite almost identical amino acid sequences, we identified an antibody with unique TRBC1 specificity. Flow cytometry (FACS) of T-cells in normal donors (n = 27) and patients with T-cell cancers (n = 18) revealed all subjects had TRBC1 and 2 cells in both CD4 and CD8 compartments, with median TRBC1 expression of 35% (range 25-47%). In addition, we examined viral-specific T-cells in healthy volunteers, by generation of Epstein Barr virus-specific primary cytotoxic T-cell lines (3 donors) or by identification of cytomegalovirus-specific (3 donors) or adenovirus-specific (5 donors) T-cells by peptide stimulation. We demonstrated similar TRBC1: 2 ratios in viral-specific cells, suggesting that depletion of either subset would not remove viral immunity. Next, using FACS and immunohistochemistry, we showed that TCR+ cell lines (n = 8) and primary T-cell lymphomas and leukemias (n = 55) across a wide range of histological subtypes were entirely restricted to one compartment (34% TRBC1). As proof of concept for TRBC-selective therapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T-cells. After retroviral transduction of healthy donor T-cells, comprising mixed TRBC1/2 populations, 90% of T-cells expressed CAR on the cell surface. No detectable TRBC1 T-cells remained in the culture, suggesting selective depletion of this population. Anti-TRBC1 CAR T-cells secreted interferon-γ in response to TRBC1-expressing target cell lines (p<0.001) or autologous normal TRBC1+ cells (p<0.001), and not TRBC2 cell lines or autologous normal TRBC2 cells. Anti-TRBC1 CAR killed multiple TRBC1 cell lines (p<0.001) and autologous normal TRBC1 cells (p<0.001), and not TRBC2 cell lines or autologous normal TRBC2 cells. These cell-line based findings were confirmed using primary cells from two patients with TRBC1+ adult T-cell leukaemia/lymphoma. We demonstrated specific tumour kill by allogeneic or autologous T-cells in vitro, despite partial downregulation of surface TCR by tumour cells. We developed a xenograft murine model of disseminated T-cell leukemia by engrafting engineered firefly luciferase+ TRBC1+ Jurkat cells in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Bioluminescent imaging and FACS of marrow at 5 days following IV T-cell injection showed that while mice treated with untransduced T-cells progressed, mice receving anti-TRBC1 CAR T-cells had disease clearance (p<0.0001). In a further model, mice were engrafted with equal proportions of TRBC1-Jurkat and TRBC2-Jurkat cells. FACS analysis of bone marrow at 5 days following T-cell injection demonstrated specific eradication of TRBC1 and not TRBC2 tumour by anti-TRBC1 CAR (p<0.001). In summary, we have demonstrated a novel approach to investigation and targeting of T-cell malignancies by distinguishing between two possible TCR β-chain constant regions. Using CART-cells targeting TRBC1 we have demonstrated proof of concept for anti-TRBC immunotherapy. Unlike non-selective approaches targeting the entire T-cell population, TRBC targeting could eradicate a T-cell tumour while preserving sufficient normal T-cells to maintain cellular immunity. Disclosures Maciocia: Autolus: Equity Ownership, Patents & Royalties: TRBC1 and 2 Targeting for the Diagnosis and Treatment of T-cell Malignancies. Philip:Autolus: Equity Ownership. Onuoha:Autolus: Employment, Equity Ownership. Pule:Amgen: Honoraria; Roche: Honoraria; UCL Business: Patents & Royalties; Autolus Ltd: Employment, Equity Ownership, Research Funding.

scholarly journals Normal Myeloid Cells Are Required for Sustained CAR T Cell Activity Against Myeloid Tumor in a Humanized Mouse Model

