anti-CD19 CAR-T Research Articles

PIT565, a First-in-Class Anti-CD19, Anti-CD3, Anti-CD2 Trispecific Antibody for the Treatment of B Cell Malignancies

The robust expression of the CD19 antigen in ALL and NHL has resulted in the development of highly effective therapeutics targeting CD19 by leveraging redirected T cells, which include the bispecific T-cell engager blinatumomab and CD19-directed CAR-T therapies axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene maraleucel. However, T cell exhaustion contributes to treatment failure following anti-CD19 bispecific T-cell engager and CAR-T therapies. To circumvent this issue, we have developed a first-in-class anti-CD19, anti-CD3, anti-CD2 IgG-like trispecific antibody, PIT565, that simultaneously engages CD19+ on tumor cells, CD3 (TCR signaling component) and CD2 (a costimulatory receptor) on T cells, which leads to redirected T-cell cytotoxicity towards CD19-positive malignant B cells. CD2 signaling has been associated with non-exhausted T cell phenotype. The interaction between CD2 and its ligand, CD58, has been shown to be important for tumor cell killing by CD19 CAR-T cells, while the loss of CD58 expression on lymphoma cells correlates with resistance and relapse in patients treated with CD19 CAR-T therapy. Therefore, CD2 co-stimulation via PIT565 may overcome T cell exhaustion and increase the depth and duration of response in patients compared to CD3 bispecifics. A redirected T cell cytotoxicity (RTCC) in vitro assay, employing co-cultures of CD19+ tumor cells and primary T-cells, demonstrated dose-dependent T-cell proliferation, cytokine production, and tumor cell lysis, which was more potent than the bispecific counterpart. Furthermore, in repeat-challenge RTCC assays, PIT565 mediated more sustained T cell activity in tumor cell killing and proliferation compared to control bispecifics. In addition, PIT565 demonstrated a more robust anti-tumor response than a control bispecific against an established diffuse large B cell lymphoma tumor in the human peripheral blood mononuclear cell adoptive transfer model. In safety and pharmacology studies, PIT565 showed IgG-like pharmacokinetics and an expected pharmacology and tolerability profile similar to CD3 bispecifics in cynomolgus monkey. Taken together, the findings from the preclinical studies suggest that PIT565 may achieve deeper and more durable responses compared to competitor CD3 bispecifics. First-in-human trial of PIT565 (NCT05397496) has been initiated and will be conducted in patients who are diagnosed with relapsed and/or refractory adult NHL after receiving two or more lines of chemotherapy and patients with relapsed and/or refractory B-ALL.

Anti CD19 CART Therapy Following Allogenic Hematopoietic Stem Cell Transplantation Improved the Survival of Patients with BCR::ABL1-like Acute Lymphoblastic Leukemia

Anti CD19 CART Therapy Following Allogenic Hematopoietic Stem Cell Transplantation Improved the Survival of Patients with BCR::ABL1-like Acute Lymphoblastic Leukemia

Novel Protease-Mediated Double Antigen Recognizing Chimeric Antigen Receptor (CAR) Enhances Directionality of CAR-T Cell Activity and Improves Target Cell Specificity

Recently, Chimeric Antigen Receptor (CAR)-T cell therapies show excellent therapeutic effect against various hematologic malignancies, including B-cell leukemia, B-cell lymphoma, and multiple myeloma. Expansion of target diseases of this attractive immunotherapy is one of the most important issues. To identify the novel specific tumor antigen as CAR-T cell target is the standard way to accomplish this goal. However, recent CAR-T cells recognize single tumor antigen, so normal tissues expressing the target antigen are possible to be attacked by CAR-T cells. For example, anti-CD19 CAR-T cells against B-cell malignancies kill not only tumor cells but also normal B-cells, so patients receiving the CAR-T cells showed severe immune deficiency. Improvement of target cell recognition system of CAR-T cells minimizes this adverse effect and contributes to expand target diseases. To improve target-cell-specificity of CAR-T cell, we established a protease-mediated regulatory CAR, named "Scissors-CAR" (panel A). This regulatory CAR is constituted with extracellular antigen recognition portion and cytoplasmic Human Immunodeficiency Virus protease (HIVPR) catalytic domain. When CAR-T cells expressing both anti-A Scissors-CAR and anti-B Counterpart-CAR that has HIVPR recognition polypeptide contact with target tumor expressing both antigen-A and B, these two CARs interact on cell membrane. This interaction induces Scissors-CAR-mediated cleavage of target polypeptide and structural change of Counterpart-CAR. Previously, we reported double antigen recognition system using Scissors-CAR. Previous Counterpart-CAR named "Signal-CAR" had HIVPR recognition polypeptide upstream of CD3-zeta, so Scissors-CAR-mediated cleavage broke the CAR protein structure and suppressed CAR-T cell activity (panel B, named "Negative system"). This system allowed CAR-T cells to directional attack against target cells expressing only antigen-A and protect the cells expressing both antigen-A and B. Here, we established another double-antigen recognition system using Scissors-CAR. In many cases including both hematologic malignancies and solid tumors, additional ectopic antigens are expressed on malignant cells. If CAR-T cells empowered to directional attack against target cells expressing both antigen-A and B, much more tumors can be treated with this tumor immunotherapy (panel C, named "Positive system"). To establish this directional CAR-T cells, we constructed novel Counterpart-CAR named as "Activating-CAR" constituted with target recognition portion, co-stimulatory domain, CD3-zeta, and protein destabilization motif known as degron. Between CD3-zeta and degron motif, HIVPR recognition polypeptide was inserted. In vitro analysis revealed that this degron motif destabilized Activating-CAR and HIVPR-mediated cleavage and that release of degron motif increased Activating-CAR stability. Furthermore, cell proliferation of Jurkat cells expressing both anti-CD19 Activating-CAR and anti-HER2 Scissors-CAR was enhanced co-cultivated with target cells expressing both CD19 and HER2. From these results, combined use of Activating-CAR and Scissors-CAR allowed CAR-T cells to directionally activate against target cells expressing two antigens recognized by each CAR. By choosing the Counterpart-CAR from "Signal-CAR" or "Activating-CAR", this protease-mediated double antigen recognition system could optimize target-cell-specificity of CAR-T cells for the individual tumor and contribute to expand the target tumors of CAR-T cell therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DHODH: A Promising New Therapeutic Target in Pediatric T-ALL

Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in children and young adults. When compared to B-ALL, T-ALL has a higher rate of induction failure as well as relapse, with very poor overall survival in both scenarios. T-ALL has not benefitted from the same immune-mediated therapies (e.g., anti-CD19 CAR-T cells) and poses an ongoing treatment challenge as well as an unmet therapeutic need. Here, we demonstrate that inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme that converts dihydroorotate to orotate as part of the de novo synthesis of uridine, has a robust anti-leukemia effect in both in vitro and in vivo models of T-ALL. DHODH is ubiquitously-expressed, and inhibition of DHODH (DHODHi) leads to rapid depletion of pyrimidine ribo- and deoxyribonucleotides. A cell's ability to tolerate periods of pyrimidine starvation is dependent on a variety of alternative salvage pathways and is not well-understood. Clinical grade DHODH inhibitors are currently under investigation in trials assessing their effect in myeloid malignancies, where they are also known to have a pre-clinical anti-leukemic effect. We sought to understand the mechanism underlying the sensitivity of T-lymphoblasts to DHODHi, and to develop new DHODHi therapeutic combinations for patients with T-ALL. It is known that the intracellular concentration of nucleotides is high in activated T-lymphocytes, underlying the fact that two low-potency DHODHi's are approved for the treatment of autoimmune T-cell diseases (e.g., teriflunomide for multiple sclerosis and leflunomide for rheumatoid arthritis). We hypothesized that T-cells may have a particular addiction to de novo nucleotide synthesis and that this can be exploited as a vulnerability by the small molecule inhibition of DHODH. We demonstrated that T-ALL is highly sensitive to both chemical and genetic DHODHi using a variety of in vitro models (Figure A). Furthermore, we show that T-lymphoblasts respond to DHODHi by undergoing rapid cell cycle arrest and apoptosis. Additionally, we quantified the changes in intracellular nucleotides using PCA extraction and high-performance liquid chromatography (HPLC). The abundance of UTP, CTP, ATP, and GTP were determined across a panel of T-ALL cell lines at baseline and following treatment with a small molecular inhibitor of DHODH. The depletion of intracellular pyrimidines is rapid, falling to ~30% of baseline at 6 hours and confirming that the extracellular salvage is not able to meet the nucleotide demands of these cells even in the presence of complete media. Of note, the concentration of ATP and GTP are unaffected, confirming the specificity of DHODH on de novo pyrimidine synthesis as well as the viability of the cells over the 24-hour treatment. In several in vivo T-ALL models, we demonstrated that DHODH inhibition (specifically using the small molecule inhibitor brequinar) led to rapid, robust disease response (Figure B). These single agent treatment studies (brequinar, 50 mg/kg given every 3-days) were initiated between day 7 - 14 when the leukemias had engrafted and were established with detectable disease in the bone marrow. Three model systems were used: (1) a genetically engineered mouse model (GEMM) of immature T-ALL in which an activated intracellular NOTCH is expressed by retroviral transduction, (2) a transgenic mouse model of mature T-cell leukemia/lymphoma driven by the HTLV1 protein Tax, and (3) a human PDX from a young man with relapsed/refractory T-ALL. DHODH inhibitor therapy was effective in all three models, though clearly and most dramatically prolonged the survival in the GEMM model, driven by dysregulated NOTCH. We are now focused on elucidating the mechanisms by which T-lymphoblasts are so incredibly sensitive to inhibition of DHODH. New data suggests that changes in mitochondrial membrane potential and mitochondrial mass following exposure to DHODHi may offer additional insights into this sensitivity. We are also studying DHODHi in combination with other approved and experimental agents toward the goal of a new therapeutic combination that is safe, well-tolerated, and efficacious for this patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Making Anti-CD19 CAR-T Cell Therapy Accessible and Affordable: First-in-Human Phase I Clinical Trial Experience from India

