Chimeric Antigen Receptor-engineered T-cell Research Articles

Novel Cancer Immunotherapy Using Cell-Derived Membrane Vesicles Orchestrates Multimodal Antitumor Immunity

Cancer immunotherapy has emerged as a promising therapy for hematological malignancies. Although multiple immunotherapeutics are currently available in the clinic such as chimeric antigen receptor-engineered T-cell therapy (CAR-T cell therapy) and bispecific T-cell engager (BiTE), their long-term efficacy is still insufficient in most of the cases. Simultaneous inputs of multiple signals may provide more optimal T-cell activation and overcome the immunosuppressive tumor microenvironment (TME), which results in a durable therapeutic effect. However, integrating multifaceted immunomodulatory signals into a single drug or cell is technically difficult. To overcome these limitations, we developed nanosized cell-derived membrane vesicles (MVs) that orchestrate multimodal antigen-specific antitumor immunity in endogenous T cells. We isolated MVs with 100-150 nm in size and intact membrane-bound proteins from the HLA-negative leukemia cell line K562 by mechanical homogenization followed by isolation of the plasma membrane fraction using magnetic beads and ultracentrifugation. MVs can be loaded with a desired combination of immunomodulatory molecules on the surface by preparing cells stably transduced with those proteins. First, K562 cells were genetically engineered with a membrane-bound form of the anti-CD3/CD19 BiTE and homogenized to form BiTE-expressing MVs. When co-cultured with T cells and CD19-positive leukemia cell line NALM-6, MVs loaded with anti-CD3/CD19 BiTE induced potent cytotoxic activity in nonspecific T cells in a dose-dependent manner. To provide optimal T-cell activation, we further loaded MVs with a variety of immunostimulatory molecules including costimulatory ligands (CD80 and 4-1BBL) and cytokines (IL-7 and IL-15) on the surface. The generated antitumor MVs simultaneously induced multiple immunostimulatory signals in T cells, resulting in superior proliferation, cytokine production, and cytolytic functions compared with MVs loaded with BiTE alone. Intriguingly, MVs activated T cells only in the presence of target tumor cells, which is considered to provide a safety advantage in clinical settings. Changing the antitumor mAb sequences of the BiTE enabled to generate MVs against any type of antigen including B cell maturation antigen (BCMA), EGFR, and mesothelin. To test functions of our MVs in vivo, NSG mice were transplanted with NALM-6 and then infused with human peripheral blood T cells. While infusion of T cells alone did not contribute to controlling leukemia progression, infusion of anti-CD19 MVs with CD80, 4-1BBL, IL-7, and Il-15 induced potent antitumor response. Repeated infusion of MVs further augmented their therapeutic efficacy. To overcome the immunosuppressive TME, we additionally loaded MVs with immune checkpoint inhibitors (anti-PD-1 and anti-CTLA-4 antibody), TGF-b receptors to trap TGF-b, and inflammatory cytokines (IL-12 and IL-18) that activate both T cells and myeloid cells in the TME. Immunocompetent Balb/c mice were subcutaneously inoculated with colon carcinoma cell line CT26 ectopically expressed with CD19 and treated with MVs targeting CD19. The injected MVs converted tumor-associated M2-type macrophages into an inflammatory phenotype and promoted CD8+ T cell infiltration within the TME, resulting in better control of tumor progression in in vivo solid tumor mouse models. K562 cells are highly sensitive to NK cells because they endogenously express several ligands for NK cell activation receptors such as MIC-A and MIC-B, and lack the expression of HLA class I that represses NK cell activation. K562-derived MVs induced potent cytotoxic activity in NK cells against NK-cell-resistant myeloma cell line MM1S in an antigen-specific manner. In summary, this novel technology enables to deliver desired combinations of antitumor immune signals to not only T cells but also NK cells and other types of immune cells, which can transmit superior therapeutic efficacy against the tumor cells that are resistant to the present immunotherapeutics.