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 734-734
Author(s):  
Miriam Y Kim ◽  
Matthew L Cooper ◽  
Julie K Ritchey ◽  
Julia Hollaway ◽  
John F. DiPersio
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Activity ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T cells are effective against B cell malignancies and multiple myeloma, but their efficacy has been limited to date for acute myeloid leukemia (AML). We sought to investigate whether there were fundamental differences in targeting B cell antigens as compared to myeloid antigens with CAR T cells, that may shed light on the mechanism of CAR T cell resistance in patients with AML. For these studies, we utilized human CAR T cells targeting CD19 (CART19) and CD33 (CART33), canonical B cell and myeloid cell antigens, respectively. To ensure that the potency of the two CAR constructs were equivalent, we generated dual CD19 and CD33 expressing cell lines, by adding CD33 to Ramos, a CD19+ B lymphoblastic cell line, and adding CD19 to THP-1, a CD33+ myeloid cell line. We confirmed that CART19 and CART33 were equally potent against CD33+Ramos and CD19+THP-1 cells. To investigate the influence of normal hematopoietic cells on CAR T cell behavior, we incubated CD19+THP-1 cells with CART19 and CART33 in the presence of peripheral blood (PB) or bone marrow (BM) mononuclear cells. We found that both PB and BM enhanced tumor clearance to a similar degree for each CAR construct. Additionally, IL-6 was detected in the supernatant of PB or BM co-cultured with CART19 and CART33, and these levels were markedly increased in the presence of tumor cells. Notably, THP-1 cells by themselves produced high levels of IL-6 upon exposure to CAR T cells, likely reflecting the myeloid origin of this cell line, while Ramos cultured with these same CAR T cells did not produce IL-6. We assessed other myeloid cell lines (U937, KG-1, Kasumi-3, Molm13, HL-60, and K562) and also noted IL-6 production when co-cultured with CART33, although the levels were significantly lower than that produced by THP-1. Of note, IL-6 levels were slightly but consistently higher with CART19 than with CART33 in these in vitro assays, which we attribute to the loss of normal myeloid cells from CART33-mediated killing. To study the effects of normal hematopoiesis on human CAR T cell activity in vivo, we injected NSGS mice with human cord blood CD34+ hematopoietic stem cells (HSCs) to generate a human hematopoietic system in these mice, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from T cells of the same cord blood product as the CD34+ cells. We confirmed the expected loss of human CD19+ B cells and CD33+ myeloid cells in the peripheral blood after CART19 and CART33 treatment, respectively. Surprisingly, we found that only CART33 treatment led to elevated plasma human IL-6 levels in this model. We then injected CD19+THP-1 cells to the mice after HSC engraftment, to assess the anti-tumor activity of the CAR T cells and to increase the potential for toxicity. Consistent with our in vitro data, mice with a human hematopoietic system cleared tumor more efficiently than mice without prior HSC engraftment after treatment with CART19 or CART33. However, while we observed mild weight loss and IL-6 elevation in mice after CART19 treatment, this effect was much more pronounced in mice that received CART33. We hypothesized that the presence of antigen on normal myeloid cells both increased the toxicity and decreased the efficacy of CART33, due to a massive release of inflammatory cytokines from myeloid cells in the immediate aftermath of CART33 treatment, followed by loss of the augmentation of CAR T cell activity mediated by myeloid cells in the long term. To test this hypothesis, we engrafted mice with either control HSCs or CD33 KO HSCs, followed by injection of THP-1 and CART33. Only mice with CD33 KO HSCs maintained myeloid cells after CART33, as expected. CD33 KO HSC-engrafted mice exhibited less toxicity after CART33 treatment than mice with control HSCs, in that they did not lose weight or demonstrate elevated IL-6 levels. Furthermore, absence of CD33 on myeloid cells led to enhanced CAR T cell expansion and persistence, that resulted in better long-term tumor control. In summary, our data suggests that targeting myeloid antigens with CAR T cells may be intrinsically self-defeating due to loss of myeloid cells that are required for sustained CAR T cell activity. These studies illuminate the challenges when extending CAR T cell therapy to myeloid malignancies, and highlight the importance of normal myeloid cells in augmenting T cell-based immunotherapies. Figure 1 Figure 1. Disclosures Kim: Tmunity: Patents & Royalties; NeoImmune Tech: Patents & Royalties. Cooper: RiverVest: Consultancy; Wugen: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties; NeoImmune Tech: Patents & Royalties.

