Dual Chimeric Antigen Receptor Research Articles

Abstract LB074: Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies

Abstract Autologous chimeric antigen receptor (CAR)-T therapies have elicited high rates of durable complete responses in B cell lymphomas. Unfortunately, disease progression, manufacturing failures, and prohibitive costs highlight the need for an alternative source of cells that would provide a consistent and readily available therapy for patients. The “Appia Cells Utilized for Allogeneic” (ACUA) platform is an innovative process that differentiates cord blood-derived CD34+ hematopoietic stem and progenitor cells into large numbers of natural killer T (NKT) cells to support off-the-shelf treatment. NKT cells express an invariant T cell receptor (iTCR) and are naturally non-alloreactive, making them an ideal cell type for an allogeneic product. The ACUA platform circumvents the challenge of expanding NKT cells from the periphery where the cells comprise less than 1% of T cells. API-192 is an ACUA-derived, NKT cell product engineered to express dual CARs targeting CD19 and CD20 to prevent tumor antigen escape. At research scale, API-192 is a highly pure product (>90% iTCR+ 19CAR+ 20CAR+) that secretes soluble interleukin-15 for improved expansion and persistence. The transgenic iTCR is not expressed above physiologic levels and upon engagement, API-192 proliferates in response to and is cytotoxic against CD1d+ tumor cells in a ligand-dependent manner. In response to Raji and Nalm6 tumor cells, API-192 secretes pro-inflammatory cytokines, proliferates, and is cytotoxic in a dose- and CAR-dependent manner. API-192 can also control multiple rounds of tumor cell rechallenge in vitro. Using human tumor xenograft mouse models, we show that API-192 expands, controls tumor growth, and prolongs survival. Furthermore, API-192 persists in the periphery, enabling control of a tumor rechallenge at 70 days after initial dosing of effector cells. Indicative of a favorable immunogenic profile, API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less interferon (IFN)-γ release by NK and T cells in a mixed lymphocyte reaction (MLR) assay. To address a theoretical concern that endogenous TCRαβ rearrangement could occur during the ACUA process, we performed genomic DNA based TCR sequencing to quantify the level of TCRα or TCRβ rearrangement and preliminary data suggested negligible levels (<0.2%). Using a MLR assay, we demonstrate that compared to conventional CAR-T cells, API-192 secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with a low graft vs host risk. In an autonomous growth assay, API-192 expands at a low level and subsequently contracts, suggesting a lack of dysregulated growth. Collectively, these results demonstrate the favorable manufacturability, potency, and safety of API-192 and its potential as a novel allogeneic therapy. Citation Format: Lanny Gov, Jiaji Yu, Maha Qubain, Saige L. Pompura, Sean D. Allen, Michael A. Christopher, Jeff Wiezorek. Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB074.

Magnetic CAR T cell purification using an anti-G4S linker antibody

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system

BackgroundA bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy.MethodsWe developed a second-generation CAR directing CD19 and...

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Dual Chimeric Antigen Receptor T Cells Targeting CD38 and SLAMF7 with Independent Signaling Demonstrate Preclinical Efficacy and Safety in Multiple Myeloma.

Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (BCMA) induces high overall response rates. However, relapse still occurs and novel strategies for targeting multiple myeloma cells using CAR T-cell therapy are needed. SLAMF7 (also known as CS1) and CD38 on tumor plasma cells represent potential alternative targets for CAR T-cell therapy in multiple myeloma, but their expression on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such treatments. Here, we used CRISPR/Cas9 deletion of the CD38 gene in T cells and developed DCAR, a double CAR system targeting CD38 and CS1 through activation and costimulation receptors, respectively. Inactivation of CD38 enhanced the anti-multiple myeloma activity of DCAR T in vitro. Edited DCAR T cells showed strong in vitro and in vivo responses specifically against target cells expressing both CD38 and CS1. Furthermore, we provide evidence that, unlike anti-CD38 CAR T-cell therapy, which elicited a rapid immune reaction against hematopoietic cells in a humanized mouse model, DCAR T cells showed no signs of toxicity. Thus, DCAR T cells could provide a safe and efficient alternative to anti-BCMA CAR T-cell therapy to treat patients with multiple myeloma.

HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity

BackgroundOne of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens...

