CD19 Chimeric Antigen Receptor Research Articles

An assessment model for the efficacy of autologous CD19 chimeric antigen receptor T-cell therapy and relapse or refractory diffuse large B-cell lymphoma risk.

An assessment model for the efficacy of autologous CD19 chimeric antigen receptor T-cell therapy and relapse or refractory diffuse large B-cell lymphoma risk.

Les cellules CAR-T anti-CD19 : prototypes du médicament vivant

Chimeric antigen receptors (CARs) are synthetic receptors that determine the specificity and functions of transduced T cells. They bind cell surface antigens through their extracellular domain and initiate via their intracellular domain cytolytic functions, cytokine secretion and proliferation. The CAR thus specifies the properties of a living drug that is programmed to exert anti-tumor and immunoregulatory effects.After establishing a method to stably integrate foreign genes in primary T lymphocytes in the early 1990s, we identified combinatorial signaling modules that could sustain a prolonged T cell function [17], which underpins an essential feature of a living drug. We further identified CD19 as a promising molecular target, which, based on encouraging preclinical results in mice [30], paved the way for clinical trials.The first clinical studies in patients with relapsed, refractory leukemias and lymphomas, which all made use of CAR T cells produced in the academic setting, yielded remarkable clinical response that were soon confirmed at several centers [2]. CAR T cells targeting CD19 were the first living drugs to be approved by the US Food and Drug Administration in 2017 and the European Medicines Agency in 2018.CD19 CARs have thus spawned a new class of drugs and inspired new applications to treat cancer and autoimmune diseases with engineered immune cells, warranting sustained efforts to lower their cost and increase their availability in equitable fashion.

Aggressive Lymphoma after CD19 CAR T-Cell Therapy

SummaryThe development of a fatal, clonal, autonomously proliferating CD4−CD8− chimeric antigen receptor (CAR)+ peripheral T-cell lymphoma (PTCL) occurred 1 month after a patient received treatment with tisagenlecleucel for relapsed primary central nervous system lymphoma. The PTCL had a clonal T-cell receptor rearrangement, which was already detectable in the apheresis product for CAR T-cell manufacturing and 7 months earlier for autologous transplantation. Somatic DNMT3A and TET2 mutations in CD34+ stem cells and their progeny were detected in the PTCL, in the apheresis specimen that was obtained for CAR T-cell production, and in the autotransplant. The PTCL harbored an additional somatic TET2 mutation, which was already detectable in the CAR T-cell apheresis product and the final CAR T-cell product at very low frequencies, providing evidence that clonal hematopoiesis had contributed to lymphomagenesis.

Shift from Widespread to Tailored Antifungal Prophylaxis in Lymphoma Patients Treated with CD19 CAR T Cell Therapy: Results from a Large Retrospective Cohort

BackgroundPatients undergoing CD19 chimeric antigen receptor (CAR) T-cell therapy exhibit multiple immune deficits that may increase their susceptibility to infections. Invasive fungal infections (IFI) are life-threatening events in the setting of hematological diseases. However, there is ongoing debate regarding the optimal role and duration of antifungal prophylaxis in this specific patient population. ObjectivesThe objective of this study was to provide a comprehensive overview of the evolution of IFI prophylactic strategies over time and to assess IFI incidence rates in a cohort of patients with relapsed or refractory (R/R) lymphoma treated with CAR-T cell therapy. Study DesignSingle-center, retrospective study from a cohort of patients with R/R B-cell lymphoma treated with CD19 CAR-T cell therapy between April 2016 and March 2023. Group A (April 2016- August 2020) consisted of patients primarily treated with fluconazole, irrespective of their individual IFI risk profile. In Group B (September 2020- March 2023) antifungal prophylaxis was recommended only for high-risk patients. ResultsOverall, 330 patients were included. Antifungal prophylaxis was prescribed to 119/142 (84%) patients in Group A and 58/188 (31%) in Group B (p<0.001). Anti-mold azoles were prescribed to 8 (5.6%) patients in Group A and 21 (11.2%) patients in Group B. In Group A, 42 (29%) patients switched to another antifungal, 9 (21%) because of toxicity, with 6 cases of transaminitis and 3 cases of prolonged QTc. In Group B, 21 (11.2%) patients switched the antifungal drug, mainly from fluconazole or micafungin to a mold-active agent following revised guidelines. No difference was found in liver toxicity between the two groups at infusion, day 10, and day 30. No significant differences were observed between the groups. IFI following CAR T-cell therapy were rare, with one case of cryptococcal meningoencephalitis in group A (0.7%) and one case of invasive aspergillosis in group B (0.5%), both occurring in patients on micafungin prophylaxis. ConclusionIn this large single-center cohort of patients with R/R lymphoma treated with CAR T-cells, we show that individualized prophylaxis, alongside careful management of CAR T-cell related toxicities like CRS, was associated with a very low IFI rate, avoiding the risk of unnecessary toxicities, drug-drug interactions, and high costs.