scholarly journals Engineered T Cell Receptor-Mimic Antibody, (TCRm) Chimeric Antigen Receptor (CAR) T Cells Against the Intracellular Protein Wilms Tumor-1 (WT1) for Treatment of Hematologic and Solid Cancers

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2155-2155 ◽  
Author(s):  
Sarwish Rafiq ◽  
Tao Dao ◽  
Cheng Liu ◽  
David A. Scheinberg ◽  
Renier J Brentjens
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Primary Cells ◽  
Intracellular Protein ◽  
Car T Cells ◽  
Car T

Abstract Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. Disclosures Dao: Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

scholarly journals CD7 CAR for the Treatment of Acute Myeloid and Lymphoid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4555-4555 ◽  
Author(s):  
Diogo Silva ◽  
Haruko Tashiro ◽  
Madhuwanti Srinivasan ◽  
Malcolm K. Brenner ◽  
Maksim Mamonkin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Leukemia ◽  
Car T Cells ◽  
Car T ◽  
Target Ratio ◽  
Acute Myeloid ◽  
Myeloid Progenitors

Abstract CD7 is a T- and NK-cell marker highly expressed in T cell leukemia and in 30% of AML cases, where its presence correlates with chemoresistance and worse prognosis. We created CD7-directed chimeric antigen receptors (CARs) using single chain variable fragment (scFv) sequences derived from three different CD7-specific mAb clones and evaluated the feasibility and potency of using CD7 CAR T cells for the treatment of the CD7+ hematologic malignancies. Since normal T cells themselves express CD7, their transduction with CD7 CARs limited their expansion in vitro, resulting in approximately 30-fold fewer T cells after 14 days of culture compared to control CAR-transduced cells. To circumvent this problem of fratricide, we used the CRISPR/Cas9 system to disrupt the CD7 gene in T cells prior to retroviral transduction with CD7 CAR. The CD7 gene was successfully disrupted in 90% of T cells, minimizing fratricide of CD7 CAR T cells, and leading to a robust T cell expansion, similar to that of control T cells transduced with an irrelevant CAR (310-fold vs 380-fold expansion after 14 days, respectively). Lack of CD7 did not compromise CAR T cell effector function, as we observed equal cytotoxicity and expansion in CD7+ and CD7- CD19 CAR T cells upon coculture with a CD19+ cell line Raji (P=0.99 and P=0.86, respectively). Expanded CD7-deficient CD7 CAR T cells demonstrated robust cytotoxicity against CD7+ AML cell lines KG-1a and Kasumi-3 and T-ALL cell line CCRF-CEM, resulting on average in a 19-fold expansion of CAR T cells and elimination of malignant cells (92-99.9%) after 7 days of co-culture at a 1:4 initial effector-to-target ratio. This cytotoxicity was CD7-specific, as CD7 CAR T cells showed no activity against a CD7-negative cell line Raji. We also observed potent activity of CD7 CAR T cells against primary AML cells, leading to an 80% reduction in blast counts after 48 hours of co-culture at a 1:1 effector-to-target ratio, compared with control CAR T cells. Moreover, a 5-hour co-culture of CD7 CAR T cells with primary AML blasts and subsequent culture on a methylcellulose medium for 12 days resulted on average in a 26-fold reduction in leukemic colonies; these data suggest that the CD7 CAR T cells can eliminate primitive leukemic progenitors. Because CD7 is expressed on some myeloid progenitors, we assessed the activity of CD7 CAR T cells against normal myeloid precursors. After coculturing cord blood cells with CD7 CAR T cells at a 10:1 effector-to-target ratio and subsequently expanding myeloid colonies on a methylcellulose medium for 14 days, we observed no significant differences in the numbers of monocytic, erythrocytic and granulocytic colonies compared to a coculture with control CAR T cells. Hence, CD7 CAR T cells appear non-toxic to normal myeloid progenitor cells. In summary, we show that CD7 CAR T cells can be efficiently generated and expanded following genomic disruption of the CD7 gene by CRISPR/Cas9. Expanded CD7 CAR T cells produce robust cytotoxic activity against AML and T-ALL cell lines as well as primary AML blasts, and show no toxicity against normal myeloid progenitors. These results demonstrate the potency and support the feasibility of using CD7 CAR T cells for the targeted therapy of acute myeloid and lymphoid leukemia. Disclosures Brenner: Viracyte: Equity Ownership; Cell Medica: Patents & Royalties.