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (<1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (>90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

A second-generation M1-polarized CAR macrophage with antitumor efficacy.

Chimeric antigen receptor (CAR) T cell therapies have successfully treated hematological malignancies. Macrophages have also gained attention as an immunotherapy owing to their immunomodulatory capacity and ability to infiltrate solid tumors and phagocytize tumor cells. The first-generation CD3ζ-based CAR-macrophages could phagocytose tumor cells in an antigen-dependent manner. Here we engineered induced pluripotent stem cell-derived macrophages (iMACs) with toll-like receptor 4 intracellular toll/IL-1R (TIR) domain-containing CARs resulting in a markedly enhanced antitumor effect over first-generation CAR-macrophages. Moreover, the design of a tandem CD3ζ-TIR dual signaling CAR endows iMACs with both target engulfment capacity and antigen-dependent M1 polarization and M2 resistance in a nuclear factor kappa B (NF-κB)-dependent manner, as well as the capacity to modulate the tumor microenvironment. We also outline a mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we provide a second-generation CAR-iMAC with an ability for orthogonal phagocytosis and polarization and superior antitumor functions in treating solid tumors relative to first-generation CAR-macrophages.

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM. Methods: CD3 + T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments. Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was > 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 + T cells, while the proportion of CD4 + T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 + primary myeloma cells. Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.

Preclinical Evidence of an Allogeneic Dual CD20xCD22 CAR to Target a Broad Spectrum of Patients with B-cell Malignancies.

Despite the remarkable success of autologous chimeric antigen receptor (CAR) T cells, some patients relapse due to tumor antigen escape and low or uneven antigen expression, among other mechanisms. Therapeutic options after relapse are limited, emphasizing the need to optimize current approaches. In addition, there is a need to develop allogeneic "off-the-shelf" therapies from healthy donors that are readily available at the time of treatment decision and can overcome limitations of current autologous approaches. To address both challenges simultaneously, we generated a CD20xCD22 dual allogeneic CAR T cell. Herein, we demonstrate that allogeneic CD20x22 CAR T cells display robust, sustained and dose-dependent activity in vitro and in vivo, while efficiently targeting primary B-cell non-Hodgkin lymphoma (B-NHL) samples with heterogeneous levels of CD22 and CD20. Altogether, we provide preclinical proof-of-concept data for an allogeneic dual CAR T cell to overcome current mechanisms of resistance to CAR T-cell therapies in B-NHL, while providing a potential alternative to CD19 targeting.

CAR-T cell development for Cutaneous T cell Lymphoma: current limitations and potential treatment strategies.

Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell malignancies, however, its application for T cell lymphoma, particularly cutaneous T cell lymphoma (CTCL), has been limited. Barriers to effective CAR-T cell therapy in treating CTCL include T cell aplasia in autologous transplants, CAR-T product contamination with leukemic T cells, CAR-T fratricide (when the target antigen is present on normal T cells), and tumor heterogeneity. To address these critical challenges, innovative CAR engineering by targeting multiple antigens to strike a balance between efficacy and safety of the therapy is necessary. In this review, we discuss the current obstacles to CAR-T cell therapy and highlight potential targets in treating CTCL. Looking forward, we propose strategies to develop more powerful dual CARs that are advancing towards the clinic in CTCL therapy.

CD19/BAFF-R dual-targeted CAR T cells for the treatment of mixed antigen-negative variants of acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent anti-tumor effects in B cell malignancies including acute lymphoblastic leukemia (ALL), but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve durability of remission, we developed a dual-targeting approach using an optimized, bispecific CAR construct that targets both CD19 and BAFF-R. CD19-BAFF-R dual CAR T cells exhibited antigen-specific cytokine release, degranulation and cytotoxicity against both CD19−/− and BAFF-R−/− variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19−/− and BAFF-R−/− variant ALL cells and treated with a single dose of CD19-BAFF-R dual CAR T cells experienced complete eradication of both CD19−/− and BAFF-R−/− ALL variants, while mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors, respectively. Further, CD19-BAFF-R dual CAR T cells showed prolonged in vivo persistence, raising the possibility that these cells may have potential to promote durable remissions. Together, our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL.