Background: Chimeric Antigen Receptor (CAR) T cells therapy has shown remarkable success in haematological malignancies. However, complexity of the manufacturing platform and exorbitant costs of the available therapy limits their accessibility in limited resource settings including India. Recently, we developed a novel humanized anti-CD19 CAR-T cell therapy (HCAR19) and showed its efficacy in preclinical models (Dwivedi et al, Mol Cancer Ther. 2021). Here we report our experience of HCAR19 manufacturing in an integrated platform for first-in-human Phase-I clinical trial. Further, we performed the cost analysis of CAR-T delivery from development to clinic. Methods: Lymphocytes were collected from 17 patients of r/r B cell malignancies (DLBCL, transformed follicular lymphoma, PMBCL and B-ALL) through apheresis (Fresenius Fomtek or Spectra Optia) upon receiving the regulatory approvals from central and local agencies. CD3+ T cells were enriched and activated using CTS™Dynabeads™ CD3/CD28 for 36-48 hours followed by consecutive double transduction with CD19 encoding lentivirus vector. Final product was harvested, cryopreserved and stored in ultra-low temperature until further use. All steps were carried out in closed gas permeable bags. Final product quality control (QC) assays were performed by flowcytometry, ELISA, analytical and cell-based assays. The cost of CAR T cell manufacturing was calculated for unit economics on fully scale production model by standard costing methodology. The final unit economics included cost of goods and services (COGS), overhead costs, financing costs of infrastructure, R&D and clinical trials costs. The model was scaled towards a 200 patient per annum in a centralized manufacturing facility. Results: Of the 19 patients enrolled in the study, only 17 patients underwent apheresis procedure for harvest of total 5-10 X 109 mononuclear cells. HCAR19 cells from all 17 patients were manufactured successfully in functionally closed integrated manufacturing platform. The required HCAR19 dose was achieved within 8 days of culture (dosage range: 1.78 x 106 - 17 x 106 viable HCAR19/kg). The transduction efficiency of HCAR19 in r/r DLBCL, r/r PMBCL and r/r B-ALL was 37.7 ± 6.64%, 28.8 ± 8.81% and 34.6 ± 9.72% (Mean ± SEM) respectively . The HCAR19 showed a robust expansion (~20-fold) ex vivo and maintained the presence of potent anti-tumor subsets like naïve and central memory T cells (Figure 1a). Final product from all 17 patients qualified the QC release criteria for safety, identity, purity and potency as per specifications. Finally, the unit economics in fully-scaled production model indicated the estimated cost of CAR-T cell delivery was approximately 30,000 USD (or 25,00,000 INR) per dosage including logistics, lentiviral vector cost and quality control (Figure 1b). The major costs incurred were towards consumables, overhead and financing costs of clinical trials and infrastructure. There was very minor components of logistics and utility costs. More importantly, the major contributors of overall low cost of CAR-T delivery in our settings were frugal R&D costs in an academic setting, scalable integrated manufacturing platform, and highly qualified yet economical manpower. Conclusions: We developed a robust, scalable and highly affordable CAR-T cell therapy platform (product and process development, and first-in-human clinical study), and proven its clinical delivery successfully with our first indigenous HCAR19 product. Given the benefit of affordable platform to broader socio-economic base of patients, the cell and gene therapy will be poised for a transformational change as a mass therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A First-in-Human Phase 1, Multicenter, Open-Label Study of CB-010, a Next-Generation CRISPR-Edited Allogeneic Anti-CD19 CAR-T Cell Therapy with a PD-1 Knockout, in Patients with Relapsed/Refractory B Cell Non-Hodgkin Lymphoma (ANTLER Study)

Background: Autologous CAR-T cell therapies have shown significant benefit in the treatment of adults with relapsed/refractory B cell non-Hodgkin lymphoma (r/r B-NHL). However, these CAR-T cell therapies have inherent challenges including functional deficiencies in the patient's T cells that could yield an inconsistent or impaired product. In addition, the extended time and complexity in manufacturing the product can limit patient access and potentially require bridging therapy. CB-010 is an allogeneic, off-the-shelf anti-CD19 CAR-T cell therapy derived from healthy donor T cells; it is the lead program in Caribou's allogeneic CAR-T cell therapy platform. Patients (pts) receiving CB-010 do not require leukapheresis or bridging therapy. A next-generation CRISPR-Cas9 genome-editing technology developed at Caribou, which uses Cas9 and CRISPR hybrid RNA-DNA (chRDNA) guides to significantly reduce off-target editing, was implemented to generate 3 genome edits in the manufacture of CB-010: (i) knockout of the TRAC gene to eliminate TCR expression to reduce the risk of graft-versus-host disease (GvHD), (ii)site-specific insertion of a CD19-specific CAR into the TRAC locus, and (iii) knockout of the gene encoding PD-1, designed to limit premature CAR-T cell exhaustion and enhance antitumor activity. Statistically significant preclinical survival benefit across B-NHL subtypes and longer-duration efficacy in vivo compared to similar CAR-T cells without a PD-1 knockout support the clinical evaluation of CB-010. Methods and Study Design: CB-010 is being investigated in a multicenter, Phase 1 clinical trial in pts with r/r B-NHL. A 3+3 dose escalation design followed by expansion at the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) is being utilized. Primary objectives are to determine the safety and tolerability of CB-010 and the RP2D. Additional key objectives include preliminary antitumor activity and pharmacokinetics (PK). After serially receiving lymphodepletion therapy with cyclophosphamide (60mg/kg/day for 2 days) and fludarabine (25mg/m2/day for 5 days), pts receive a single dose infusion of CB-010 and are followed for safety and efficacy. Initial antitumor activity, as well as time to progression, overall survival, and disease response as measured by Cheson criteria will be evaluated. The study is actively enrolling patients. Cohort 1 at an initial single dose of 40 million CAR-T cells has been completed successfully with 6 patients. Dose escalation of CB-010 is ongoing, and patients are being enrolled in cohort 2 to receive a single dose of 80 million CAR-T cells. The ANTLER Phase 1 trial is registered on clinicaltrials.gov (NCT04637763).