Novel Humanised ROR1 Chimeric Antigen Receptors for the Treatment of Haematological Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3361-3361
Author(s):  
Satyen Harish Gohil ◽  
Marco Della Peruta ◽  
Solange R Paredes-Moscosso ◽  
Micaela Harrasser ◽  
Gordon Weng-Kit Cheung ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lines ◽  
Target Cell ◽  
Reciprocal Relationship ◽  
Target Cells ◽  
Single Chain ◽  
Car T Cells ◽  
Solid Malignancies ◽  
Car T

Abstract Introduction: Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) is a surface antigen expressed on a range of haematological and solid malignancies including Chronic Lymphocytic Leukaemia (CLL). Although expressed during embryogenesis, its virtual absence on normal adult tissues makes it an attractive target for immunotherapy, especially with Chimeric Antigen Receptor modified T-cells (CAR T-cells). We have generated novel fully humanised ROR1CAR constructs for the treatment of CLL and other ROR1 positive malignancies. Results: Following a rat immunisation programme 38 oligloclonal hybridoma clones were single cell sorted and subjected to 5'RACE. Of 13 novel anti-ROR1 antibodies isolated, 10 retained specific binding when cloned into a heavy-linker-light single chain variable fragment (scFv) format. These scFvs in combination with a second generation CAR architecture comprising CD3zeta and 4-1BB demonstrated specific toxicity against ROR1 positive cell lines after T-cell transduction using lentiviral vectors. We found cytotoxicity with ROR1CAR T-cells was dependent on target cell ROR1 density. In order to ensure our screening assays allowed us to select which of the 10 binders was most suitable for targeting primary CLL, we assessed the antigen density of ROR1 and CD19 on CLL cells. Median expression of ROR1 was 2304 molecules/cell (Range 800-4828), compared to CD19, which had a much higher density of 12,583 (Range 5894-23,652). In view of this, subsequent functional assessment was focused on SKW and Jeko1 cell lines with constitutive ROR1 expression at levels similar to CLL cells, as opposed to those transduced to express supra-physiological levels. Our initial optimisation focused on modifying the CAR extracellular spacer region. We demonstrated a reciprocal relationship between cytotoxicity and the distance between T-cells and target cells. This was assessed by using clones that bound either the membrane-distal immunoglobulin domain or a more membrane-proximal frizzled domain of ROR1. The use of an optimum spacer enhanced cytotoxicity of all scFv constructs but yielded two lead candidates: Clones A & F. These showed consistently superior cytotoxicity against target cell lines compared to the other isolated clones. In addition epitope mapping revealed binding sites unique from the previously described rabbit R12 and murine 4A5 anti-ROR1 CAR T-cells. One of the advantages of targeting ROR1 as opposed to CD19 is sparing the normal B-cell compartment from CAR mediated eradication. However this comes with the consequent risk of B-cell mediated immune responses against rat-derived scFvs. To minimise immunogenicity we undertook a humanisation programme and grafted the complementary determining regions (CDR) of the heavy and light chains of Clone A and F into 5 acceptor human germline VH and VL sequences, generating 25 potential scFvs for each. Binding assessment showed seventeen successfully humanised binders for Clone A and three for Clone F. Of these, 5/17 and 3/3 showed activity in a CAR format against target cells. A final selection was made based on specific cytotoxicity, enhanced cytokine secretion (Interleukin-2 and Interferon gamma) and proliferation compared to the parental clones resulting in 2 unique constructs targeting different extracellular domains of ROR1. In addition, we have demonstrated cytotoxicity against a panel of ROR1 positive solid cancer cell lines to demonstrate their wider applicability. Conclusion: ROR1CAR T-cells have the potential to be an effective therapeutic not just for CLL but also Acute Lymphoblastic Leukaemia, Mantle Cell Lymphoma and solid malignancies. We have described the first humanised ROR1 CARs, which target novel epitopes and have proved effective in relevant pre-clinical assays. Although other ROR1 CARs have been described, we believe the unique properties of these constructs merits further investigation and comparison in the preclinical and clinical setting. Disclosures Gohil: UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Paredes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Pule:Roche: Honoraria; UCL Business: Patents & Royalties; Autolus Ltd: Employment, Equity Ownership, Research Funding; Amgen: Honoraria. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.