Evaluation of Central Nervous System Involvement in Patients undergoing Chimeric antigen receptor-engineered T-cell Therapy by Magnetic Resonance Imaging

Chimeric antigen receptor-engineered (CAR) T-cell therapy is an effective immunotherapy for aggressive hematologic cancers. However, it can lead to complications such as immune effector cell-associated neurotoxicity syndrome (ICANS), or complications related to the immunosuppressive status of these patients. The role of imaging in this context is essential to help in ICANS diagnosis and to rule out other potential diagnosis, such as central nervous system infections. Two cases are presented to illustrate this clinical problem. Case 1describes a 38-year-old patient with diffuse large B-cell lymphoma who developed ICANS after CAR T-cell therapy. MRI revealed signs of leukoencephalopathy. Case 2 involves a 57-year-old patient with mantle-cell lymphoma who presented neurologic symptoms -clinically suggestive of ICANS- after CAR-T therapy. MRI showed signs indicative of limbic encephalitis.These two cases highlight the importance of MRI in clinical practice after CAR T-Cell Therapy underscoring the role of MRI in the diagnosis of complications in patients with neuropsychiatric symptoms.

Evaluación mediante resonancia magnética de la afectación del sistema nervioso central en pacientes sometidos a terapia de células T con receptores quiméricos de antígenos

Chimeric antigen receptor-engineered (CAR) T-cell therapy is an effective immunotherapy for aggressive hematologic cancers. However, it can lead to complications such as immune effector cell-associated neurotoxicity syndrome (ICANS), or complications related to the immunosuppressive status of these patients. The role of imaging in this context is essential to help in ICANS diagnosis and to rule out other potential diagnosis, such as central nervous system infections. Two cases are presented to illustrate this clinical problem.Case 1: describes a 38-year-old patient with diffuse large B-cell lymphoma who developed ICANS after CAR T-cell therapy. MRI revealed signs of leukoencephalopathy. Case 2:involves a 57-year-old patient with mantle-cell lymphoma who presented neurologic symptoms -clinically suggestive of ICANS- after CAR-T therapy. MRI showed signs indicative of limbic encephalitis.These two cases highlight the importance of MRI in clinical practice after CAR T-Cell Therapy underscoring the role of MRI in the diagnosis of complications in patients with neuropsychiatric symptoms.

Advancements in CAR-NK therapy: lessons to be learned from CAR-T therapy.

Advancements in chimeric antigen receptor engineered T-cell (CAR-T) therapy have revolutionized treatment for several cancer types over the past decade. Despite this success, obstacles including the high price tag, manufacturing complexity, and treatment-associated toxicities have limited the broad application of this therapy. Chimeric antigen receptor engineered natural killer cell (CAR-NK) therapy offers a potential opportunity for a simpler and more affordable "off-the-shelf" treatment, likely with fewer toxicities. Unlike CAR-T, CAR-NK therapies are still in early development, with few clinical trials yet reported. Given the challenges experienced through the development of CAR-T therapies, this review explores what lessons we can apply to build better CAR-NK therapies. In particular, we explore the importance of optimizing the immunochemical properties of the CAR construct, understanding factors leading to cell product persistence, enhancing trafficking of transferred cells to the tumor, ensuring the metabolic fitness of the transferred product, and strategies to avoid tumor escape through antigen loss. We also review trogocytosis, an important emerging challenge that likely equally applies to CAR-T and CAR-NK cells. Finally, we discuss how these limitations are already being addressed in CAR-NK therapies, and what future directions may be possible.

Genetic engineering strategies to enhance antitumor reactivity and reduce alloreactivity for allogeneic cell-based cancer therapy.

The realm of cell-based immunotherapy holds untapped potential for the development of next-generation cancer treatment through genetic engineering of chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapies for targeted eradication of cancerous malignancies. Such allogeneic "off-the-shelf" cell products can be advantageously manufactured in large quantities, stored for extended periods, and easily distributed to treat an exponential number of cancer patients. At current, patient risk of graft-versus-host disease (GvHD) and host-versus-graft (HvG) allorejection severely restrict the development of allogeneic CAR-T cell products. To address these limitations, a variety of genetic engineering strategies have been implemented to enhance antitumor efficacy, reduce GvHD and HvG onset, and improve the overall safety profile of T-cell based immunotherapies. In this review, we summarize these genetic engineering strategies and discuss the challenges and prospects these approaches provide to expedite progression of translational and clinical studies for adoption of a universal cell-based cancer immunotherapy.

Antitumor effect of CAR-T cells targeting transmembrane tumor necrosis factor alpha combined with PD-1 mAb on breast cancers

BackgroundOur previous study showed that transmembrane tumor necrosis factor alpha (tmTNF-α) is overexpressed in primary breast cancers including triple-negative breast cancers (TNBCs). Chimeric antigen receptor engineered-T (CAR-T) cells have been...