High Safety and Efficacy of CRISPR-Based Non-Viral PD-1 Locus Specific Integrated Anti-CD19 CAR-T Cells (BRL-201) in Treating Relapsed or Refractory Non-Hodgkin's Lymphoma: First-in-Human Phase I Study

High Safety and Efficacy of CRISPR-Based Non-Viral PD-1 Locus Specific Integrated Anti-CD19 CAR-T Cells (BRL-201) in Treating Relapsed or Refractory Non-Hodgkin's Lymphoma: First-in-Human Phase I Study

The Safety and Efficacy of p CAR-19B, a Scfv Humanlized CD19-Directed CAR-T for Patients with Relapsed/Refactory (R/R) CD19+ B-ALL

Introduction: Two murine anti-CD19 scFv based chimeric antigen receptors T cell (CAR-T) products have been approved by FDA for children and adults R/R B-ALL patients, with about 50%-70% complete remission (CR) rates. Humoral and/or cellular immune responses against murine scFv may attenuate the efficacy. To reduce immunogenicity of murine scFv, we developed an optimal humanized anti-CD19 CAR-T product named pCAR-19B, which have a CAR structure including humanized CD19 specific scFv following 4-1BB and CD3ζ cytoplasmic domain. Here we report the preliminary safety and efficacy data for the adults R/R B-ALL patients in a phase I clinical trials. Methods: In the phase Istudy (NCT04888442), Eligible patients were histologically confirmed CD19+ B-ALL, KPS＞60, relapsed or refractory to standard therapies. Patients received single-dose of pCAR-19B at dose level 1 (DL1) (0.6×106 CAR/kg), level 2 (DL2) (2×106 CAR/kg) after fludarabine (25-30 mg/m2 × 3 days) and cyclophosphamide (10-20mg/ kg× 3 days) based pre-condition. Toxicity was graded by CTCAE, CRS and ICANS were graded by ASTCT criteria. Primary endpoint is the incidence of dose-limiting toxicities (DLTs) in the first 28 days. Secondary endpoints are cellular kinetics, overall response rate (CR/CRi), duration of response, and overall survival. Results: As of June 30, 2022, nine patients (3 in DL1, 6 in DL2) were infused with pCAR-19B and all patients have finished at least 6 months follow up visit. Demographics, baseline disease, prior treatment and special concerned adverse events are presented in the table. CAR-T-related adverse events occurred in 9 patients (9/9, 100%), no adverse events leading to patient withdrawal from the study occurred. 8 patients (8/9, 89%) experienced cytokine release syndrome (CRS), including 5 (5/9, 56%) grade 1, 3 (3/9, 33%) grade 2, and no grade 3 or higher CRS occurred. Two patients (2/9, 22%) experienced immune effector cell-associated neurotoxicity syndrome (ICANS), including 1 (1/9, 11%) grade 1, 1 (1/9, 11%) grade 2, and no grade 3 or higher ICANS occurred. No DLTs were reported. 100% (9/9) CR/CRi was achieved in the first month after CAR-T infusion. The relapse-free survival (RFS) is presented in the figure. The median OS and RFS is not reached yet. No anti-CAR antibody was detected in any patients in 1M and 3M after CAR-T infusion. Conclusion: pCAR-19B has demonstrated a good safe profile and 100% CR rate in phase I study for adults R/R CD19+ B-ALL. The muti-center pivotal study is going to recruit patients soon in China. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Subsequent Secondary CAR-T Cell Therapy Was Superior to Chemotherapy in Patients with Refractory/Relapsed B-Cell Lymphoma after Failure of Anti-CD19 CAR-T Cell Therapy

Subsequent Secondary CAR-T Cell Therapy Was Superior to Chemotherapy in Patients with Refractory/Relapsed B-Cell Lymphoma after Failure of Anti-CD19 CAR-T Cell Therapy

Safety and Feasibility of Anti-CD19 CAR-T Expressing IL-7 and CCL19 in Patients with Relapsed or Refractory Large B-Cell Lymphoma

Chimeric antigen receptor (CAR) T cell therapy provides a potential curative option for patients with relapsed or refractory large B-cell lymphoma (R/R LBCL). However, there are major limitations of this therapy which result in treatment resistance in B-cell malignancies, including inadequate CAR-T persistence, poor trafficking and tumor infiltration, the immunosuppressive microenvironment and frequent tumor antigen escape. Recently, several armored CAR T-cells are being developed for potential improved efficacy relative to conventional CAR T cells. Here, we engineered a CD19-spectific CAR-T cell capable of constitutive secretion of interleukin (IL)-7 and chemokine (C-C motif) ligand (CCL)-19 (7 × 19 CAR-T) and performed a multicenter phase 1b clinical trial of 7 × 19 CAR-T cell therapy in patients with R/R LBCL (NCT03929107 and NCT04833504). The 7 × 19 CAR-T cells showed that a favorable safety profile, with grade 3 cytokine release syndrome occurred in 5 (12.8%) patients and grade 3 or higher neurotoxicity occurred in 4 (10.3%) patients. The overall response rate evaluated at 3 months post-single infusion was 79.5% (complete remission, 56.4%; partial response, 23.1%). With a median follow-up of 32 months, the median progression free survival was 13 months and median overall survival was not reached, with the estimated rate of 53.8% (95% CI, 40.3% to 72.0%) at two years. Together, this long-term follow-up data from a multicenter study suggests that 7 × 19 CAR-T cells can induce durable responses with a median overall survival of greater than 2 years, and has a manageable safety profile in patients with R/R LBCL.