scholarly journals c-Met-Specific Chimeric Antigen Receptor T Cells Demonstrate Anti-Tumor Effect in c-Met Positive Gastric Cancer

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5738
Author(s):  
Chung Hyo Kang ◽  
Yeongrin Kim ◽  
Da Yeon Lee ◽  
Sang Un Choi ◽  
Heung Kyoung Lee ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) technology has been highlighted in recent years as a new therapeutic approach for cancer treatment. Although the impressive efficacy of CAR-based T cell adoptive immunotherapy has been observed in hematologic cancers, limited effect has been reported on solid tumors. Approximately 20% of gastric cancer (GC) patients exhibit a high expression of c-Met. We have generated an anti c-Met CAR construct that is composed of a single-chain variable fragment (scFv) of c-Met antibody and signaling domains consisting of CD28 and CD3ζ. To test the CAR construct, we used two cell lines: the Jurkat and KHYG-1 cell lines. These are convenient cell lines, compared to primary T cells, to culture and to test CAR constructs. We transduced CAR constructs into Jurkat cells by electroporation. c-Met CAR Jurkat cells secreted interleukin-2 (IL-2) only when incubated with c-Met positive GC cells. To confirm the lytic function of CAR, the CAR construct was transduced into KHYG-1, a NK/T cell line, using lentiviral particles. c-Met CAR KHYG-1 showed cytotoxic effect on c-Met positive GC cells, while c-Met negative GC cell lines were not eradicated by c-Met CAR KHYG-1. Based on these data, we created c-Met CAR T cells from primary T cells, which showed high IL-2 and IFN-γ secretion when incubated with the c-Met positive cancer cell line. In an in vivo xenograft assay with NSG bearing MKN-45, a c-Met positive GC cell line, c-Met CAR T cells effectively inhibited the tumor growth of MKN-45. Our results show that the c-Met CAR T cell therapy can be effective on GC.

EXTH-59. GENERATION OF A THIRD GENERATION CAR T CELL THAT SIMULTANEOUSLY TARGETS WILDTYPE EGFR AND ITS MUTANT ISOFORM EGFRVIII FOR TREATMENT OF GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi176-vi176
Author(s):  
Daniel Wilkinson ◽  
Katherine Ryan ◽  
Vidyalakshmi Chandramohan ◽  
Daniel Landi ◽  
Darell Bigner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Solid Tumors ◽  
Third Generation ◽  
Single Chain ◽  
Car T Cells ◽  
Car T Cell ◽  
Hematological Cancers ◽  
Car T

