scholarly journals A single-chain antibody generation system yielding CAR-T cells with superior antitumor function

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshiki Ochi ◽  
Masaki Maruta ◽  
Kazushi Tanimoto ◽  
Fumitake Kondo ◽  
Toshihiro Yamamoto ◽  
...  
Keyword(s):  
T Cells ◽  
Variable Region ◽  
Target Cells ◽  
Single Chain ◽  
Generation System ◽  
Single Chain Antibody ◽  
Car T Cells ◽  
Car T ◽  
Scfv Library

AbstractCancer immunotherapy using T cells redirected with chimeric antigen receptor (CAR) has shown a lot of promise. We have established a single-chain antibody (scFv) generation system in which scFv library-expressing CAR-T cells can be screened appropriately based on their antitumor functions. A variable region library containing the variable and J regions of the human immunoglobulin light or heavy chain was fused with the variable region of a heavy or light chain encoded by an existing tumor-specific antibody to generate a new scFv library. Then, scFv library-expressing CAR-T cells were generated and stimulated with target cells to concentrate the antigen-specific population. Using this system, target-specific recognition of CAR-T cells appeared to be finely tuned by selecting a new variable region. Importantly, we have demonstrated that the newly optimized scFv-expressing CAR-T cells had better proliferation capacity and durable phenotypes, enabling superior reactivity against advanced tumors in vivo in comparison with the original CAR-T cells. Therefore, the optimization of an scFv is needed to maximize the in vivo antitumor functions of CAR-T cells. This system may allow us to adjust an immunological synapse formed by an scFv expressed by CAR-T cells and a target antigen, representing an ideal form of CAR-T-cell immunotherapy.

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

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

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

scholarly journals Bispecific c-Met/PD-L1 CAR-T Cells Have Enhanced Therapeutic Effects on Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Wei Jiang ◽  
Tao Li ◽  
Jiaojiao Guo ◽  
Jingjing Wang ◽  
Lizhou Jia ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Carcinoma Cells ◽  
Therapeutic Effects ◽  
Single Chain ◽  
Antitumor Activities ◽  
Hepatocellular Carcinoma Cells ◽  
Car T Cells ◽  
Car T

T cells expressing chimeric antigen receptors, especially CD19 CAR-T cells have exhibited effective antitumor activities in B cell malignancies, but due to several factors such as antigen escape effects and tumor microenvironment, their curative potential in hepatocellular carcinoma has not been encouraging. To reduce the antigen escape risk of hepatocellular carcinoma, this study was to design and construct a bispecific CAR targeting c-Met and PD-L1. c-Met/PD-L1 CAR-T cells were obtained by lentiviral transfection, and the transfection efficiency was monitored by flow cytometry analysis. LDH release assays were used to elucidate the efficacy of c-Met/PD-L1 CAR-T cells on hepatocellular carcinoma cells. In addition, xenograft models bearing human hepatocellular carcinoma were constructed to detect the antitumor effect of c-Met/PD-L1 CAR-T cells in vivo. The results shown that this bispecific CAR was manufactured successfully, T cells modified with this bispecific CAR demonstrated improved antitumor activities against c-Met and PD-L1 positive hepatocellular carcinoma cells when compared with those of monovalent c-Met CAR-T cells or PD-L1 CAR-T cells but shown no distinct cytotoxicity on hepatocytes in vitro. In vivo experiments shown that c-Met/PD-L1 CAR-T cells significantly inhibited tumor growth and improve survival persistence compared with other groups. These results suggested that the design of single-chain, bi-specific c-Met/PD-L1 CAR-T is more effective than that of monovalent c-Met CAR-T for the treatment of hepatocellular carcinoma., and this bi-specific c-Met/PD-L1 CAR is rational and implementable with current T-cell engineering technology.

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.

scholarly journals Novel CS1 CAR-T Cells and Bispecific CS1-BCMA CAR-T Cells Effectively Target Multiple Myeloma

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1422
Author(s):  
Vita Golubovskaya ◽  
Hua Zhou ◽  
Feng Li ◽  
Robert Berahovich ◽  
Jinying Sun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Ifn Gamma ◽  
Myeloma Cells ◽  
Car T Cells ◽  
Car T ◽  
First Time