SC-gtCAR19, a Non-Viral Genome Targeting Anti-CD19 CAR-T Candidate in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia: Updated Results from Long-Term Follow-up

Introduction It has been made great commercial success in hematological malignancies by chimeric antigen receptor (CAR) T cell therapy. However, high rate of adverse events (AEs) still remains an issue. Non-viral Genome Targeting CAR (gtCAR) delivery technology, which uses CRISPR-Cas9 to insert CAR DNA to TRAC locus specifically in human T cells, produces moderate CAR-T cells compared with conventional ones. Earlier results of SC-gtCAR19, an anti-CD19 gtCAR-T candidate from an investigator initiated Phase I clinical trial (ChiCTR2000031942) demonstrated a favorable benefit-risk profile in relapsed/refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL) patients. Reported here are updated long-term follow-up results. Objective The primary objective is to assess safety. Secondary objective is to evaluate efficacy. Level of CAR-T cell in blood was explored. Method This is a single center, open-label and single-arm trial. Patients or allogeneic donors underwent leukapheresis and their CD3+ T cells were selected and modified by non-viral vector to produce SC-gtCAR19. A standard lymphodepletion chemotherapy was performed. The median dose was 3.89×106CAR+ cells/kg (0.58-8.31×106/kg). AEs were graded according to CTCAE v5.0. Cytokine Release Syndrome (CRS) and neurological events (NEs) were graded by ASTCT consensus grading. Relapse defined as bone marrow (BM) blast or B-ALL related mutation gene positive from nonexistence regardless of specific value, and extramedullary disease (EMD) occurrence. Result As of May 1st 2022, twenty-two r/r B-ALL patients received SC-gtCAR19 (Table 1). The median age was 29 (13-61), and the median number of previous regimen was 5 (2-16). The median baseline BM blast after preconditioning was 44.1% (0.3-99%), particularly 73% of patents with high disease burden (>25% leukemic blast). Five patients (23%) once had stem cell transplantation (SCT), and 14 (64%) have B-ALL related chromosomal and molecular abnormalities. One patient has EMD. No patient has been treated by any other CD19 related therapy before enrollment. The most common AEs of any grade were anaemia (100%), pyrexia (91%), neutrophil count decreased (91%), and white blood cell count decreased (86%) (Table 2). CRS occurred in 20 patients (91%), and 4 of them (18%) with grade 3 CRS. The median time to CRS onset was 7 days (IQR 7-14), and the median duration of any grade CRS was 3 days (IQR 2-5). Three patents developed NEs, one was grade 2 and 2 were grade 3 (temporary seizure, related to infection and patient's cerebral infarction before enrollment). No fatal or life-threatening events happened. No Tocilizumab administered, four patients used vasopressors and 2 steroids for treatment of CRS. All patients were eligible for efficacy analysis. At Day 28, the overall response rate was 100%, with 9 (41%) patients achieving complete remission (CR), ten (45%) achieving complete remission with incomplete haematological recovery (CRi), and 3 (14%) with blast-free hypoplastic. Among them, twenty patients (91%) achieved minimum residual disease (MRD) negative CR, and those 2 patients with MRD positive CR were assessed undetectable BM MRD in Day 90. Patient with EMD, the nodal lesions in her breast disappeared at Day 60. At a median follow-up of 19.9 months (4.5-26.6), nineteen patients relapsed, within which 7 (36.8%) antigen escape relapse. The median duration of remission for 19 patients achieving CR/CRi was 8 months (2.5-NE). All patients received a consolidation treatment by Lenalidomide 3 months after infusion on condition of CR status until disease progression/death, and all of them did not receive SCT afterwards before relapse. As of the data cutoff, two patients (9%) had ongoing CR with median follow-up of 22.4 months. The median relapse-free survival for 19 patients was 8.9 months (2.9-NE) (Figure 1). The median OS was 24.2 months (3.7-NE) (Figure 2). CAR T cell level measured by cell copies percentage per peripheral blood mononuclear cells peaked at median 12 days (IQR 10-13) (Figure 3). The median peak percentage of copies was 53% (IQR 26.5-72.1%). Conclusion This study is the first clinical trial applied a gtCAR-T candidate for r/r B-ALL treatment. SC-gtCAR19 has a better risk-tolerance. Despite TCR gene knocked-out, gtCAR-T cell proliferation and its function both short and long term may not be influenced. We illustrate that this novel approach is feasible in terms of manufacture and clinical application. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deriving Predictive Features of Severe CRS from Pre-Infusion Clinical Data in CAR T-Cell Therapies