Dual Chimeric Antigen Receptor Approach Combining Novel Tumor Targeting Strategies Circumvents Antigen Escape in Multiple Myeloma

Chimeric antigen receptor (CAR) T-cell therapy has proven highly effective in patients with hematological malignancies. However, resistance to CAR-T cell therapy arising from target protein shedding and other forms of antigen downregulation can lead to CAR-resistant disease relapse. Tumor escape may be successfully prevented through the simultaneous targeting of multiple tumor antigens. The ability to target multiple antigens with a single therapeutic modality offers the potential for anti-tumor responses, broader coverage of heterogeneous tumor populations, and the potential to prevent antigen escape, potentially inducing durable clinical remission. Multiple myeloma (MM) presents an ideal case to employ a dual-CAR approach, as BCMA-targeting cell therapies have shown impressive efficacy to date, but curative treatment remains elusive. Additionally, the oligoclonal nature of MM may contribute to antigen escape and clonal resistance. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of MM. We further demonstrate the efficacy in an induced pluripotent stem cell (iPSC) platform, where a master engineered iPSC line is used as the starting material for mass production of off-the-shelf, dual-CAR immune effector cells.We selected B cell maturation antigen (BCMA), a well-defined TAA in MM, as the first antigen target. To develop the CAR-BCMA motif, we utilized our previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). As shown previously, the designed CAR-BCMA demonstrates potent and selective cellular cytotoxicity against MM (Figure 1a, left panel). BCMA has been observed to be actively cleaved from the surface of MM cells though, resulting in reduced efficacy and clinical relapse. To circumvent BCMA antigen escape, we developed a companion CAR targeting the pan-TAAs, MICA and MICB, which are expressed on MM plasma cells as well as monoclonal gammopathy of undetermined significance (MGUS) plasma cells. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding and drive anti-tumor immunity (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against an array of cancers, including the MM.1S cancer cell line (Figure 1a, right panel). To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression patterns of BCMA and MICA/B antigens on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden targeting approach of malignant plasma cells (Figure 1b). Initial studies to evaluate the dual CAR approach in MM were performed by generating anti-BCMA and anti-MICA/B-α3 dual-CAR (MM dual-CAR) T-cells. MM dual-CAR T cells showed antigen-specific activation, degranulation and cytotoxicity against both antigens in an additive manner, consistent with the initial antibody staining on target cells and illustrating that co-targeting MICA/B and BCMA may increase the activity against MM (Figure 1c). Similar trends were observed in a series of cytotoxicity assays against several MM lines. Preliminary studies are ongoing in induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing MM dual-CARs as a unique off-the-shelf cell therapy targeting both BCMA and MICA/B. Since MM dual-CAR iNK cells also express CD16, which mediates antibody-dependent cellular cytotoxicity, combination with therapeutic antibodies, such as anti-CD38 antibodies, can be deployed to target three TAAs for a complete therapeutic approach in MM. The data highlights the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. [Display omitted] DisclosuresLee: Fate Therapeutics, Inc.: Current Employment. Wucherpfennig: Novartis: Research Funding; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Bispecific CD19-CD20 and CD19-CD22 CAR-T Cells with Glycogen Synthase Kinase (GSK)-3β Inhibitor TWS119 Treatment Have Superior Therapeutic Effects on Mantle Cell Lymphoma

