Bispecific Chimeric Antigen Receptor Research Articles

Targeting Primary Pre-B Cell Acute Lymphoblastic Leukemia and CD19-Negative Relapses Using Trivalent CAR T Cells

B-Acute Lymphoblastic Leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19, have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens.Here, we hypothesize that targeting three distinct leukemia antigens including CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse.We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR), and the second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). All CARs are fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta (ζ) chain (2nd generation). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target (E:T) ratios. TriCAR and SideCAR T cells secreted similar levels of IFN-γ/TNF-α when compared to CD19 CAR T cells suggesting a safety profile similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-γ and TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Finally, we interrogated immune synapse (IS) microcluster formation and actin dynamics during interactions between CAR T cells and primary B-ALL cells by quantitative imaging flow cytometry. All CAR T cells exhibited higher actin polymerization compared to non-transduced T cell controls. TriCAR T cells formed significantly higher number of IS microclusters with different morphologies of actin polymerization compared to CD19 CAR T cells, suggesting distinct remodeling and dynamics of T cell polarization and immunoactivity, when interacting with TXL-2 primary B-ALL cells. TriCAR T cells formed IS microclusters, while CD19 CAR T cells failed to form IS with CD19 knockout TXL-2 cells.In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells with varying antigen profiles and at mitigating CD19-negative relapse. This strategy has the potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. DisclosuresNo relevant conflicts of interest to declare.

Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation

Immunotherapy has raised high expectations in the treatment of virtually every cancer. Many current efforts are focused on ensuring the efficient delivery of active cytotoxic cells to tumors. It is assumed that, once these active cytotoxic cells are correctly engaged to cancer cells, they will unfailingly eliminate the latter, provided that inhibitory factors are in check. T cell bispecific antibodies (TCBs) and chimeric antigen receptors (CARs) offer an opportunity to test this assumption. Using TCB and CARs directed against HER2, here we show that disruption of interferon-gamma signaling confers resistance to killing by active T lymphocytes. The kinase JAK2, which transduces the signal initiated by interferon-gamma, is a component repeatedly disrupted in several independently generated resistant models. Our results unveil a seemingly widespread strategy used by cancer cells to resist clearance by redirected lymphocytes. In addition, they open the possibility that long-term inhibition of interferon-gamma signaling may impair the elimination phase of immunoediting and, thus, promote tumor progression.

Successful treatment of relapsed acute B-cell lymphoblastic leukemia with CD20/CD22 bispecific chimeric antigen receptor T-cell therapy

Successful treatment of relapsed acute B-cell lymphoblastic leukemia with CD20/CD22 bispecific chimeric antigen receptor T-cell therapy

Developing a Novel Anti-CD19/CD20 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cell Therapy for Relapsed/Refractory (r/r) B-Cell NHL