Background Cytokine release syndrome (CRS) is a life-threatening toxicity of chimeric antigen receptor (CAR) T-cell therapy that limits the widespread use of this lifesaving therapy. While only a portion of CAR T-cell therapy patients experience CRS, there is a limited understanding of its risk factors (Fajgenbaum et al. NEJM 2020). Known markers of severe CRS lack specificity or require central lab facilities, making them unsuitable for safety surveillance during trials or real-time clinical decision making. Earlier work by the authors suggests that patterns in longitudinal measurements of common laboratory markers distinguish patients who develop severe CRS after CAR-T infusion from others(Strait et al. JCO 40, 2022). Using pre-infusion clinical data from anti-CD19 CAR-T trials from the Medidata Enterprise Data Store (MEDS), we derived features that capture longitudinal patterns in common laboratory markers and vitals before and after the start of lymphodepleting chemotherapy (LDC). We fit penalized logistic regression models to these derived features using the occurrence of severe CRS (grade 3 and higher) as a binary response variable. Below we describe these novel features and their importance in predicting severe CRS following CAR-T therapy. Method Subjects in the anti-CD19 CAR-T trials obtained from MEDS had relapsed or refractory disease in one of the following: B Acute Lymphoblastic Leukemia (B-ALL), Mantle Cell Lymphoma (MCL), Non-Hodgkin Lymphoma (NHL), or Diffuse Large B-cell Lymphoma (DLBCL). Laboratory markers with repeated measurements between screening and the start of LDC as well as between LDC start and CAR-T infusion that were present in all studies were used to compute the pre and post LDC features respectively. Only post LDC features were derived for heart rate, systolic blood pressure, weight and temperature (vitals) measured between LDC start and CAR-T infusion. Age, weight and ECOG at enrollment, race, number of prior therapies, response to last therapy and prior allogeneic or autologous hematopoietic stem cell transplant were included as baseline features. Since the prevalence of grade 3+ CRS varies between the studies, we split the data into training (80%) and test (20%) partitions by stratifying on grade 3+ CRS per each study. Penalized logistic regression models were fit on the training partition and evaluated on the test partition. Results The dataset consisted of 361 patients: 19.1% B-ALL, 39.9% DLBCL, 21.6% MCL and 19.4% comprising patients with Transformed Follicular Lymphoma, Primary Mediastinal B-cell Lymphoma and High Grade B-cell Lymphoma; mean age 55.8 (SD=14.1) years, 69.5% males. 25.2% had CRS 3+ with a median time-to-event of 4 days. At enrollment, all patients had an ECOG score ≤ 1 with 47.7% patients having an ECOG = 0. During their last therapy prior to CAR-T cell therapy, 51.5% patients had progressive disease and 23.3% patients had a response followed by relapse. Feature importance values in the pre and post LDC intervals are shown in Figure 1. Pre-LDC, the levels of leukocyte count, hemoglobin, aspartate aminotransferase and the change in alanine aminotransferase, albumin, and platelets were the strongest predictors of developing grade 3+ CRS. Post-LDC but pre-CAR-T therapy, the levels of alanine aminotransferase, alkaline phosphatase, hemoglobin, platelets and albumin and the change in platelets, albumin, and creatinine were the strongest predictors. High levels of Chloride, both pre and post LDC correlate with lower risk of grade 3+ CRS. Among vitals, heart rate, temperature and systolic blood pressure, as well as the rate of change in weight post LDC are the strongest predictors of grade 3+ CRS. While partial response or disease progression in prior therapy is correlated with grade 3+ CRS, the model with baseline features alone performed worst on the test partition. Adding predictors based on laboratory markers and vital signs, results in about 10% improvement in the AUROC over the baseline model (Table 1). Conclusions Absolute levels as well as the rate of change in laboratory markers and vitals may indicate immune pathway activity that correlates with a future grade 3+ CRS event. Modeling the pre and post LDC kinetics can yield novel predictors for grade 3+ CRS.The association between pre-infusion temporal patterns in clinical variables and grade 3+ CRS implies that dynamic tracking of patient status will be key to effective mitigation of grade 3+ CRS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CNCT19 CAR T-Cell Therapy in Relapsed or Refractory B-Cell Lymphoma: Long-Term Results of a Pilot Prospective Clinical Study