Introduction: Chimeric antigen receptor (CAR) - expressing T cells have shown breakthrough clinical successes to hematological malignancies such as diffuse large B cell lymphoma. Intensive development on CAR-T cell therapies are being developed in many common lymphomas. However, the development of CAR-T cell therapies in mantle cell lymphoma (MCL), a rare lymphoma has lagged behind. So far only CD19 CAR-T has been applied to MCL treatment, and MCL frequent relapse from prior CD19 CAR-T therapy with CD19 antigen loss. Bispecific CAR-T cells target two tumor antigens at once could reduce the risk of relapse. In addition, immune checkpoint blockade such as GSK3β and PD-1inhibition has been successfully used to enhance CAR-T cell therapies. Therefore, we attempt to develop a dual CAR for mantle cell lymphoma with checkpoint blockade.Methods: CD19-CD20 and CD19-CD22 bispecific CAR-T constructs were generated by subcloning the fragments in-frame into the pSFG retroviral vector. The BiCAR-T cells were cultured with/without a small molecule GSK3β inhibitor TWS119. Then, we performed a serial of experiments using CARs. 1, We assayed the CAR expression and cell expansion rates in vitro. 2, We assessed the tumor-killing capacity in vitro using MCL cell lines and primary MCL patient samples. 3, We performed phenotypic analyses by determination of proteins expression profiles of exhaustion markers and stem cell markers by flow cytometry. 4, We examined in vivo antitumor efficacy using preclinical models of MCL cell line-derived xenograft (CDX) .Results: Firstly, we found CD19-CD20 and CD19-CD22 bispecific CAR-T cells showed similar CAR expression levels (55%-72% CAR+ ) and similar expansion rate as single CD19 or CD20 CAR. Secondly, bispecific CAR- T cells potently and specifically lysed tumor cells in vitro, and showed the better killing of MCL tumor than CD19 or CD20 single CAR after challenge with MCL cell lines in vitro. Finally, we also confirmed these effects in CDX mice models derived from MCL cell line, Jeko-1 in vivo. CD19-CD20 and CD19-CD22 bispecific CAR successfully eliminated MCL tumor, showed better antitumor efficacy, and prolonged mouse survival than single CAR treated groups (P < 0.001). We also found that CD19-CD20 and CD19-CD22 bispecific-CAR T cells treated with TWS119 express significantly fewer exhaustion markers, such as PD-1 (P < 0.001), and LAG3 (P < 0.001), and showed better antitumor efficacy than the nontreated control group and single CAR groups in vivo. Furthermore, CAR-T cells expressing bispecific CD19-CD20 and CD19-CD22 CAR with TWS119 treatment had superior anti-MCL activity in vitro. In total, significantly better anti-MCL effects were observed for CD19-CD20 and CD19-CD22 bispecific CARs than single CAR. Their anti-MCL functions were further improved by TWS119 treatment.Conclusion: Altogether, these findings suggest that bispecific CD19-CD20 and CD19-CD22 CAR-T cells are potent for treating MCL relapse and drug resistant patients. In addition, bispecific CD19-CD20 and CD19-CD22 CAR-T cells with TWS119 treatment will further bring up the full power of CARs. These data support the therapeutic potential of bispecific CARs in patients with MCL. DisclosuresWang: Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; BGICS: Honoraria; Bayer Healthcare: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Clinical Care Options: Honoraria; Lilly: Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; Epizyme: Consultancy, Honoraria; Dava Oncology: Honoraria; Chinese Medical Association: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Molecular Templates: Research Funding; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; VelosBio: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; CStone: Consultancy; Scripps: Honoraria; OMI: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Oncternal: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding.