Background: Aiming to improve both the response rate and durability of response while limiting antigen escape of CD-19 following anti-CD-19 CAR-T therapy, C-CAR039 has been developed as a 2nd generation 4-1BB novel bi-specific chimeric antigen receptor T (CAR-T) therapy targeting both CD19 and CD20 antigens. Methods: NCT04317885 is a single arm, single-center, non-randomized phase I clinical trial designed to evaluate the safety and efficacy of C-CAR039 in treatment of relapsed or refractory NHL (r/r NHL) patients. The primary objective of the study is to evaluate incidence and severity of treatment emergent adverse events. The secondary objectives include determining overall response rate (ORR), progression-free survival, and overall survival. Results: In preclinical studies, human T cells transduced with the lentiviral vector encoding C-CAR039in vitroshowed CAR-T proliferation, cytokine production, cytotoxicity to CD19, and CD20 single positive and double positive tumor cells. C-CAR039 can eradicate CD19/CD20 positive tumor cellsin vivobased on our animal model study. A Phase 1 trial was conducted in Shanghai Tongji Hospital in patients with r/r NHL to assess the safety and efficacy of C-CAR039 (NCT04317885). Following apheresis to harvest T cells, C-CAR039 was manufactured and infused as a single intravenous dose after a standard 3-day cyclophosphamide/fludarabine conditioning regimen. C-CAR039 was manufactured in a serum free, semi-automated, and digitally closed system with median vein to vein time of 18 days. The manufacturing success rate was 100%. As of Aug 3, 2020, 16 patients were infused with C-CAR039 with a dose range of 1.0 x 106 to 5.0 x 106 CAR-T cells/kg. 14 patients had at least one-month evaluable safety data and 13 patients (11 DLBCL, 2 FL patients) had one-month or longer efficacy data. C-CAR039 treatment was well tolerated with no grade 3 or higher cytokine release syndrome(CRS) and no neurotoxicity event. Reversible grade 1~2 CRS was observed in 12 (86%) of patients. Cytopenias due to the conditioning regimen were common and reversible. At the one-month evaluation, 12/13 patients showed clinical improvement (ORR=92%) and 11/11 of DLBCL patients responded to the treatment (ORR=100%). Median follow-up was 70 days (range: 35-257 days). The best overall response (BOR) includes 10 complete responses (CRs) and 2 partial responses (PRs). Furthermore, C-CAR039 proliferation and expansion in the peripheral blood positively correlated with tumor regression. Conclusion: C-CAR039 shows promising efficacy and a favorable safety profile in the early clinical trial in patients with r/r NHL. The early clinical efficacy signal is encouraging and compares favorably to anti-CD19 CAR-T therapies. These findings need to be evaluated in more patients with longer follow-up to confirm safety, efficacy and duration of response. Disclosures Huang: Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Zhu:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Yao:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Zhu:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Zheng:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Chen:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Li:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Lan:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Chen:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Guo:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Zhang:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Zheng:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Wang:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Hong:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Ren:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Zhang:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Humphries:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.Yao:Cellular Biomedicine Group Inc:Current Employment, Current equity holder in publicly-traded company.

Bispecific Chimeric Antigen Receptor T Cell Therapy for B Cell Malignancies and Multiple Myeloma.

Simple SummaryIn relapsed, refractory B cell malignancies and multiple myeloma, chimeric antigen receptor (CAR) T cell therapy has represented a major scientific advancement with high response rates and durable responses for many. Nonetheless, target antigen downregulation in tumor cells can lead to poor responses and relapsed disease. Current FDA approved CAR T cell therapies only target a single B-cell specific cell marker. While effective, single targeted CAR T cell therapy can lead selective pressure against the target antigen leading to loss of expression and tumor cell escape. Simultaneous dual antigen targeting CAR therapy has been evaluated in multiple early phase clinical trials in response to these clinical challenges in hopes of improving response rates and preventing relapse. This article discusses the limitations of single targeted CAR T cell therapy, approaches to dual antigen targeting, and the results of early phase trials utilizing dual antigen targeting CAR T cell therapy.Chimeric antigen receptor (CAR) modified T cell therapy offers a targeted immunotherapeutic approach to patients with refractory hematological malignancies. This technology is most advanced in B cell malignancies and multiple myeloma and is rapidly evolving as more data become available regarding clinical efficacy and response durability. Despite excellent initial response rates with single antigen targeting CARs, failure to respond to therapy and relapse due to target antigen downregulation remain clinical challenges. To mitigate immunophenotypic selective pressures, simultaneous dual antigen targeting with bispecific CAR T cells or multiple administration of different populations of CAR T cells may prevent relapse by addressing one resistance mechanism attributed to antigenic loss. This article will review recently published data on the use of dual targeting with CAR T cells from early phase clinical trials aimed at treating B cell malignancies and multiple myeloma.