Background: Chimeric antigen receptor (CAR) T-cell targeting tumor cells depends on the single chain variable fragment (scFv) against the specific tumor antigen. Most anti-CD19 CAR-T products have the scFv deriving from hybridoma clone FMC63. We developed a new hybridoma clone HI19α which binds to distinct CD19 episodes. CNCT19 was a second-generation CAR T-cell with scFv derived from clone HI19α and 4-1BB/CD3-ζ costimulatory domain. The results of CNCT19 in pediatric and adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia had previously been reported [Runxia Gu et al. J Hematol Oncol. 2020 Sep 7;13(1):122]. Herein, we reported the outcome and long-term follow-up of a pilot study administrating CNCT19 in relapsed or refractory B-cell lymphoma. This trial was registered at www.clinicaltrials.gov as NCT03029338. Methods: Patients with R/R CD19-positive B-cell lymphoma were eligible for this study. Subjects with active central nervous system involvement were excluded. Lymphodepleting chemotherapy comprising fludarabine (25~30mg/m2, day -4,-3,-2) and cyclophosphamide (350mg/m2 day -4, -2) was administrated once the CNCT19 product was available. We infused CNCT19 cells in three regimens: three-day split dose (10%-30%-60%, patient (P)1-P10), two-day split dose (40%-60%, P11-P17), and single dose (P18-20). Results: From May 2017 through March 2019, 20 patients were enrolled. Diagnosis of histological subtypes included diffuse large B-cell lymphoma not otherwise specified (DLBCL, NOS) in 14 (70%) patients, DLBCL transformed from chronic lymphocytic leukemia in 1 (5%) patient, follicular lymphoma (FL) grade 3b in 1 (5%) patient, and mantle cell lymphoma (MCL) in 4 (20%) patients. Considering the differences in efficacy and survival between the population with MCL and those with LBCL, we here reported the results of 16 patients who had diseases of DLBCL or FL 3b. The median age was 51 (range 16 to 69), and 62.5% were male. The majority (87.5%) of patients had advanced stage disease, and 11 (68.8%) had non-GCB immunophenotype. Double expressor lymphoma (DEL, cut-off for MYC, >=40%, BCL2, >=50%) was identified in 11 (68.8%) patients, with one of them having MYC and BCL2 rearrangement. TP53 mutation and/or deletion were detected in 5 of 6 (83.3%) patients. The patients had received a median of 3 (range 2 to 7) lines of prior therapy. Thirteen (81.3%) patients had diseases refractory to last-line therapy. The median dose of infused CNCT19 cells was 2.48×106 (range 1.87 to 5.06×106) per kilogram of body weight. Cytokine release syndrome (CRS) occurred in 11 (68.8%) patients, all of whom were grade 1. Only one patient experienced CAR-T-cell-related encephalopathy syndrome (CRES), manifesting as distal muscle weakness of the bilateral upper limb, and he recovered completely after corticosteroids treatment. Twelve (75.0%) patients achieved a response after CNCT19 infusion, with a complete response (CR) rate of 37.5%. The ORR and CR rate at 3 months was 62.5% and 31.3%, respectively. Two patients with ongoing partial remission underwent allogeneic hematopoietic stem-cell transplantation, and both of them died of progressive disease at 6 months and 11 months post CNCT19 infusion, respectively. As of July 5, 2022, the median follow-up from CNCT19 infusion to the data-cutoff date was 44.5 months. The median progression-free survival and overall survival were 6.5 (95% CI 1.0 to NA) months and 12.7 (95% CI 2.9 to NA) months, respectively. Six (37.5%) patients remained alive by data cut-off. Conclusions: CNCT19 cells exhibited comparable efficacy and favorable safety in patients with R/R DLBCL and FL3b. A phase 2 clinical trial (NCT04586478) of administrating CNCT19 in R/R large B-cell lymphoma and high-grade B-cell lymphoma is underway.

Targeted immunotherapy efficacy analysis in patients with relapsed/refractory B cell acute lymphocytic leukemia

Objective: Comparison of conventional chemotherapy and immunotargeted therapy efficacy in patients with relapsed/refractory (R/R) acute B cell leukemia (B-ALL) . Methods: The clinical data of 212 patients with R/R B-ALL in the Affiliaed Cancer Hospital of Zhengzhou University from January 2008 to July 2020 were analyzed retrospectively to compare the response rate and survival time difference between conventional chemotherapy and immunotargeted therapies (antiCD19 CAR-T and CD3CD19 bi-specific antibody blinatumomab) , and to explore the related factors affecting prognosis. Results: The CR rate of patients with R/R B-ALL treated with anti-CD19 CAR-T cells was 80.4% , patients treated with blinatumomab was 62.5% , and patients treated with chemotherapy was 38.6% . There was significant difference in the CR rate among the three therapies (P<0.001) . CAR-T cells 1-year OS rate was 41.5% , which was significantly higher than that of the chemotherapy group (10.3% ) (P<0.001) . The 1-year PFS rate of CAR-T cells (30.1% ) was also significantly higher than that of the chemotherapy group (9.7% ) (P<0.001) . The median OS of patients with bridging allo-HSCT after CR treatment by CAR-T cells was 18.5 months, which was higher than that of patients without allo-HSCT (8 months) (P=0.027) . The median PFS of patients with allo-HSCT was 17 months, which was higher than that of patients without allo-HSCT (4 months) (P=0.001) . The 1-year OS rate of patients treated with blinatumomab was 14.3% , which was higher than that of the chemotherapy group (10.3% ) (P=0.018) . The 1-year PFS rate (14.6% ) was also higher than that of the chemotherapy group (9.7% ) (P=0.046) . The median OS and median PFS of patients with bridging allo-HSCT were 13 and 11 months, respectively, which was higher than that of patients without allo-HSCT (9.5 and 6 months) . The cytokine release syndrome (CRS) incidence in patients with R/R B-ALL treated with anti-CAR-T cells was 89.8% . Grades 3-4, grade 2, and grade 1 CRS were experienced by 30.2% , 11.3% and 58.5% patients, respectively. Only three patients (37.5% ) with blinatumomab developed CRS, all of which were grade 1. Conclusion: The response rate and survival rate of patients with R/R B-ALL treated with CD19 CAR-T cells and blinatumomab were significantly better than those treated with conventional chemotherapy.

CAR-T cells: from cancer to autoimmunity

Charlotte Barker, Editor, BioInsights,&#160;speaks with&#160;Dimitrios Mougiakakos, Professor &amp; MD,&#160;Department of&#160;Hematology &amp; Oncology, University Hospital Magdeburg&#160;Professor Dimitrios Mougiakakos, MD has been the director of the Hematology and Oncology department at the Otto-von-Guericke University in Magdeburg, Germany since November 2021. He studied medicine in Hannover and performed his residency at the Universities of Freiburg, Regensburg, and Erlangen, Germany. During his postdoctoral studies at the Karolinska Institute in Stockholm, Sweden he focused on immunotherapeutic concepts against malignancies and against hyper-inflammatory conditions. Moreover, he has a special interest in understanding the metabolic perspective of heterocellular crosstalk and to use these insights for improving immunotherapies. Following his return to Erlangen he was strongly involved in developing and leading the local cell therapy program, where patients with refractory SLE were treated with anti-CD19 CAR-T cells for the first time.Dimitrios Mougiakakos joins us to discuss the exciting potential for CAR-T cell therapy for autoimmune disease and how his team approached GMP manufacturing in the clinical setting.