CD123 and CD33 Co-Targeting By Balanced Signaling on CAR-CIK Cells Reduces Potential Off-Target Toxicity While Preserving the Anti-Leukemic Activity of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated a high therapeutic potential in B-acute lymphoblastic leukemia, but translation in AML is limited by the absence of an ideal tumor-specific antigen. CD123 (known as IL-3 receptor alfa) and CD33 satisfy several features of ideal target antigens as they are expressed in almost all AML patients (mainly overexpressed in NPM1 and FLT-3 mutated AML), and conserved at disease relapse . Despite that, CD33 expression on hemopoietic stem/progenitor cells (HSPC) induced prolonged myelotoxicity after anti-CD33 CAR-T cell therapy and CD123 expression on endothelium was responsible for severe capillary leak syndrome after anti-CD123 CAR therapy. With the aim to improve selectivity for CD123+/CD33+ AML cells while minimizing toxicity against healthy cells, we probe a dual targeting model by Cytokine Induced Killer (CIK) cells co-expressing a first generation low affinity anti-CD123 IL-3 zetakine (IL3z.CAR) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR). This trans-signaling strategy could allow: 1) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; 2) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; 3) full CAR-CIK activation only against double expressing CD123 + / CD33 + leukemic cells.Fresh and frozen peripheral blood mononuclear cells were transduced with retroviral vectors during the CIK cell differentiation process . The functional activity of CAR-CIK cells was assessed in vitro by means of short- and long- term cytotoxicity and cytokine production assays upon challenge with different CD123/CD33 positive AML cell lines (THP-1 and KG-1) or with a CD123 positive human endothelial cell line (TIME). To assess the myelotoxicity against HSPC, CD34+ cells were sorted after 24h co-culture with CAR-CIK cells and mixed with methylcellulose-based semisolid medium to evaluate Colony Forming Units (CFU) after 14 days through an automated imaging and counting system for hematopoietic colonies (Stemvision). The in vivo anti-leukemic activity was evaluated by monitoring luciferase-expressing KG1 AML cell line growth over time in NSG mice treated with 3 infusions of all different CAR-CIK conditions.Dual CAR-CIK cells (IL-3z.CAR/CD33.CCR) display a potent and specific in vitro anti-leukemic efficacy against all the AML cell lines tested, compared to single targeting IL-3z.CAR and non-transduced CIK cells. To further minimize toxicities against healthy cells exploiting differences in CD123 antigen density between leukemic and healthy cells, we generated a low affinity dual CAR decreasing the IL3z.CAR binding affinity to CD123, by site-directed mutagenesis. Low affinity dual CAR-CIK cells show irrelevant cytotoxicity against the TIME endothelial cell line, comparable to non-transduced CIK cells, while preserving the efficacy against THP1 and KG1 AML cell lines. As a confirm of the reduced reactivity against endothelial cells, low affinity dual CAR-CIK cells produce the same levels of IL-2 and IFNγ either alone or in the presence of TIME cell line. Low affinity dual CAR-CIK cells also preserve the in vitro CFU-E, CFU-GM and CFU-GEMM colony forming capacity as compared to wild type (wt) dual CAR-CIK and wt IL-3z.CAR-CIK cells, highlighting the possibility to find a therapeutic window to minimize toxicity on healthy cells. The anti-leukemic activity of low affinity dual CAR-CIK cells has been confirmed also in vivo, with a significant suppression of leukemic growth against KG1 AML cell line. Mice treated with 3 infusions of low affinity dual CAR-CIK cells exhibited a significant better anti-leukemic profile, when compared to IL-3-single targeting CAR-CIK cells in terms of tumor growth and overall survival.These preclinical data demonstrate a powerful antitumor efficacy mediated by low affinity dual targeting IL-3z.CAR/CD33.CCR CIK cells against AML targets without any relevant in vitro toxicity on HSPC and endothelial cells, offering a proof-of-concept strategy to increase selectivity for CD123+/CD33+ AML cells whilst reducing the risk of “on-target off-tumor toxicity”. DisclosuresPerriello: Novartis: Other: Advisory Board. Martelli: Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals: Honoraria. Falini: Rasna Therapeutics: Honoraria. Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria.

CAR T\u2009cells with dual targeting of CD19 and CD22 in pediatric and young adult patients with relapsed or refractory B\u2009cell acute lymphoblastic leukemia: a phase 1 trial

Chimeric antigen receptor (CAR) T cells targeting CD19 or CD22 have shown remarkable activity in B cell acute lymphoblastic leukemia (B-ALL). The major cause of treatment failure is antigen downregulation or loss. Dual antigen targeting could potentially prevent this, but the clinical safety and efficacy of CAR T cells targeting both CD19 and CD22 remain unclear. We conducted a phase 1 trial in pediatric and young adult patients with relapsed or refractory B-ALL (n = 15) to test AUTO3, autologous transduced T cells expressing both anti-CD19 and anti-CD22 CARs (AMELIA trial, EUDRA CT 2016-004680-39). The primary endpoints were the incidence of grade 3–5 toxicity in the dose-limiting toxicity period and the frequency of dose-limiting toxicities. Secondary endpoints included the rate of morphological remission (complete response or complete response with incomplete bone marrow recovery) with minimal residual disease-negative response, as well as the frequency and severity of adverse events, expansion and persistence of AUTO3, duration of B cell aplasia, and overall and event-free survival. The study endpoints were met. AUTO3 showed a favorable safety profile, with no dose-limiting toxicities or cases of AUTO3-related severe cytokine release syndrome or neurotoxicity reported. At 1 month after treatment the remission rate (that is, complete response or complete response with incomplete bone marrow recovery) was 86% (13 of 15 patients). The 1 year overall and event-free survival rates were 60% and 32%, respectively. Relapses were probably due to limited long-term AUTO3 persistence. Strategies to improve CAR T cell persistence are needed to fully realize the potential of dual targeting CAR T cell therapy in B-ALL.