Multiple myeloma (MM) is a hematological cancer caused by abnormal proliferation of plasma cells in the bone marrow, and novel types of treatment are needed for this deadly disease. In this study, we aimed to develop novel CS1 CAR-T cells and bispecific CS1-BCMA CAR-T cells to specifically target multiple myeloma. We generated a new CS1 (CD319, SLAM-7) antibody, clone (7A8D5), which specifically recognized the CS1 antigen, and we applied it for the generation of CS1-CAR. CS1-CAR-T cells caused specific killing of CHO-CS1 target cells with secretion of IFN-gamma and targeted multiple myeloma cells. In addition, bispecific CS1-BCMA-41BB-CD3 CAR-T cells effectively killed CHO-CS1 and CHO-BCMA target cells, killed CS1/BCMA-positive multiple myeloma cells, and secreted IFN-gamma. Moreover, CS1-CAR-T cells and bispecific CS1-BCMA CAR-T cells effectively blocked MM1S multiple myeloma tumor growth in vivo. These data for the first time demonstrate that novel CS1 and bispecific CS1-BCMA-CAR-T cells are effective in targeting MM cells and provide a basis for future clinical trials.

scholarly journals Engineered IL-7 Receptor Enhances the Therapeutic Effect of AXL-CAR-T Cells on Triple-Negative Breast Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Zhenhui Zhao ◽  
Yan Li ◽  
Wei Liu ◽  
Xun Li
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Xenograft Model ◽  
Target Cells ◽  
Car T Cells ◽  
Car T

Triple-negative breast cancer (TNBC) is a very aggressive malignant type of tumor that currently lacks effective targeted therapies. In hematological malignancies, chimeric antigen receptor T (CAR-T) cells have shown very significant antitumor ability; however, in solid tumors, the efficacy is poor. In order to apply CAR-T cells in the treatment of TNBC, in this study, constitutively activated IL-7 receptor (C7R) that has been reported is used to enhance the antitumor function of constructed CAR-T cells by ourselves. Using in vitro coincubation experiments with target cells and in vivo antitumor experiments in mice, we found that the coexpressed C7R can significantly improve the activation, cell proliferation, and cytotoxicity of CAR-T cells. In addition, the in vivo experiments suggested that the enhanced CAR-T cells displayed significant antitumor activity in a TNBC subcutaneous xenograft model, in which in vivo, the survival time of CAR-T cells was prolonged. Together, these results indicated that CAR-T cells that coexpress C7R may be a novel therapeutic strategy for TNBC.

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 DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

Cancers ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 54
Author(s):  
Sripathi M. Sureban ◽  
Robert Berahovich ◽  
Hua Zhou ◽  
Shirley Xu ◽  
Lijun Wu ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Strategy ◽  
Cell Surface Expression ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Car T Cells ◽  
Hematological Cancers ◽  
Car T ◽  
Ifn Γ

CAR-T (chimeric antigen receptor T cells) immunotherapy is effective in many hematological cancers; however, efficacy in solid tumors is disappointing. Doublecortin-like kinase 1 (DCLK1) labels tumor stem cells (TSCs) in genetic mouse models of colorectal cancer (CRC). Here, we describe a novel CAR-T targeting DCLK1 (CBT-511; with our proprietary DCLK1 single-chain antibody variable fragment) as a treatment strategy to eradicate CRC TSCs. The cell surface expression of DCLK1 and cytotoxicity of CBT-511 were assessed in CRC cells (HT29, HCT116, and LoVo). LoVo-derived tumor xenografts in NOD Scid gamma (NSG™) mice were treated with CBT-511 or mock CAR-T cells. Adherent CRC cells express surface DCLK1 (two-dimensional, 2D). A 4.5-fold increase in surface DCLK1 was observed when HT29 cells were grown as spheroids (three-dimensional, 3D). CBT-511 induced cytotoxicity (2D; p < 0.0001), and increased Interferon gamma (IFN-γ) release in CRC cells (2D) compared to mock CAR-T (p < 0.0001). Moreover, an even greater increase in IFN-γ release was observed when cells were grown in 3D. CBT-511 reduced tumor growth by approximately 50 percent compared to mock CAR-T. These data suggest that CRC cells with increased clonogenic capacity express increased surface DCLK1. A DCLK1-targeted CAR-T can induce cytotoxicity in vitro and inhibit xenograft growth in vivo.

scholarly journals Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors

2021 ◽  
Vol 9 (4) ◽  
pp. e002173
Author(s):  
Guanmeng Wang ◽  
Xin Zhou ◽  
Giovanni Fucà ◽  
Elena Dukhovlinova ◽  
Peishun Shou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Antigen Expression ◽  
Human Antibody ◽  
Single Chain ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs.MethodsWe used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs.ResultsHuman VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression.ConclusionsFully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

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 105 4–1BB and optimized CD28 co-stimulation enhances function of human mono- and bi-specific third-generation CAR T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A115-A116
Author(s):  
Emiliano Roselli ◽  
Justin Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Kristen Spitler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
The Other ◽  
Target Cells ◽  
Third Generation ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).Abstract 105 Figure 1A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).ConclusionsThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono- and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

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