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.

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.

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.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12 ◽  
Author(s):  
Nikhil Hebbar ◽  
Rebecca Epperly ◽  
Abishek Vaidya ◽  
Sujuan Huang ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Cells ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p<0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p<0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

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.

Construction and Molecular Characterization Of a T-Cell Receptor-Like Antibody and CAR-T Cells Specific For Minor Histocompatibility Antigen HA-1H

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4490-4490
Author(s):  
Yoko Inaguma ◽  
Yasushi Akahori ◽  
Yoshiki Akatsuka ◽  
Yuko Murayama ◽  
Keiko Shiraishi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Immunotherapy ◽  
Conflicts Of Interest ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Minimal Risk ◽  
Single Chain ◽  
Car T Cells ◽  
Car T

Selective graft-versus-tumor (GVT) reactivity with minimal risk of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation is thought to be induced by targeting minor histocompatibility (H) antigens (Ags) expressed only on patients’ hematopoietic cells. Among HLA-A* 02:01 positive patients, minor H Ags such as HA-1 and HA-2 have been shown to be associated with anti-tumor responses with minimal GVHD and explored for application to adoptive immunotherapy. Because preparation of Ag-specific cytotoxic T cell clones (CTLs) or lines for adoptive immunotherapy is labor-intensive and time consuming, the genetic transfer of T-cell receptors (TCRs) directed toward target Ags into T lymphocytes has been used to efficiently generate anti-tumor T cells without the need for in vitro induction and expansion. Alternatively, T cells could be gene-modified with a chimeric antigen receptor (CAR) harnessing a single chain antibody moiety (scFv). The conventional CAR strategy has the limitation of only targeting cell surface Ags on target cells. One possible way to attain intracellular Ag targeting with a CAR is to generate a TCR-like monoclonal antibody (mAb) as a source of scFv. In this study, we sought to generate highly specific mAbs specific for HA-1H minor H Ag by immunizing mice with tetramerized recombinant HLA-A2 incorporating HA-1H minor H Ag peptides and β2-microglobulin (HA-1H/HLA-A2). We hypothesized that the use of HLA-A2 transgenic mice, which should be tolerant to human HLA-A2, would facilitate efficient induction of mAbs specific for peptides presented on HLA-A2. Phage libraries were generated from splenic B cells and screened by panning for clones reactive to plate-bound HA-1H/HLA-A2 in the presence of free MAGEA4/HLA-A2 for competition. Candidate scFv encoded by obtained phage clones were transformed to scFv tetrameric Ab form or introduced into T cells as CAR coupled to CD28 transmembrane and CD3ζ domains (CD28-ζ). A total of 144 clones were randomly selected from 8.1×108 clones that had been recovered after the third panning. Among 144 clones, 18 (12.5%) showed preferential binding to HA-1/HLA-A2, 137 showed similar binding to both pMHC complexes, and 7 showed reactivity to neither of them. One of 18 scFv Abs, clone #131, demonstrated high affinity (KD = 8.34nM) for the HA-1H/HLA-A2 complex. Primary human CD8 T cells transduced with #131 scFv-CD28-ζ were stained with HA-1H/HLA-A2 tetramers as strongly as a CTL clone, EH6, specific for endogenously HLA-A2- and HA-1H-positive cells. Unexpectedly, however, #131 scFv-CD28-ζ CAR-T cells required ∼100-fold higher Ag density when pulsed exogenously to exert cytotoxicity than did the cognate EH6-CTL. In addition, mAb blocking experiments demonstrated that #131 scFv-CD28-ζCAR-T cells were less sensitive to CD8 blockade when they were completely blocked with HA-1H/HLA-A2 tetramer. These data suggest that T cells with higher affinity antigen receptors than TCRs (average KD ranging between 1μM∼100μM) are less able to recognize low density peptide/MHC antigens as reported in the case of affinity-matured TCR or CAR, and that CD8+ CAR-T cells may not be necessarily CD8-dependent possibly due to failure to form complexes with CD3. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distribution of chimeric antigen receptor-modified T cells against CD19 in B-cell malignancies

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhitao Ying ◽  
Ting He ◽  
Xiaopei Wang ◽  
Wen Zheng ◽  
Ningjing Lin ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Target Cells ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Over Time