Abstract Glioblastoma (GBM) is the most aggressive primary brain cancer with a median survival of less than 16 months. This dire prognosis signifies the urgent need for improved treatment options, such as immunotherapy. Chimeric antigen receptor (CAR) T cells have helped revolutionize immunotherapy, achieving considerable success in eliminating hematological cancers but generally failing to control solid tumors. One major hindrance to CAR T cell success in solid tumors is tumor heterogeneity. Tumor-associated or tumor-specific antigens (TAA or TSA, respectively) are rarely expressed by all malignant cells within a tumor. As a specific example in GBM, the most prevalent TSA, EGFRvIII, is present in just 30% of tumors, and then on only 30-50% of cells. Our pre-clinical and clinical experiences with CAR T cells reveal that tumors possessing as few as 5-10% EGFRvIII-negative cells will easily escape EGFRvIII-targeted CARs. Tumor cells that lack EGFRvIII expression often overexpress the wildtype isoform of EGFR (EGFRwt). Notably, EGFR is absent on normal brain. Therefore, a superior approach would be to simultaneously target EGFRvIII and EGFRwt, an approach that would bypass EGFR heterogeneity in EGFRwt/EGFRvIII-expressing tumors. Here, we generated a third generation CAR using the D2C7 single-chain variable fragment (scFv) targeting moiety that recognizes an epitope present in EGFRwt and EGFRvIII. Initial in vitro characterization of D2C7 CAR validated the specificity and function of D2C7 CAR, as it potently killed murine cell lines engineered to express either EGFRwt or EGFRvIII, but not a cell line expressing neither. Concomitant IFN-γ release supported these conclusions. Additionally, D2C7 CAR killed the human-derived GBM cell line U87 and vIII-transfected U87, U87vIII. Importantly, intracranially-administered D2C7 CAR significantly prolonged survival of mice bearing orthotopic U87vIII or U87/U87vIII heterogeneous tumors compared to mock-treated controls. Altogether, these data provide evidence that D2C7 CAR T cells represent a viable therapeutic option for EGFRwt/EGFRvIII heterogeneous tumors.

scholarly journals Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 743
Author(s):  
Aleksei Titov ◽  
Ekaterina Zmievskaya ◽  
Irina Ganeeva ◽  
Aygul Valiullina ◽  
Alexey Petukhov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
Adoptive Immunotherapy ◽  
Γδ T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T

Adoptive cell immunotherapy (ACT) is a vibrant field of cancer treatment that began progressive development in the 1980s. One of the most prominent and promising examples is chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of B-cell hematologic malignancies. Despite success in the treatment of B-cell lymphomas and leukemia, CAR T-cell therapy remains mostly ineffective for solid tumors. This is due to several reasons, such as the heterogeneity of the cellular composition in solid tumors, the need for directed migration and penetration of CAR T-cells against the pressure gradient in the tumor stroma, and the immunosuppressive microenvironment. To substantially improve the clinical efficacy of ACT against solid tumors, researchers might need to look closer into recent developments in the other branches of adoptive immunotherapy, both traditional and innovative. In this review, we describe the variety of adoptive cell therapies beyond CAR T-cell technology, i.e., exploitation of alternative cell sources with a high therapeutic potential against solid tumors (e.g., CAR M-cells) or aiming to be universal allogeneic (e.g., CAR NK-cells, γδ T-cells), tumor-infiltrating lymphocytes (TILs), and transgenic T-cell receptor (TCR) T-cell immunotherapies. In addition, we discuss the strategies for selection and validation of neoantigens to achieve efficiency and safety. We provide an overview of non-conventional TCRs and CARs, and address the problem of mispairing between the cognate and transgenic TCRs. Finally, we summarize existing and emerging approaches for manufacturing of the therapeutic cell products in traditional, semi-automated and fully automated Point-of-Care (PoC) systems.

scholarly journals Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory

2018 ◽  
Vol 115 (46) ◽  
pp. E10898-E10906 ◽  
Author(s):  
Sophie Viaud ◽  
Jennifer S. Y. Ma ◽  
Ian R. Hardy ◽  
Eric N. Hampton ◽  
Brent Benish ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Rest Period ◽  
Cell System ◽  
Cellular Activity ◽  
Dosing Regimen ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the “rest” period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.

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