Abstract 6605: Functional screening of bispecific CAR ectodomains targeting HER2 and GD2 in melanoma

Abstract We describe the development of targeted immunotherapy using a bispecific chimeric antigen receptor (CAR) modified T-cell directed against the tumor associated antigens, human epidermal growth factor receptor 2 (HER2) and the disialoganglioside GD2, surface expressed in a large proportion of melanoma. Melanoma remains the most aggressive of skin cancers with a 5-year survival rate less than 20% in those with metastatic disease. For these patients, immunotherapy has offered an alternative promising approach in the recent years. We designed bispecific CAR molecule ectodomains (n=8) using different permutations of single chain variable fragments (scFv) recognizing HER2 and GD2 in tandem arrangements. HER2 recognizing scFvs were derived from FRP5 and 4D5 (Trastuzumab), and GD2 recognizing scFvs were derived from 14G2a and humanized 3F8. The ectodomain containing the tandem arrangements of two distinct scFvs was joined to a signaling endodomain of the co-stimulatory molecules, CD28 and OX40, and a T-cell receptor zeta-chain. Primary human T cells were successfully transduced using a retroviral system achieving 37-73% efficiency of surface expression among various bispecific CAR constructs, as determined by flow cytometry. The function of bispecific CAR T cells were evaluated in a series of immunoassays against melanoma cell lines with variable levels of HER2 and GD2 expression. T cells expressing CARs with single specificity (HER2 or GD2) and non-transduced T cells from the same donor were used as controls. T cells transduced with bispecific TanCAR molecules proliferated after co-culture with melanoma cells. Upon evaluation of the cytokine release after encountering melanoma cells, 14G2a/FRP5 derived bispecific CAR T cells demonstrated the highest level of interferon-γ release with 14G2a-FRP5 (distal-proximal) tandem arrangement exhibiting the most robust activation. Further, consistent cytotoxicity against melanoma cells was observed in 4-hour 51Cr-release assay across donors for T cells expressing CAR ectodomain with 4D5-14G2a, 14G2a-FRP5, FRP5-14G2a and hu3F8-FRP5 tandem arrangements. Of these, T cells expressing 14G2a-FRP5, FRP5-14G2a and hu3F8-FRP5 tandem ectodomain arrangements demonstrated long-term tumor control in an impedance based in vitro tumor cell killing assay. In conclusion, we have rationally designed and generated HER2/GD2 bispecific CAR T cells with the intent of targeting the antigenic heterogeneity in melanoma. We have successfully screened multiple iterations of bispecific CAR ectodomains against melanoma demonstrating that the scFv as well as their sequence of tandem arrangement influences the antitumor function of modified T cells. The antitumor function of the top three candidate bispecific ectodomains identified will be further evaluated in pre-clinical animal models to determine the optimal construct for clinical development. Citation Format: Sujith K. Joseph, Shoba A. Navai, Zakaria Grada, Kristen Fousek, Pretty R. Mathew, Ankita Shree, Amanda Wakefield, Nabil Ahmed, Meenakshi Hegde. Functional screening of bispecific CAR ectodomains targeting HER2 and GD2 in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6605.

A Trispecific Anti-HIV Chimeric Antigen Receptor Containing the CCR5 N-Terminal Region.

Anti-HIV chimeric antigen receptors (CARs) promote direct killing of infected cells, thus offering a therapeutic approach aimed at durable suppression of infection emerging from viral reservoirs. CD4-based CARs represent a favored option, since they target the essential conserved primary receptor binding site on the HIV envelope glycoprotein (Env). We have previously shown that adding a second Env-binding moiety, such as the carbohydrate recognition domain of human mannose-binding lectin (MBL) that recognizes the highly conserved oligomannose patch on gp120, increases CAR potency in an in vitro HIV suppression assay; moreover it reduces the undesired capacity for the CD4 of the CAR molecule to act as an entry receptor, thereby rendering CAR-expressing CD8+ T cells susceptible to infection. Here, we further improve the bispecific CD4-MBL CAR by adding a third targeting moiety against a distinct conserved Env determinant, i.e. a polypeptide sequence derived from the N-terminus of the HIV coreceptor CCR5. The trispecific CD4-MBL-R5Nt CAR displays enhanced in vitro anti-HIV potency compared to the CD4-MBL CAR, as well as undetectable HIV entry receptor activity. The high anti-HIV potency of the CD4-MBL-R5Nt CAR, coupled with its all-human composition and absence of immunogenic variable regions associated with antibody-based CARs, offer promise for the trispecific construct in therapeutic approaches seeking durable drug-free HIV remission.