Dual-Target CAR-Ts with On- and Off-Tumour Activity May Override Immune Suppression in Solid Cancers: A Mathematical Proof of Concept.

Simple Summary(CAR)-T cell-based therapies have achieved substantial success against different haematological malignancies. However, results for solid tumours have been limited up to now, in part due to the fact that the immunosuppressive tumour microenvironment inactivates CAR-T cell clones. In this paper we study mathematically the competition of CAR-T and tumour cells, taking into account their immunosuppressive capacity. Using computer simulations, we show that the use of large numbers of CAR-T cells targetting the solid tumour antigens could overcome the immunosuppressive potential of cancer. To achieve such high levels of CAR-T cells we propose, and study in silico, the manufacture and injection of CAR-T cells targetting two antigens: CD19 and a tumour-associated antigen. This strategy lead in our simulations to the expansion of the CAR-T cells injected and the production of a massive army of CAR-T cells targetting the solid tumour, and potentially overcoming its immune suppression capabilities. Thus, our proposed strategy could provide a way to develop successful CAR-T cell therapies against solid tumours.Chimeric antigen receptor (CAR)-T cell-based therapies have achieved substantial success against B-cell malignancies, which has led to a growing scientific and clinical interest in extending their use to solid cancers. However, results for solid tumours have been limited up to now, in part due to the immunosuppressive tumour microenvironment, which is able to inactivate CAR-T cell clones. In this paper we put forward a mathematical model describing the competition of CAR-T and tumour cells, taking into account their immunosuppressive capacity. Using the mathematical model, we show that the use of large numbers of CAR-T cells targetting the solid tumour antigens could overcome the immunosuppressive potential of cancer. To achieve such high levels of CAR-T cells we propose, and study computationally, the manufacture and injection of CAR-T cells targetting two antigens: CD19 and a tumour-associated antigen. We study in silico the resulting dynamics of the disease after the injection of this product and find that the expansion of the CAR-T cell population in the blood and lymphopoietic organs could lead to the massive production of an army of CAR-T cells targetting the solid tumour, and potentially overcoming its immune suppression capabilities. This strategy could benefit from the combination with PD-1 inhibitors and low tumour loads. Our computational results provide theoretical support for the treatment of different types of solid tumours using T cells engineered with combination treatments of dual CARs with on- and off-tumour activity and anti-PD-1 drugs after completion of classical cytoreductive treatments.

Comprehensive meta-analysis of anti-BCMA chimeric antigen receptor T-cell therapy in relapsed or refractory multiple myeloma

Background Chimeric antigen receptor (CAR) T-cell therapy shows impressive results in clinical trials. We conducted a meta-analysis based on the most recent data to systematically describe the efficacy and safety of anti-BCMA CAR T therapy for patients with relapsed or refractory multiple myeloma (R/R MM). Methods PubMed, Embase, Web of Science, Cochrane library, ClinicalTrials.gov, China Biology Medicine disc (CBM disc) and Wanfang Data were searched on 8 November 2020. Registration number of PROSPERO was CRD42020219127. Results From 763 articles, we identified 22 appropriate studies with 681 patients. The pooled overall response rate (ORR) was 85.2% (95%CI 0.797–0.910), complete response rate (CRR) was 47.0% (95%CI 0.378–0.583), and minimal residual disease (MRD) negativity rate was 97.8% (95%CI 0.935–1.022). The pooled incidence of grade 3–4 cytokine release syndrome was 6.6% (95%CI 0.036–0.096) and neurotoxicity was 2.2% (95%CI 0.006–0.038). The median progression-free survival (PFS) was 14.0 months and median overall survival (OS) was 24.0 months. Subgroup analysis showed dual epitope-binding CAR T cells achieved the best therapy outcomes and humanized CAR T cells had the best safety profile. Patients who were older, heavily pre-treated or received lower dose of CAR T cells had worse ORR. There was no significant difference in ORR, CRR and PFS between patients with and without high-risk cytogenetic features. The PFS and CRR of non-extramedullary disease (EMD) group was superior to those of EMD group. Conclusion Anti-BCMA CAR T therapy is effective and safe for patients with R/R MM. It can improve the prognosis of patients with high-risk cytogenetic features while the prognosis of patients with EMD remains poor. Moreover, patients are likely to benefit from an earlier use of CAR T therapy and human-derived CAR T cells have obvious advantages based on the existing data.