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells.

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR FMC63 as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.

Automated manufacturing and characterization of clinical grade autologous CD20 CAR T cells for the treatment of patients with stage III/IV melanoma.

Point-of-care (POC) manufacturing of chimeric antigen receptor (CAR) modified T cell has expanded rapidly over the last decade. In addition to the use of CD19 CAR T cells for hematological diseases, there is a growing interest in targeting a variety of tumor-associated epitopes. Here, we report the manufacturing and characterization of autologous anti-CD20 CAR T cells from melanoma patients within phase I clinical trial (NCT03893019). Using a second-generation lentiviral vector for the production of the CD20 CAR T cells on the CliniMACS Prodigy®. We demonstrated consistency in cell composition and functionality of the products manufactured at two different production sites. The T cell purity was >98.5%, a CD4/CD8 ratio between 2.5 and 5.5 and transduction rate between 34% and 61% on day 12 (harvest). Median expansion rate was 53-fold (range, 42-65-fold) with 1.7-3.8×109 CAR T cells at harvest, a sufficient number for the planned dose escalation steps (1×105/kg, 1×106/kg, 1×107/kg BW). Complementary research of some of the products pointed out that the CAR+ cells expressed mainly central memory T-cell phenotype. All tested CAR T cell products were capable to translate into T cell activation upon engagement of CAR target cells, indicated by the increase in pro-inflammatory cytokine release and by the increase in CAR T cell amplification. Notably, there were some interindividual, cell-intrinsic differences at the level of cytokine release and amplification. CAR-mediated T cell activation depended on the level of CAR cognate antigen. In conclusion, the CliniMACS Prodigy® platform is well suited for decentralized POC manufacturing of anti-CD20 CAR T cells and may be likewise applicable for the rapid and automated manufacturing of CAR T cells directed against other targets. https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1, identifier NCT03893019.

Chimeric Antigen Receptor T Cells Targeting CD19 and GCC in Metastatic Colorectal Cancer