Enhanced cytotoxicity against solid tumors by bispecific antibody-armed CD19 CAR T cells: a proof-of-concept study

Although adoptive cell therapy with chimeric antigen receptor (CAR)-engineered T cells has shown durable clinical efficacy in patients with CD19+ B cell malignancies, the application of this approach to solid tumors is challenging. The goal of this proof-of-concept study was to investigate whether loading of CD19-CAR T cells (CART19) with anti-HER2 or anti-EGFR bispecific antibodies (BiAb) will target HER2+/EGFR+ CD19- targets and signal the intracellular domain of CAR without engaging antigen-specific CD19 ScFv of CAR T cells. We used CART19 armed with anti-CD3 (OKT3)×anti-HER2 BiAb (HER2Bi) or anti-CD3 (OKT3)×anti-EGFR BiAb (EGFRBi) to evaluate the cytotoxicity directed at HER2 or EGFR expressing cancer cell lines compared with unarmed CART19 measured by short-term 51Cr release assay and long-term real-time cell analysis usingxCelligence. We also determined the differences in exhaustion or effector phenotypes and cytokine profiles during the short- and long-term cytotoxicity assays. Specific cytotoxicity was exhibited by CART19 armed with HER2Bi or EGFRBi against multiple tumor cell lines. Armed CART19 and armed activated T cells (ATC) showed comparable specific cytotoxicity that ranged between 10 and 90% against breast, pancreatic, ovarian, prostate, and lung cancer cell lines at 10:1 E/T ratio. Serial killing (repeated killing) by HER2Bi-armed CART19 ranged between 80 and 100% at 10:1 E/T ratio against MCF-7 cells up to 19days (up to 4th round of repeated killing) measured by a real-time cell analysis without CART19 becoming exhausted. HER2Bi- or EGFRBi-armed CART19 exhibited specific cytotoxicity against multiple HER2+/EGFR+/CD19- tumor targets in overnight and long-term serial killing assays. CART19 showed improved survival and were resistant to exhaustion after prolonged repeated exposure to tumor cells.

Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies, yet marked vulnerability to antigen escape and tumor relapse exists. Here we report the rational design and optimization of bispecific CAR-T cells with robust activity against heterogeneous multiple myeloma (MM) that is resistant to conventional CAR-T cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CAR-T cells exhibit superior CAR expression and function compared to T cells that co-express individual BCMA and CS1 CARs. Combination therapy with anti–PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, while CAR-T cell treatment alone achieves durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CAR-T cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

Bispecific CAR T cells for multiple myeloma: natural ligand compared to tandem scFv design

Abstract One of the most promising candidates for Chimeric Antigen Receptor (CAR) T cell therapy beyond CAR19 is treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen (BCMA). However, current reports of BCMA CAR in the clinic have a median progression free survival of 11.8 months, suggesting that targeting BCMA alone may not be sufficient. Evidence suggests patients treated with BCMA-targeted therapies may be thwarted by BCMA negative relapse. We have developed a novel second generation CAR T cell against another multiple myeloma target: transmembrane activator and CAML interactor (TACI). Mice were immunized against TACI and we designed a new scFv targeting TACI based on the resulting antibodies. Anti-TACI CAR T cells are cytotoxic in vitro and in vivo against multiple myeloma. To overcome single antigen loss, we designed bispecific tandem scFv CAR T cells targeting BCMA and TACI. These two antigens share a natural ligand, A Proliferation-Inducing Ligand (APRIL), which has also been used to design a dual-targeting CAR called TriPRIL. We show that these dual targeting CARs, based on tandem scFv or natural ligand design, have similar efficacy against wild type multiple myeloma models. However, this changes in the context of single antigen loss. We have characterized the proliferative (population doublings) and activation capability (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotype (PD-1, TIM-3, LAG-3), with long term exposure to single antigen. Our studies show that sensitivity to antigen density differs between the tandem bispecifics and the natural ligand CAR. These data provide insight into how structural differences between dual-targeting CAR T cells affects their function.