Abstract Background The unprecedented efficacy of chimeric antigen receptor T (CAR-T) cell immunotherapy of CD19+ B-cell malignancies has opened a new and useful way for the treatment of malignant tumors. 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 NALM-6, a human B-cell acute lymphoblastic leukemia (B-ALL) cell line, was used as target cells. CAR-T cells were injected into a mice model with or without target cells. Then we measured the distribution of CAR-T cells in mice. In addition, an exploratory clinical trial was conducted in 13 r/r B-cell non-Hodgkin lymphoma (B-NHL) patients, who received CAR-T cell infusion. The dynamic changes in patient blood parameters over time after infusion were detected by qPCR and flow cytometry. Results CAR-T cells still proliferated over time after being infused into the mice without target cells within 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at week 6. In the clinical trial, we found that CAR-T cells peaked at 7–21 days after infusion and lasted for 420 days in peripheral blood of patients. Simultaneously, mild side effects were observed, which could be effectively controlled within 2 months in these patients. Conclusions CAR-T cells can expand themselves with or without target cells in mice, and persist for a long time in NHL patients without serious side effects. Trial registration The registration date of the clinical trial is May 17, 2018 and the trial registration numbers is NCT03528421.

scholarly journals 5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5812-5812
Author(s):  
Alla Dolnikov ◽  
Swapna Rossi ◽  
Ning Xu ◽  
Guy Klamer ◽  
Sylvie Shen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Target Cells ◽  
Car T Cells ◽  
Anti Tumour Activity ◽  
Car T ◽  
Pre Treatment ◽  
Tumour Activity

Abstract T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals CAR T cell therapy in B-cell acute lymphoblastic leukaemia: Insights from mathematical models

2020 ◽  
Author(s):  
Odelaisy León-Triana ◽  
Soukaina Sabir ◽  
Gabriel F. Calvo ◽  
Juan Belmonte-Beitia ◽  
Salvador Chulián ◽  
...  
Keyword(s):  
Mathematical Model ◽  
T Cells ◽  
T Cell ◽  
Side Effects ◽  
B Cell ◽  
Target Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractImmunotherapies use components of the patient immune system to selectively target cancer cells. The use of CAR T cells to treat B-cell malignancies – leukaemias and lymphomas– is one of the most successful examples, with many patients experiencing long-lasting complete responses to this therapy. This treatment works by extracting the patient’s T cells and adding them the CAR group, which enables them to recognize and target cells carrying the antigen CD19+, that is expressed in these haematological tumors.Here we put forward a mathematical model describing the time response of leukaemias to the injection of CAR T-cells. The model accounts for mature and progenitor B-cells, tumor cells, CAR T cells and side effects by incorporating the main biological processes involved. The model explains the early post-injection dynamics of the different compartments and the fact that the number of CAR T cells injected does not critically affect the treatment outcome. An explicit formula is found that provides the maximum CAR T cell expansion in-vivo and the severity of side effects. Our mathematical model captures other known features of the response to this immunotherapy. It also predicts that CD19+ tumor relapses could be the result of the competition between tumor and CAR T cells analogous to predator-prey dynamics. We discuss this fact on the light of available evidences and the possibility of controlling relapses by early re-challenging of the tumor with stored CAR T cells.

scholarly journals HIV-1-Specific CAR-T Cells With Cell-Intrinsic PD-1 Checkpoint Blockade Enhance Anti-HIV Efficacy in vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhengtao Jiang ◽  
Huitong Liang ◽  
Hanyu Pan ◽  
Yue Liang ◽  
Hua Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Neutralizing Antibody ◽  
Single Chain ◽  
Functional Cure ◽  
Car T Cells ◽  
Car T ◽  
Broadly Neutralizing Antibody ◽  
Anti Hiv ◽  
Hiv 1

Adoptive cellular immunotherapy therapy using broadly neutralizing antibody-based chimeric antigen receptor-T cells (bNAb-based CAR-T) has shown great potency and safety for the functional cure of HIV. The efficacy of bNAb-based CAR-T cells could be compromised by adaptive resistance during HIV chronic infection according to the phenomenon that cellular exhaustion was observed in endogenous cytotoxic T-lymphocytes (CTLs) along with upregulated expression of PD−1. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a 3BNC117-DNR CAR (3BD CAR) construct that enables the expression of PD-1 dominant negative receptor (DNR) and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). Compared with HIV CAR expression alone, 3BD CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells. Moreover, 3BD CAR-T cells can kill HIV-latently-infected cell lines, which are reactivated by the secretory cytokines of effector cells followed by contact with initial HIV-expressing fraction. Furthermore, bioluminescence imaging indicated that 3BD CAR-T cells displayed superior anti-HIV function in an HIV NCG mouse model of transplanting Env+/PD-L1+ cells (LEL6). These studies suggested that our proposed combinational strategy of HIV CAR-T therapy with PD-1 blockade therapy is feasible and potent, making it a promising therapeutic candidate for HIV functional cure.