Decentralized manufacturing of anti CD19 CAR-T cells using CliniMACS Prodigy®: real-world experience and cost analysis in India.

Chimeric Antigen Receptor (CAR) T cell therapy is an accepted standard of care for relapsed/refractory B cell malignancies. However, the high cost of existing industry-driven centralized production makes this therapy unaffordable in low and middle-income countries. Decentralized or point of care manufacturing has the potential to overcome some of these challenges. Here we demonstrate a decentralized manufacturing process for anti-CD19-CAR-T cells using a fully automated closed system (Miltenyi CliniMACS Prodigy®) is feasible in a developing country setting. Validation run data, as part of a pre-clinical trial safety evaluation, demonstrates the successful and robust manufacturing of anti-CD19 CAR-T cells with T cell expansion of 25 to 47-fold. The median transduction efficiency was 48.8%, with a median viability of 98% and fulfillment of all standard release criteria assays for clinical application. Evaluation of production costs in an academic, not for profit setting in India provide a benchmark for low and middle-income pricing which could greatly increase access to this therapy. Based on our analysis, the cost per product would be approximately $35,107 US dollars. Our data highlights the safety, efficacy, and reproducibility of the process for use in planned future clinical trials.

Allogeneic Hematopoietic Cell Transplantation after Chimeric Antigen Receptor T Cell Therapy in Large B Cell Lymphoma

Anti-CD19 chimeric antigen receptor T cell (CAR-T) therapy has transformed the care of patients with relapsed/refractory large B cell lymphoma (LBCL). However, approximately 60% of CAR-T recipients ultimately will experience disease recurrence or progression. Salvage therapies after CAR-T treatment failures are of limited efficacy and have a short duration of response. The objective of the present study was to evaluate the role of allogeneic hematopoietic cell transplantation (allo-HCT) after CAR-T therapy in LBCL patients. This was a multicenter observational study reporting the outcome of 39 adult LBCL patients who underwent allo-HCT following anti-CD19 CAR-T therapy. The median patient age was 47 years (range, 20 to 68 years). HLA-matched sibling, HLA-matched unrelated, and alternative donors were used in 36%, 36%, and 28% of transplantations, respectively. Conditioning regimens were primarily of low or intermediate intensity. Disease status at allo-HCT was complete response in 41%, partial response in 38%, and progressive disease in 21%. Allo-HCT was performed at a median of 127 days (range, 82 to 206 days) after CAR-T therapy. A high incidence of hepatic toxicity (28%), including sinusoidal obstruction syndrome (15.4%; 95% confidence interval; [CI], 6.2% to 28.5%), was observed. The 1-year cumulative incidence of grade II-IV and grade III-IV acute graft-versus-host disease (GVHD) was 38.5% (95% CI, 23.2% to 53.6%) and 15.4% (95% CI, 6.1% to 28.5%), respectively. The 2-year cumulative incidence of moderate-severe chronic GVHD was 11.1% (95% CI, 3.3% to 24.3%). Overall, 2-year nonrelapse mortality and relapse/progression incidence were 26% (95% CI, 13% to 41%) and 43% (95% CI, 27% to 59%), respectively. With a median follow-up of 32 months, the 2-year overall survival (OS) and progression-free survival (PFS) were 45% (95% CI, 31% to 66%) and 31% (95% CI, 19% to 50%), respectively. In multivariable analyses, pre-HCT elevated lactate dehydrogenase level and transformed lymphoma were predictive of OS and PFS, respectively. Our data suggest that allo-HCT after anti-CD19 CAR-T treatment failure is feasible with a relatively promising efficacy but possibly high toxicity rate.

Treatment of SLE with anti-CD19 CAR-T cells.

Treatment of SLE with anti-CD19 CAR-T cells.

A polyamine-centric, blood-based metabolite panel predictive of poor response to CAR-T cell therapy in large B cell lymphoma

Anti-CD19 chimeric antigen receptor (CAR) Tcell therapy for relapsed or refractory (r/r) large B cell lymphoma (LBCL) results in durable response in only a subset of patients. MYC overexpression in LBCL tumors is associated with poor response to treatment. We tested whether an MYC-driven polyamine signature, as a liquid biopsy, is predictive of response to anti-CD19 CAR-T therapy in patients with r/r LBCL. Elevated plasma acetylated polyamines were associated with non-durable response. Concordantly, increased expression of spermidine synthase, a key enzyme that regulates levels of acetylated spermidine, was prognostic for survival in r/r LBCL. A broad metabolite screen identified additional markers that resulted in a 6-marker panel (6MetP) consisting of acetylspermidine, diacetylspermidine, and lysophospholipids, which was validated in an independent set from another institution as predictive of non-durable response to CAR-T therapy. A polyamine centric metabolomics liquid biopsy panel has predictive value for response to CAR-T therapy in r/r LBCL.

208MO Immune related adverse events and biomarkers after Anbal-cel, novel anti-CD19 CAR-T therapy with dual silencing of PD-1 and TIGIT: Updates from phase I study

Anbal-cel is a novel 2nd generation autologous CD19 CAR-T cell therapy which has been knock-downed for PD-1 and TIGIT using OVIS platform and demonstrated the superior T-cell functionality compared with CD19 CAR-T cells at preclinical studies.