Novel car design reversing the PD-1 signal boosts the efficacy of car-T cells with low cytokine release and no off-target effect

Background & Aim Purpose The immunosuppressive tumor microenvironment (TIME) and cytokine release syndrome (CRS) hinder the progression of chimeric antigen receptor (CAR) T cell therapy. Strategies that block or reverse PD-1/PD-L1 inhibitory signals through CAR structural redesign to overcome TIME are extensively studied but still need be improved. Targeting the widely expressed PD-L1 without increasing the off-tumor effect while reduce cytokines release are critical challenges. Based on the above considerations, we engineered a novel bispecific CAR T cell type targeting CD19 and PD-L1 by incorporating the PD-1 extracellular domain next to the transmembrane domain (TMD) of CD19-targeting CAR domain, which we named EMD.PD-1 CAR. Methods, Results & Conclusion Experimental design In this study, we firstly engineered a novel CD19/PD-L1 bispecific CARs by incorporating the PD-1 domain into CD19 CAR and constructed second-generation CAR as control. Second, to assess the function of the novel anti-CD19 CAR T cells via antigen-specific stimulation, the antitumor functions, cytokine release, and off-target effects were studied in vitro and in vivo. We demonstrated that PD-L1 stimulation alone is not sufficient to activate EMD.PD-1 CAR T cells for the production of a antigen-specific immune response, including specific lysis, cytokines release, and proliferation. When challenged with CD19+PDL1+ tumor cells, EMD.PD-1 CARs showed a stronger specific immune response than 2nd CAR in vitro. Moreover, EMD.PD-1 CAR T cells exhibited only moderate specific cytokine release compared with 2nd CAR T cells when challenged with target tumor cells. In xenograft NOD/SCID/γc−/−(NSG) mouse model bearing CD19+PD-L1+ tumor cells, our results demonstrated that EMD.PD-1 CAR prolonged the overall survival and decreased the tumor burden compared with 2nd CAR. In addition, EMD.PD-1 CAR T cells showed a higher fraction of stem cell memory (TSCM) and central memory (TCM) T cell phenotype and lower PD-L1, TIM-3, CTLA-4, and intrinsic PD-1 expression in flow cytometry, CAR T cells demonstrated decreased differentiation and exhaustion marker expression. In conclusion, when faced with heterogeneous tumors with high expression of immunosuppressive signals, traditional second-generation CAR T cells are insufficient. The novel CAR, called EMD.PD-1 CAR with a low-avidity affinity to PD-L1 and moderate cytokine release can address this critical challenge.

Commentary on Some Recent Theses Relevant to Combating Aging: April 2020.

Theses reviewed in this issue include "Advanced Imaging Technologies for Combined Biomedical Ultrasound and Photoacoustic Imaging," "Engineering Bispecific Chimeric Antigen Receptors to Improve the Efficacy of Adoptive T-Cell Therapy," "Il-36 Gamma Promotes Anti-Tumor Immunity Through Therapeutic Induction of Tumor-Associated Tertiary Lymphoid Structures," "Investigating the Role of Matrix Vesicles During Aortic Valve Interstitial Cell Calcification," "Local Delivery of Cyclosporine and Erythropoietin Promotes Functional Recovery in a Rodent Model of Stroke Injury by Endogenous Tissue Repair," and "Targeting Primary Cilia-Mediated Mechanotransduction to Promote Whole Bone Formation."

Harnessing T Cells to Target Pediatric Acute Myeloid Leukemia: CARs, BiTEs, and Beyond.

Outcomes for pediatric patients with acute myeloid leukemia (AML) remain poor, highlighting the need for improved targeted therapies. Building on the success of CD19-directed immune therapy for acute lymphocytic leukemia (ALL), efforts are ongoing to develop similar strategies for AML. Identifying target antigens for AML is challenging because of the high expression overlap in hematopoietic cells and normal tissues. Despite this, CD123 and CD33 antigen targeted therapies, among others, have emerged as promising candidates. In this review we focus on AML-specific T cell engaging bispecific antibodies and chimeric antigen receptor (CAR) T cells. We review antigens being explored for T cell-based immunotherapy in AML, describe the landscape of clinical trials upcoming for bispecific antibodies and CAR T cells, and highlight strategies to overcome additional challenges facing translation of T cell-based immunotherapy for AML.