scholarly journals Design and in Vitro Evaluation of a CAR-T Prototype (ARI-0003) Targeting CD123 for Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4799-4799
Author(s):  
Alex Bataller Torralba ◽  
Néstor Tirado ◽  
Diego Sánchez-Martínez ◽  
Talía Velasco-Hernández ◽  
Aina Oliver-Caldés ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Target Cell ◽  
In Vitro Assays ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Abstract Introduction The outcome of patients with Acute Myeloid Leukemia (AML) is still dismal, especially in patients with a relapsed or refractory (R/R) disease. In these patients, innovative treatment strategies must be considered in order to improve survival. Chimeric Antigen Receptor T-cell (CART) immunotherapy has demonstrated its efficacy and safety in diverse B-cell neoplasms and therefore is being investigated in other hematological malignancies. In AML, CART development is challenging due to the absence of a universal target antigen across AML subtypes, as well as on-target/off-tumor toxicity in healthy tissues. Among all explored AML antigens, CD123 seems to be a safe antigen to target, given its expresion in a high proportion of bulk and leukemic AML stem cells and with a higher level compared to normal hematopoietic progenitors cells. Herein we detail the generation and the in vitro assays of a CD123 CAR-T model (ARI-0003) for AML. Methods We developed an anti-CD123 antibody-secreting hybridoma and thereafter identified the variable domains of heavy and light chains of the immunoglobulin to create the scFv domain. The CAR (ARI-0003) genetic sequence was designed and cloned into a pCCL plasmid, and together with a VSV-G, RRE and REV plasmids we transfected HEK 293T cells to obtain 3 rd generation lentivirus (Figure 1A). T-lymphocytes were obtained from healthy volunteers and were transduced with lentiviral vectors to generate CAR-T cells. CAR expression in infected T-cells was monitored with flow cytometry using anti-F(ab) 2 antibodies. To test in vitro activity against AML, non-transduced T-lymphocytes (NT) and CAR-T cells were incubated with AML cell lines and AML primary samples from diverse genetic subtypes, and cytotoxicity was evaluated by flow cytometry at 24 and 48 hours using CD123 and CD33 antibodies. Results At the time of co-incubation of target cells and lymphocytes, the mean scFv expression of ARI-0003 in CAR-T cells was 37% (range, 20-57). Cytotoxicity assays with AML cell lines (THP-1, Kasumi1) showed higher target cell mortality with CAR-T cells, compared to NT cells (mean percentage of alive cells at 48h, obtained with an effector-target cell ratio of 1:1, of 0 and 1% [CAR] vs 45 and 57 [NT] in THP-1 and Kasumi1, respectively; Figure 1B-C). This differential cytotoxic activity was maintained using distinct effector ratio, being statistically significant in higher ratios (namely, 1:1, 1:2 and 1:4). Moreover, cytotoxicity induced by CAR-T cells was higher at 48 hours compared to 24 hours. To demonstrate antigen specificity of ARI-0003 CAR-T cells, CD123 negative cell lines (Raji, 697) were used, and target cell mortality was not statistically significant between CAR-T and NT in CD123-negative cell lines. Finally, ARI-0003 CAR-T efficacy was tested against 8 primary AML samples with a distinct genetic risk, including 5 unfavorable cases according to the European LeukemiaNet classification and 3 AML samples refractory to multiple high-intensity regimens (Figure 1F). Interestingly, a significant cytotoxic effect was observed against all 8 samples after incubation with ARI-0003 CAR-T cells at higher E:T ratio (p &lt; 0.05 for ratios 1:1, 1:2 and 1:4; Figure D-E). Conclusions In vitro assays showed that immunotherapy with CAR-T cells ARI0003 against CD123 could be an effective approach for R/R AML. In vivo experiments are needed in order to confirm these results before translating this therapy to clinical phases. Moreover, on-target/off-tumor toxicity induced by ARI-0003 CAR-T needs to be explored, especially myelotoxicity due to target antigen expression in hematopoietic precursor cells. Figure legend A) Structure of the ARI0003 CAR. B) Cytotoxicity of CAR-T cells against cell lines (normalized to background toxicity observed with NT cells). C) Flow cytometry of THP1 cells against NT and CAR-T cells. D) Cytotoxicity of CAR-T cells against AML primary samples (normalized to NT percentage). E) Absolut number of remaining AML cells after 48h of coincubation with CAR or NT cells. F) Characteristics of AML primary samples used for in vitro assays. Figure 1 Figure 1. Disclosures Diaz-Beyá: Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Esteve: Abbvie: Consultancy; Astellas: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Jazz: Consultancy; Novartis: Research Funding.

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