41 (PB031) - CAR-T cell engineering strategies aimed at safe and effective targeting of solid tumors

Adoptive cell therapy based on chimeric antigen receptor-engineered T (CAR-T) cells has proven to be life-saving for many cancer patients. However, its therapeutic efficacy has so far been restricted to only a few malignancies, with solid tumors proving to be especially recalcitrant to efficient therapy. Poor intra-tumor infiltration by T cells and “T cell dysfunction” due to an immunosuppressive microenvironment are key barriers against CAR-T cell success against solid tumors. Furthermore, low level expression of CAR-directed tumor-associated antigens (TAA) in normal tissues can result in “on-target off-tumor” cytotoxicity, raising potential safety concerns. Using our best-in-class TALEN®-based gene editing platform, we present here innovative T cell engineering strategies that can combat some of the challenges posed by CAR-T cell development for solid tumors. These allogeneic ‘Smart CAR-T’ cells are designed to integrate locus-specific synthetic genes that respond to cues localized to the solid tumor microenvironment. The subsequent expression of these genes increases CAR-T cell persistence and anti-tumor activity while staying restricted to the tumor milieu. Thus, our proof-of-concept study demonstrates the feasibility of developing CAR-T cell engineering strategies that can improve solid tumor targeting while mitigating potential safety risks, encouraging us to bring them to the clinic. No conflict of interest.

Novel CD19 chimeric antigen receptor T cells manufactured next-day for acute lymphoblastic leukemia

Chimeric antigen receptor-engineered T (CAR-T) cells have shown promising efficacy in patients with relapsed/refractory B cell acute lymphoblastic leukemia (R/R B-ALL). However, challenges remain including long manufacturing processes that need to be overcome. We presented the CD19-targeting CAR-T cell product GC007F manufactured next-day (FasTCAR-T cells) and administered to patients with R/R B-ALL. A total of 21 patients over 14 years of age with CD19+ R/R B-ALL were screened, enrolled and infused with a single infusion of GC007F CAR-T at three different dose levels. The primary objective of the study was to assess safety, secondary objectives included pharmacokinetics of GC007F cells in patients with R/R B-ALL and preliminary efficacy. We were able to demonstrate in preclinical studies that GC007F cells exhibited better proliferation and tumor killing than conventional CAR-T (C-CAR-T) cells. In this investigator-initiated study all 18 efficacy-evaluable patients achieved a complete remission (CR) (18/18, 100.00%) by day 28, with 17 of the patients (94.4%) achieving CR with minimal residual disease (MRD) negative. Fifteen (83.3%) remained disease free at the 3-month assessment, 14 patients (77.8%) maintaining MRD negative at month 3. Among all 21 enrolled patients, the median peak of CAR-T cell was on day 10, with a median peak copy number of 104899.5/µg DNA and a median persistence period of 56 days (range: 7–327 days). The incidence of cytokine release syndrome (CRS) was 95.2% (n = 20), with severe CRS occurring in 52.4% (n = 11) of the patients. Six patients (28.6%) developed neurotoxicity of any grade. GC007F demonstrated superior expansion capacity and a less exhausted phenotype as compared to (C-CAR-T) cells. Moreover, this first-in-human clinical study showed that the novel, next-day manufacturing FasTCAR-T cells was feasible with a manageable toxicity profile in patients with R/R B-ALL.

Development of next-generation chimeric antigen receptor-engineered T-cell therapy

Chimeric antigen receptor (CAR)-engineered T-cell therapy against B-cell malignancies and multiple myeloma was recently introduced for clinical use. However, the efficacy of CAR-T cell therapy is not durable in most patients, warranting the development of CAR-T cells with additional genetic modification or engineering of synthetic molecules to enhance their functions. This review will provide an overview of the molecular mechanisms underlying the functional alteration of T cells, especially transcriptional networks associated with memory formation and T cell exhaustion. In addition, methods to rationally improve CAR-T cell functions based on these mechanistic insights will be discussed.

Development of Stem Cell-Derived Immune Cells for Off-the-Shelf Cancer Immunotherapies.