Chimeric Antigen Receptor T Cell Therapy for Acute Myeloid Leukemiachimeric Antigen Receptor T Cell Therapy for Acute Myeloid Leukemia

Relapse of leukemic cells that do not express the antigen targeted by chimeric antigen receptor (CAR) is still a risk. As is the potential for targeting hematopoietic stem cells (HSCs) that share the same antigen expression, off-tumor on-target toxicity. Further, CAR T cells that bind different epitopes of the same antigen can have different tumor-killing efficacies. Therefore, we screen murine single chain variable fragment (scFv) based for indirect affinity to identify a CAR that targets Acute myeloid leukemia (AML), while minimizing toxicities. Also, recent advances in CAR design have demonstrated that the requirement of two separate tumor antigens to be ligated by CARs can increase the specificity for tumor targets. So designing a CAR that only activates a T cell when it binds two separate AML antigens will allow T cells to enhance safety. Therefore, we set out to develop a affinity based multi-antigen CAR T cell therapy that targets well described antigens for AML, including CD33 and CD123. Mice were immunized with these antigens, spleens collected, and fused with myeloma cell lines. The antibodies of fused hybridomas were screened for binding and activation against antigens by high throughput flow cytometry. After screening, we derived multiple de novo CD33, and CD123 scFvs by sequencing. We incorporated CD33 and CD123 scFvs into standard mono-specific CARs utilizing a 41BB co-stimulatory domain to validate antigen-specificity. Gene transfer assessment of CAR T cells demonstrated about 50-80% transduction efficiency for CD33 and CD123 scFvs. There were no differences in CD4 and CD8 proportions in these CAR T cells. We next examined the CARs for their cytotoxic ability using a Real-Time Cell Analysis (RTCA) system. For the CD33 CARs, 2 (6A11-1 and 27A3-1) out of 5, and for the CD123 CAR, 2 (15A12-11 and 15 A12-12) out of 8, scFv sequences transduced into T cells were highly efficacious at killing target cells and generated significant amounts of cytokines such as IFN-g, TNF- a, and IL-6. CAR T cells with these same scFv sequences were able to proliferate better in response to targeted antigen. To find the best possible combination of CD33 and CD123 scFvs we double transduced T cells with four selected CD33 or CD123 scFvs each with only one co-stimulation domain either CD3z or 4-1BB in AND gate fashion. Clear differences in cytotoxic ability and cytokine production were observed. We selected 12 combination of CD33/123 CARs bi-specific CARs to evaluate in vitro efficacy, polyfunctionality, and safety. Finally, to find the best combination of CARs that would be less toxic to HSCs, we performed a Colony Forming Unit (CFU) assay with CD34+ bone marrow stem cells and found 5 bi-specific pairs that were less toxic to HSCs . Based on the CFU assay and PSI index, we were able to select the combination of CD33/123 scFvs that would target AML but minimize the killing of HSCs.

Single variable domains from the T cell receptor \u03b2 chain function as mono- and bifunctional CARs and TCRs

Cell therapy using T cell receptors (TCRs) and chimeric antigen receptors (CARs) represents a new wave of immunotherapies garnering considerable attention and investment. Further progress in this area of medicine depends in part on improving the functional capabilities of the engineered components, while maintaining the overall size of recombinant constructs to ensure their compatibility with existing gene delivery vehicles. We describe a single-variable-domain TCR (svd TCR) that utilizes only the variable domain of the β chain (Vβ). This Vβ module not only works in TCR and CAR formats, but also can be used to create single-chain bispecific CARs and TCRs. Comparison of individual ligand-binding Vβ domains in different formats suggests that the lone Vβ sequence controls the sensitivity and a major part of the specificity of the CAR or TCR construct, regardless of signaling format, in Jurkat and primary T cells.