Chimeric Antigen Receptor T Cell Therapy in the Relapsed or Refractory Multiple Myeloma with Extramedullary Disease–a Single Institution Observation in China

Background: Extramedullary disease (EMD) is characterized as the presence of myeloma cells invasion outside the bone marrow in a patient with multiple myeloma (MM), it may be found 6%~8% in de novo patients, and 10% to 30% in relapsed or refractory (R/R) MM patients across the overall disease course (Joan Blade et al. 2011, Matthew Weinstock et al. 2013 and 2015, Cyrille Touzeau et al. 2015). EMD was considered as high-risk group of myeloma and is associated with adverse prognosis and the management is particularly challenging. Here we discuss and improve the understanding of the clinical characters and prognoses of the R/R MM with EMD in the treatment of CAR T therapy, illustrate our experience in a single institution. Methods: LCAR-B38M is a Chimeric antigen receptor (CAR) T cell therapy with 4-1BB as the co-stimulator, a dual epitope-binding CAR T cell therapy directed against 2 distinct B cell maturation antigen (BCMA) epitopes. In total of 57 patients were treated in China from The Second Affiliated Hospital of Xi'an Jiaotong University (LEGEND-2, NCT03090659), 17 patients with R/R MM with EMD were included and received the CAR T treatment. The study was initiated on March 30, 2016, and the analysis reported here is from a data cutoff date of July 31, 2019, the detailed treatment schema was presented in the former paper (Zhao et al, 2018). Results: Overall, 17 (29.8%) of 57 treated patients with EMD were included in our CAR T treatment. The median age of R/R MM patients with EMD (EMD group) and without EMD (non-EMD group) were 55 (range, 27-72) and 53.3 (range, 42-73) years old respectively, and the median number of prior therapy lines were 3 (range, 1-6 and 1-9) in both groups. There were no statistical difference in the proportion of male and female patients, median time from initial diagnosis and ISS stage in EMD group and non-EMD group. The Eastern Cooperative Oncology Group (ECOG) scores of patients receiving CAR T treatment were 0-2 points, the proportion of ECOG 1-2 points in EMD group were higher than non-EMD group (P &amp;lt; 0.05, R=0.1474, 95% CI, 0.03 to 0.73). IgG and IgA subtype were the main types in the non-EMD patients while the light chain type was more frequently seen in EMD group (P = 0.014, OR=0.1886, 95% CI, 0.05 to 0.66). The overall response rates (ORR) in the EMD group and non-EMM group were 82.4% and 90% respectively (Fig 1 A), and there were no significant difference in the median time to the first response and the best response between the EMD group and non-EMD group(P &amp;gt; 0.05). At cutoff, the median follow-up was 25 months, the median progression-free survival (PFS) in EMD group and non-EMD group were 8.1 months and 25 months (P &amp;lt; 0.001, R=0.32, 95% CI, -0.19 to 0.84), and the median overall survival (OS) were 13.9 months and not reached (P = 0.0019, R=4.833, 95% CI, 1.91 to 12.22), survival curves were shown in Fig 1 B, C . Conclusions: LCAR-B38M is a highly effective therapy in R/R MM patients with EMD and without EMD, and both groups had similar response time and reached a high ORR rate. R/R MM patients with EMD demonstrated a poor prognosis and they may lost their best response in a relatively shorter time compared with the patients without EMD. With a median follow-up of 25 months, LCARB38M manifested a relatively durable therapeutic effect in this high-risk R/R MM patients with EMD. Disclosures No relevant conflicts of interest to declare.