Cell-based cancer immunotherapy has revolutionized the treatment of hematological malignancies. Specifically, autologous chimeric antigen receptor-engineered T (CAR-T) cell therapies have received approvals for treating leukemias, lymphomas, and multiple myeloma following unprecedented clinical response rates. A critical barrier to the widespread usage of current CAR-T cell products is their autologous nature, which renders these cellular products patient-selective, costly, and challenging to manufacture. Allogeneic cell products can be scalable and readily administrable but face critical concerns of graft-versus-host disease (GvHD), a life-threatening adverse event in which therapeutic cells attack host tissues, and allorejection, in which host immune cells eliminate therapeutic cells, thereby limiting their antitumor efficacy. In this review, we discuss recent advances in developing stem cell-engineered allogeneic cell therapies that aim to overcome the limitations of current autologous and allogeneic cell therapies, with a special focus on stem cell-engineered conventional αβ T cells, unconventional T (iNKT, MAIT, and γδ T) cells, and natural killer (NK) cells.

Safety and Efficacy of Two Anakinra Dose Regimens for Refractory CRS or Icans after CAR T-Cell Therapy

Safety and Efficacy of Two Anakinra Dose Regimens for Refractory CRS or Icans after CAR T-Cell Therapy

Effects of Prior Exposure to Tec Kinase(BTK/ITK) Inhibitors on Kte-X19 Products

Introduction:Frequent and durable responses were recently reported in relapsed or refractory (R/R) mantle cell lymphoma (MCL) patients treated with KTE-X19, an autologous CD19-targeted chimeric antigen receptor-engineered T-cell (CAR-T) product (Wang et al. N Engl J Med. 2020). Most patients enrolled had received at least one line of Tec kinase inhibitor prior to KTE-X19 manufacturing, either in the form of ibrutinib, a Bruton's tyrosine kinase (BTK) and Inducible T cell kinase (ITK) inhibitor, or acalabrutinib, a more selective BTK inhibitor. Pharmacokinetic expansion of KTE-X19 was higher in ibrutinib-treated patients relative to acalabrutinib-treated patients. We previously showed that prolonged exposure to ibrutinib enhanced T cell effector function and proliferation in patients with CLL (Fraietta et al, Blood, 2016). To assess the impact of Tec kinase inhibitor on KTE-X19 products and downstream clinical outcomes, we examined the phenotype, transcriptional profile and cytokine production of KTE-X19 infusion products and post-infusion lymphocytes from patients with R/R MCL treated on the Zuma-2 study.Study Design and Methods:We evaluated biospecimens from MCL patients who enrolled on the Zuma-2 clinical trial (NCT02601313) and who were previously treated with ibrutinib (n=14) or acalabrutinib (n=6). Samples analyzed consisted of KTE-X19 CAR T products and peripheral blood mononuclear cells (PBMC) collected 7 days after infusion. Lymphocytes were assessed for CAR expression, T cell phenotype, transcriptional profile and cytokine production. In addition, CAR T cell phenotypes and cytokines were profiled following co-culture of KTE-X19 with CD19 + Toledo cells (DLBCL).Results:Flow cytometric analysis of KTE-X19 demonstrated similar distributions of CD4+ and CD8+ T cells and comparable frequencies of central and effector memory populations in the CAR+ T cells derived from patients with prior exposure to ibrutinib vs. acalabrutinib. T helper subset analysis trended towards enrichment of Th1/Th17 populations within the CAR+ CD4+ cells of the ibrutinib cohort. This finding was further supported by transcriptional profiling of sorted CAR+ T cells from infusion products, where Th1/Th17, Jak/STAT and activation-related genes were enriched in the cohort with prior ibrutinib exposure. In addition, the Th1 phenotype was more frequent in PBMC of ibrutinib-exposed patients (8/14) compared to acalabrutinib-exposed patients (1/4). Interestingly, a shift from a central memory-dominant product towards an effector memory phenotype was observed in peripheral CD4+ and CD8+ CAR T cells in the ibrutinib cohort, whereas acalabrutinib post-infusion CAR T cells maintained a central memory phenotype. In vitro stimulation of KTE-X19 CAR-T infusion products with tumor cells resulted in a significant enrichment of the Th1 population in patients who had received ibrutinib compared to those that received acalabrutinib (p=0.0058). Following stimulation, CAR-T cells from the acalabrutinib cohort produced higher levels of Th2 cytokines, including IL-4, IL-5, and IL-13 as well as GM-CSF compared to the ibrutinib cohort.Conclusions:Analysis of KTE-X19 infusion products and day 7 post-infusion PBMC demonstrated that CAR T cells from patients with prior ibrutinib exposure have a Th1 predominant phenotype, suggesting that ibrutinib but not acalabrutinib promotes Th1 differentiation and effector function, potentially through the inhibition of ITK. Furthermore, our data suggest that inhibition of non-BTK targets such as ITK may play a role in driving a Th17 phenotype. Prior exposure to ibrutinib may increase CAR T cell effector function to a greater extent than exposure to acalabrutinib to enhance clinical outcome in patients with MCL. DisclosuresBudka: Kite Pharma: Current Employment. Sowrirajan: Kite Pharma: Current Employment. Nguyen: Kite Pharma: Current Employment. Shen: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties; Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