Phase I Trial Using CD19/CD22 Bispecific CAR T Cells in Pediatric and Adult Acute Lymphoblastic Leukemia (ALL)

Phase I Trial Using CD19/CD22 Bispecific CAR T Cells in Pediatric and Adult Acute Lymphoblastic Leukemia (ALL)

IMMU-48. RATIONAL DEVELOPMENT AND CHARACTERIZATION OF MULTIVALENT TARGETING TANDEM AND PARALLEL CAR T CELLS FOR GLIOMA

Abstract High grade glioma is the most common malignant primary brain tumor. Despite the best available therapy, the median survival is limited. Chimeric antigen receptor (CAR)-T cells effectively attack target positive tumor cells, demonstrating a promising possible treatment. Based on our previous clinical and pre-clinical studies, we utilized epidermal growth factor receptor variant III (EGFRvIII) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) targeting single-chain variable fragments (scFvs) to generate bi-specific tandem CAR T cells and bi-specific parallel CAR T cells to decrease the potential of antigen escape and tumor recurrence. 5, 10, 15 or 20 flexible amino acids were utilized to link scFvs in tandem CARs. Ribosomal “skipping” 2A peptides were utilized to generate parallel CAR T cells. Flow-based IL13Rα2 staining on glioma stem cells (GSCs) showed 7 out 11 cases positive (64%) heterogeneous intratumoral expression. Tandem CAR T cells and parallel CAR T cells upregulated CD69 expression (P< 0.0001), generated IFNγ, interleukin 2 and TNFα (P< 0.0001), as well as exhibiting cytotoxic activity (P< 0.0001) when compared against un-transduced T cells, in co-culture with either single- or double-target positive GSCs. There was no significant difference between different lengths of linker in tandem CAR T cells. Parallel CAR T cells generated a larger cytokine response (P< 0.0001) and more effectively killed target positive cells (P< 0.05) than tandem CAR T cells. In summary, glioma associated target expression is heterogeneous in GSCs. Multivalent tandem CAR T cells and parallel CAR T cells effectively respond to target expressing tumor cells. The function of tandem CAR T cells is not relative with the length of linker. Parallel CAR T cells are a more promising strategy in generating gene modified multivalent targeting T cells.

CAR T cell therapy: A new era for cancer treatment (Review).

Cancer has recently been identified as the leading cause of mortality worldwide. Several conventional treatments and cytotoxic immunotherapies have been developed and made available to the market. Considering the complex behavior of tumors and the involvement of numerous genetic and cellular factors involved in tumorigenesis and metastasis, there is a need to develop a promising immunotherapy that targets tumors at both the cellular and genetic levels. Chimeric antigen receptor (CAR) Tcell therapy has emerged as a novel therapeutic T cell engineering practice, in which T cells derived from patient blood are engineered invitro to express artificial receptors targeted to a specific tumor antigen. These directly identify the tumor antigen without the involvement of the major histocompatibility complex. The use of this therapy in the last few years has been successful, with a reduction in remission rates of up to 80% for hematologic cancer, particularly for acute lymphoblastic leukemia (ALL) and non‑Hodgkin lymphomas, such as large B cell lymphoma. Recently, anti‑CD19 CAR therapy, or UCART19, has been shown to be efficacious in treating relapsed/refractory hematologic cancer. Several other cell surface tumor antigens, such as CD20 and CD22, found in the majority of leukemias and lymphomas are considered potential targets by pharmaceutical companies and research organizations, and trials have been ongoing in this direction. Although this therapeutic regimen is currently confined to treating hematologic cancer, the increasing involvement of several auxiliary techniques, such as bispecific CAR, Tan‑CAR, inhibitory‑CAR, combined antigens, the clustered regularly interspaced short palindromic repeats gene‑editing tool and nanoparticle delivery, may substantially improve its overall anticancer effects. CAR therapy has the potential to offer a rapid and safer treatment regime to treat non‑solid and solid tumors. The present review presents an insight into the advantages and the advances of CAR immunotherapy and presents the emerging discrepancy of CAR therapy over usual forms of therapy, such as chemotherapy and radiotherapy.