Significant Long-Term Benefits of CAR T-Cell Therapy Followed By a Second Allo-HSCT for Relapsed/Refractory (R/R) B-Cell Acute Lymphoblastic Leukemia (B-ALL) Patients Who Relapsed after an Initial Transplant

Introduction Current available treatments are limited once patients with B-ALL relapse following allogeneic hematopoietic stem cell transplantation (allo-HSCT). While chimeric antigen receptor (CAR) T-cell therapy offers a chance of remission, long-term outcomes for these patients remain poor. The benefit of bridging into a second transplant after CAR T-cell therapy remains inconclusive and available data are limited. Here, we report the long-term outcomes of 23 B-ALL patients who chose to undergo a second allo-HSCT after achieving complete remission (CR) from CAR T-cell therapy. Methods From April 2017 to April 2020, 23 R/R B-ALL patients (median age of 20 years, ranging from 3 to 58 years) who relapsed after first allo-HSCT received CAR T-cell therapy. The data were aggregated from seven different clinical trials (www.clinicaltrials.gov NCT03173417, NCT02546739 NCT03825718, NCT03825731, NCT03952923, NCT04100187 and www.chictr.org.cn ChiCTR1800016541). Patients' first transplant sources were HLA-identical sibling (n=5), matched-unrelated donor (MUD) (n=1), and haploidentical donors (haplo) (n=17). Eight of the 23 patients had disease relapse within 6 months following the first transplant. The median time from first transplant to CAR T-cell infusion was 261days (range: 117~2181 days). Before CAR T-cell infusion, patients' median bone marrow (BM) blasts by morphology were about 72.5% (1.5%-94.5%) including 12 patients with BM blasts &amp;gt;70% (5 with BM blasts &amp;gt;90%). Three of the 23 patients (13%) had received at least one prior donor lymphocyte infusion. No patients had active graft-versus-host disease (GVHD) prior to CAR T-cell therapy. Second generation CAR T-cells were generated by using purified T-cells from transplant donors (n=15) or patients (n=8). Twenty-two patients received T-cells modified with CD19-targeting CAR T-cells containing either a 4-1BB (n=18) or a CD28 co-stimulatory domain (n=4), and one patient received CD19-CD22 dual specificity CAR T-cells. All patients received a conditioning regimen of IV fludarabine (30mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single CAR T-cell infusion with a median dose of 3×105 cells/kg (1×105-6×105 cells/kg) in 21 patients. Two patients received a second CAR T-cell infusion in 2-3 months (1/3×105 cells/kg dose). Post CAR-T therapy, all patients bridged into a consolidation second transplantation with conventional myeloablative pre-transplantation conditioning regimens including 15 patients who received total body irradiation-based and 7 patients that received a busulfan-based conditioning regimen. Cyclosporin A, short-term methotrexate, and mycophenolate mofetil were used for GVHD prophylaxis. Results Patients' characteristics are shown in Table 1. On Day 30 post CAR-T-cell infusion, 23/23 (100%) patients achieved minimal residual disease (MRD)-negative CR. A total of 16/23 (69.6%) patients developed cytokine release syndrome (CRS) of which 14/23 (60.9%) had Grade I-II and 2/23 (8.7%) had Grade III CRS. Two patients had Grade III neurotoxicity. All patients with MRD-negative status subsequently bridged into a second transplant (2 from MUD and 21 from haplo donors) with a median interval time of 67 days (39- 329 days) from CAR T-cell therapy to a second transplant. At a median follow-up time of 258 days (84-978 days), no patients relapsed, which was encouraging. Five of 23 patients (21.7%) died from transplant-related mortality (TRM) at a median time of 295 days (103-372 days) (1 from GVHD and 4 from infection). The 1-year overall survival (OS) was 68.0% and 2-year OS was 54.4% (Fig.1). While there was a trend towards a more efficacious OS for patients whose CAR T-cells were derived from donors rather than from patients themselves but the number are too small to reach statistical significance (1-year OS 83.9% vs. 64.3%, 2-year OS 83.9% vs. 42.9%, P=0.739. Fig.2). After the 2nd transplant, four patients developed GVHD. Conclusions Our study demonstrates that even for R/R B-ALL patients who have relapsed following a first allo-HSCT , an MRD-negative CR status can still be achieved through CAR T-cell cell therapy without increasing CRS or neurotoxicity, making consolidation second allo-HSCT feasible for these patients. CAR T-cell therapy combined with a consolidation second HSCT are effective for these heavily pre-treated patients with an encouraging prospect for long-term survival. Disclosures No relevant conflicts of interest to declare.