CAR T-Cell Therapy: An Update for Radiologists.

Chimeric antigen receptor-engineered (CAR) T-cell therapy is a promising novel immunotherapy that has the potential to revolutionize cancer treatment. Four CAR T-cell therapies have received FDA approval within the last 5 years, and the role of CAR T cells is anticipated to continue to evolve and expand. However, various aspects of CAR T-cell therapies remain poorly understood, and the therapies are associated with severe side effects, including cytokine release syndrome and immune effector cell-associated neurotoxicity, which require prompt diagnosis and intervention. The purposes of this review are to describe the role of imaging in diagnosing and monitoring toxicities from CAR T-cell therapies and explore the use of various imaging techniques, including PET/CT with novel radiotracers, to predict and assess treatment response and adverse effects. It is important for radiologists to recognize the imaging findings associated with each syndrome and to recognize the typical and atypical treatment response patterns associated with CAR T-cell therapy. Given the expected increase in use of CAR T cells in the near future, radiologists should familiarize themselves with the imaging findings encountered in these novel therapies so that they can provide comprehensive and up-to-date guidance for clinical management.

1372O CLDN 18.2-targeted CAR-T cell therapy in patients with cancers of the digestive system

Efficacy of chimeric antigen receptor-engineered T (CAR-T) cell therapy against solid tumors is still limited. CLDN18.2 is a promising target highly expressed in various cancer types of the digestive system. CT041, an anti-CLDN18.2 CAR-T cell therapy, showed promising anti-tumor activities in preclinical studies and an investigator-initiated study (CT041-CG4003 NCT03159819). This newly initiated phase I study was conducted to assess the safety and efficacy of CT041 produced with a modified manufacturing process. Patients with CLDN18.2+ cancers of digestive system received CT041 at doses of 2.5×108, 3.75×108 or 5×108 CAR-T cells after preconditioning chemotherapy. The objective was to assess the safety, efficacy and cytokinetic profile of CT041. As of April 8, 2021, a total of 37 patients with cancer of digestive system, including 28 with gastric cancer (GC), 5 with pancreatic ductal adenocarcinoma (PDAC) and 4 with other cancer types, received CT041 infusion and completed at least 12 weeks of follow-up after the first infusion for the last subject. Cell doses of 2.5×108, 3.75×108 and 5×108 were administrated in 28, 6 and 3 patients, respectively. With a median follow-up of 7.6 months (95%CI 5.6, 8.6), all patients experienced ≥G3 hematologic toxicity while no DLT was observed. Immune effector cell-associated neurotoxicity syndrome, treatment-related death and ≥G3 cytokine release syndrome was not observed. The overall ORR for all patients and patients with GC were 48.6% (95%CI, 31.9%, 65.6%) and 57.1% (95%CI, 37.2, 75.5) respectively. At the dose level of 2.5×108, 18 patients with GC who have failed at least 2 prior lines of therapy including 44% (8/18) exposure to anti-PD-(L)1 antibody, the overall ORR, median PFS and OS was 61.1% (11/18), 5.4 months (95%CI, 2.6, NE) and 9.5 months (95%CI, 5.2, NE) respectively. CT041 showed acceptable safety profile and promising anti-tumor activities in patients with refractory CLDN18.2+ cancers of digestive system. In patients with GC who have failed at least 2 prior lines of therapy, CT041 displayed a significantly improved efficacy compared to historical data.

Chimeric antigen receptor- and natural killer cell receptor-engineered innate killer cells in cancer immunotherapy.