Bispecific Chimeric Antigen Receptor Targeting Both CD19 and CD22 T Cell Therapy in Adults with Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

Background: Despite the impressive CR induced by CD19 CAR-T cell therapy in B-ALL, the high rate of complete responses is sometimes limited by the emergence of CD19-negative leukemia. Bispecific CAR-modified T cells targeting both CD19 and CD22 may overcome the limitation of CD19-negative relapse. Methods: Here, we report the design of a bispecific CAR that triggers robust cytolytic activity against target cells by targeting either CD19 or CD22. We performed a phase 1 trial of bispecific CAR T cell therapy in patients with relapsed/refractory precursor B-ALL at a dose that ranged from 1.7×106 to 3×106 CAR T cells per kilogram of body weight. Finding: MRD-negative CR was achieved in 6 out of 6 enrolled patients. Autologous CD19/CD22 CAR T cells proliferated in vivo and were detected in the blood, bone marrow, and cerebrospinal fluid. No neurotoxicity occurred in any of the 6 patients treated. Of note, one patient had a relapse with blast cells that no longer expressed CD19 and exhibited diminished CD22 site density approximately 5 months after treatment. Interpretation: In brief, autologous CD19/CD22 CAR T cell therapy is feasible and safe and mediates potent antileukemic activity in patients with relapsed/refractory B-ALL. Furthermore, the emergence of target antigen loss and expression downregulation highlights the critical need to anticipate antigen escape. Our study demonstrates the reliability of bispecific CD19/CD22 CAR T cell therapy in inducing remission in adult patients with relapsed/refractory B-ALL. Trial Registration: NCT03185494. Funding Statement: National Key Research and Development Program of China and National Natural Science Foundation of China. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocols were approved by the institutional review board at the Chinese PLA general hospital, and the patients provided written informed consent. This clinical investigation was conducted according to the principles of the Declaration of Helsinki.

Results of point-of-care manufacturing of bispecific chimeric antigen receptor (CAR) LV20.19 CAR-TCELLS in a phase i study for relapsed/refractory (R/R), non-hodgkin lymphoma (NHL)

Background & Aim Current approved CAR-T cell manufacturing employs off-site production, a labor-intensive process that requires several weeks and involves shipping back and forth to the production site. This production model may limit availability and accessibility to CAR-T cells and delay treatment for patient (pt) s with rapidly progressive disease. To address these limitations, we utilized the Miltenyi CliniMACS Prodigy, a GMP-compliant device, to produce CAR-T cells on-site for a first-in-human bi-specific CD19/20 (LV20.19) CAR-T Phase I trial. Methods, Results & Conclusion CAR-T cells were manufactured within our Cell Therapy Laboratory, an ISO7 facility. Pt's mononuclear cell apheresis was collected and loaded onto the Prodigy to isolate CD4/CD8 T cells. The T cells were suspended in TexMACS supplemented with human AB serum and IL-2, activated with TransACT, and then transduced by LV20.19 the next day. Culture wash and feedings were done during the production process, and the final cell product was harvested at day 14. The process was automated with limited machine/operator interactions. CAR-T expression was assessed by protein L staining. CAR-T cells were either administered fresh or cryopreserved for later infusion. Dose was escalated in a 3+3 manner from 2.5e+5 cells/kg to a target dose of 2.5e+6 cells/kg. CAR-T cells have been successfully generated for 11/11 enrolled pts thus far with no manufacture failures, and all products met release criteria. Median T cell recovery from the apheresis products was 59.5% with a median CD3+ purity of 97.2% (Figure 1). Final product CAR expression was at a median of 17.6%. A median of 4.7e+8 CAR T cells was obtained at harvest, exceeding the required target dose. CAR-T cells contained both CD4 and CD8 T cells with higher CD4 content. The majority of CAR-T cells showed an effector-memory phenotype. CAR-T cells were able to kill CD19+/20+ target cells and produced IFN-gin response to antigens. Among the 11 treated pts 8 received fresh CAR-T cells and 3 received CAR-T cells after cryopreservation. As no DLTs were observed at the 3 dose levels, a dose of 2.5e+6 cells/kg has been selected for further expansion. Clinical response shows 6 pts have achieved a CR at Day +28 with a duration of response between 2 to 14 months. In summary, initial results demonstrated feasibility for point-of-care manufacturing of bispecific LV20.19CAR-T cells with safe and promising efficacy in treating heavily pretreated pts with R/R B-cell NHL, which warrants further investigations.