Chimeric antigen receptor (CAR)-engineered T-cell (CAR-T) therapy has demonstrated impressive therapeutic efficacy against hematological malignancies, but multiple challenges have hindered its application, particularly for the eradication of solid tumors. Innate killer cells (IKCs), particularly NK cells, NKT cells, and γδ T cells, employ specific antigen-independent innate tumor recognition and cytotoxic mechanisms that simultaneously display high antitumor efficacy and prevent tumor escape caused by antigen loss or modulation. IKCs are associated with a low risk of developing GVHD, thus offering new opportunities for allogeneic "off-the-shelf" cellular therapeutic products. The unique innate features, wide tumor recognition range, and potent antitumor functions of IKCs make them potentially excellent candidates for cancer immunotherapy, particularly serving as platforms for CAR development. In this review, we first provide a brief summary of the challenges hampering CAR-T-cell therapy applications and then discuss the latest CAR-NK-cell research, covering the advantages, applications, and clinical translation of CAR- and NK-cell receptor (NKR)-engineered IKCs. Advances in synthetic biology and the development of novel genetic engineering techniques, such as gene-editing and cellular reprogramming, will enable the further optimization of IKC-based anticancer therapies.

Cellular Immunotherapy for Refractory Diffuse Large B Cell Lymphoma in the Chimeric Antigen Receptor-Engineered T Cell Era: Still a Role for Allogeneic Transplantation?

Chimeric antigen receptor-engineered T (CART) cells are a promising new treatment option for patients with multiply relapsed and refractory (R/R) diffuse large B cell lymphoma (DLBCL). Because of the favorable outcome data reported for CART cells, uncertainty is emerging if there is still a role for allogeneic hematopoietic cell transplantation (allo-HCT) in the treatment of R/R DLBCL. This article provides an overview of available evidence and theoretical considerations to put these 2 types of cellular immunotherapy (CI) into perspective. Altogether, current data suggest that CART cells are preferred now over transplantation as first-choice CI in many clinical situations. However, the majority of patients will fail CART therapy, resulting in an unmet medical need where allo-HCT could be beneficial. In contrast, employing allo-HCT instead of CART cells as first CI should be presently restricted to situations where CART cell therapy is deemed not feasible or useful, such as patients with refractory cytopenia or incipient myelodysplastic syndrome. However, allo-HCT remains a standard treatment option as first CI for patients with chemosensitive R/R DLBCL when CARTs are not available or transplantation is preferred by the patient. Continuous collection and analysis of CI outcome data by professional registries appear to be of key importance for developing rational strategies of CI allocation and sequencing.

Efficacy and Safety of CAR-T Therapy with Safety Switch Targeting Bcma for Patients with Relapsed/Refractory Multiple Myeloma in a Phase 1 Clinical Study

Background: Encouraging results are seen from several early phase clinical trials on the cellular immunotherapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) targeting B cell maturation antigen (BCMA) for the treatment of relapsed/refractory (RR) multiple myeloma (MM). We developed an anti-BCMA CAR-T cell product manufactured via gamma-retrovirus-mediated transduction of activated T cells to express a second-generation CAR with the 4-1BB costimulatory domain along with a truncated epidermal growth factor receptor (tEGFR) as a safety switch. The preclinical study confirmed its high reactivity against MM cells. Methods: A phase 1 clinical trial (NCT03093168) has been launched to evaluate the safety and feasibility of this BCMA CAR-T cell product for treating RRMM. The enrolled RRMM patients had received at least 2 prior treatment regimens, including a proteasome inhibitor and an immunomodulatory agent, or are double-refractory, and have over 5% BCMA expression on plasma cells (Nine patient with extramedullary plasmacytoma does not express BCMA). Patients were subjected to a lymphodepleting regimen with Cy (300 mg/m2, d-5 to d-3) and Flu daily for 3 days (25 mg/m2, d-5 to d-3) prior to the CAR-T infusion (d0) at a dose of 9×106CAR+ cells/kg. The efficacy was assessed by the International Uniform Response Criteria for Multiple Myeloma, and the toxicity is graded by CTCAE 4.03. Results: As of March 1th, 2019, 46 patients had been infused with this intended dose of the autologous BCMA CAR-T cells, and 44 patients had reached at least 1 month of follow-up. As of this data cut-off, the overall response rate (ORR) for the 44 evaluable patients was 79.6%, including 2sCRs, 16CRs, 8VGPRs and 8PRs, and 16 patients reached MRD-negative response. The CAR-T cell expansion and persistence were consistently observed throughout these patients. The medianPFS is 15mon, and the median OS result has not been reached (49.16% progression-free survival, and 53.95% overall survival at 24 months). Among the 44 infused patients, 22.7% had grade 1-2 Cytokine release syndrome (CRS ) and 6.8% (3 patients) had grade 3 CRS. No grade 4 CRS reactions developed and all toxicities were fully reversible. Conclusions: Our result demonstrates the high potential of this single CAR-T infusion therapy for RRMM, including 2sCRs, 16CRs and ongoing clinical responses for more than 26 months, with manageable CRS to date. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Disclosures No relevant conflicts of interest to declare.

Durable Remission Achieved from Bcma-Directed CAR-T Therapy Against Relapsed or Refractory Multiple Myeloma

Background: Promising results are seen from several early phase clinical trials on the cellular immunotherapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) targeting B cell maturation antigen (BCMA) for the treatment of relapsed/refractory (RR) multiple myeloma (MM). We developed an anti-BCMA CAR-T cell product manufactured via gamma-retrovirus-mediated transduction of activated T cells to express a second-generation CAR with the 4-1BB costimulatory domain along with a truncated epidermal growth factor receptor (tEGFR) as a safety switch. The preclinical study confirmed its high reactivity against MM cells.Methods: We initiated a phase 1 clinical trial (NCT03093168) to evaluate the safety and feasibility of this BCMA CAR-T autologous cellular therapy for treating RRMM. The enrolled RRMM patients either had received at least 3 prior treatment regimens, including a proteasome inhibitor and an immunomodulatory agent, or were double-refractory, and have over 5% BCMA expression on plasma cells (One patient with extramedullary plasmacytoma does not express BCMA). Patients were subjected to a lymphodepleting regimen with Cy (300 mg/m2, d-5 to d-3) and Flu daily for 3 days (25 mg/m2, d-5 to d-3) prior to the CAR-T infusion (d0) at a dose of 9´106 CAR+ cells/kg. The efficacy was assessed by the International Uniform Response Criteria for Multiple Myeloma, and the toxicity was graded by CTCAE 4.03.Results: As of July 6th, 2018, 17patients had been infused with this intended dose of the autologous BCMA CAR-T cells, and 14 patients had reached at least 1 month of follow-up. As of this data cut-off, the most common non-haematologicalgrade 3 adverse events were pneumonia, hypophosphatemiaand hypocalcemia(two[14%] of 14patients), and fever, cytokine release syndrome,and neurotoxicities (one[7%]of 14patients).All toxicities were fully reversible. The overall response rate (ORR) for the 14 evaluable patients was 79%, including 3 sCRs, 4 CRs and 2 MRD-negative responses (2 VGPR). The CAR-T cell expansion and persistence were consistently observed throughout these patients.The durable remission was observed for two early enrolled patients, with the ongoing objective response (1 sCR and 1 VGPR) lasting more than 15 months.Conclusions: Our result demonstrates the high potential of this single CAR-T infusion therapy for RRMM, including 3 sCRs and ongoing durable clinical responses, with only mild and manageable CRS to date. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. DisclosuresLiu:HRAIN Biotechnology: Employment. Fang:HRAIN Biotechnology: Employment. He:HRAIN Biotechnology: Employment. Xie:HRAIN Biotechnology: Employment. Chen:HRAIN Biotechnology: Employment. Wei:HRAIN Biotechnology: Employment. Tao:HRAIN Biotechnology: Employment. Wang:Immune Design: Equity Ownership; HRAIN Biotechnology: Consultancy, Equity Ownership.

GMP-Grade Manufacturing of T Cells Engineered to Express a Defined γδTCR.

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αβTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αβTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Recent advances in immunotherapy of acute leukemia via chimeric antigen receptor-engineered T cells

Chimeric antigen receptor-engineered T-cell (CAR-T) therapy is a type of adoptive cellular immunotherapy, in which T cells isolated from patients’ or donors’ peripheral blood mononuclear cells are genetically modified, expanded, and reinfused to patients. CAR-T therapy has resulted in encouraging outcomes in patients with B-cell malignancies and is currently being investigated for the treatment of other hematological malignancies. This review discusses the basic structure of CAR, the evolution of CAR structure, the advances in acute leukemia therapy, and the innovative strategies in CAR design and CAR-T production.