Chimeric antigen receptor (CAR) T-cell therapy (CART) has transformed the treatment landscape of hematologic cancer, but has negligible effects for adult solid cancers. In this trial, an autologous CAR T-cell product demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer (mCRC). To evaluate the safety and efficacy of guanylate cyclase-C (GCC19) CART in participants with metastatic colorectal cancer (mCRC). This single-arm, nonrandomized, phase 1 trial was conducted at the First Hospital of Jilin University from December 3, 2020, to April 13, 2022. Data analysis was conducted from May 2022 to April 2024. Adults with relapsed and refractory mCRC expressing GCC were treated with GCC19CART, a mixture of autologous CAR T cells transduced with lentiviral vectors expressing genes that encode either CD-19 CAR or GCC CAR. Safety and tolerability of CAR T-cell therapy targeting GCC in patients with mCRC without therapeutic options is capable of conferring a reasonable likeliness of clinical benefit. Other outcomes included objective response rate, progression-free survival, overall survival, and immune activation. Of 15 patients 9 (60%) were women, and the median (range) age was 44 (33-61) years. Treatment with GCC19CART was associated with the development of cytokine release syndrome and diarrhea in most patients, all of which were self-limited and manageable. The objective response rate was 40%, with a partial response in 2 of 8 and 4 of 7 patients treated with either 1 × 106 cells/kg or 2 × 106 cells/kg. Median overall survival was 22.8 months (95% CI, 13.4-26.1) at data cutoff; the median progress-free survival was 6.0 months in the high dose level group (95% CI, 3.0 to not available). The results of this nonrandomized clinical trial suggest that GCC19CART was safe and tolerable in heavily pretreated patients with mCRC and is the first CAR T-cell therapy known to produce objective clinical activity in refractory cancer. Given the paucity of effective therapeutics developed for colorectal cancer in recent decades, the observation that CD-19 CART target engagement can robustly induce GCC19CART target engagement sufficient to produce objective activity may serve as a foundation to develop effective cellular therapy in mCRC and other solid cancers. Chinese Clinical Trial Registry: ChiCTR2000040645.

Peripheral blood-derived PD-1/CD28-CD19-CAR-modified PD-1+ T-cell therapy in patients with solid tumors.

T cells expressing PD-1 in the peripheral blood (PB) of patients with tumors possess therapeutic potential; however, the immunosuppressive, PD1-triggered signaling pathway and limited proliferative capacity of PD-1+ T cells present challenges to their therapeutic application. Here, we observed no discernible distinction between PD-1+ and PD-1- T cells in terms of clonal overlap. However, CD8+PD-1+ T cells from PB and tumor tissues exhibited tighter clustering based on clone size. Single-cell RNA sequencing analysis showed that PD-1+ T cells from PB highly expressed cytotoxicity-related genes and were enriched for T cell activation-related pathways compared with PD-1- T cells from PB or tumor tissues. Consistent with this, PB-derived PD-1+ T cells exhibited strong cytotoxicity towards autologous tumor cells and tumor cell lines. To augment PD-1+ T-cell activity against solid tumors in vivo, we introduced a PD-1/CD28 fusion receptor combined with a CD19 chimeric antigen receptor (CAR) into PD-1+ T cells, which were then expanded in vitro. The modified PD-1+ T cells exhibited superior proliferation and antitumor abilities in vitro. In addition, four patients with cancer were infused with autologous PD-1/CD28-CD19-CAR PD-1+ T cells. None of these patients experienced severe side effects and one patient with melanoma achieved a complete response that was maintained for 6.7 months. The three other patients had stable disease. Collectively, these results suggested that cell therapy with modified PB-derived PD-1+ T cells is both safe and effective, and it may constitute a promising treatment strategy for cancer patients.

Targeting chronic lymphocytic leukemia with B-cell activating factor receptor CAR T cells.

The challenge of disease relapsed/refractory (R/R) remains a therapeutic hurdle in chimeric antigen receptor (CAR) T-cell therapy, especially for hematological diseases, with chronic lymphocytic leukemia (CLL) being particularly resistant to CD19 CAR T cells. Currently, there is no approved CAR T-cell therapy for CLL patients. In this study, we aimed to address this unmet medical need by choosing the B-cell activating factor receptor (BAFF-R) as a promising target for CAR design against CLL. BAFF-R is essential for B-cell survival and is consistently expressed on CLL tumors. Our research discovered that BAFF-R CAR T-cell therapy exerted the cytotoxic effects on both CLL cell lines and primary B cells derived from CLL patients. In addition, the CAR T cells exhibited cytotoxicity against CD19-knockout CLL cells that are resistant to CD19 CAR T therapy. Furthermore, we were able to generate BAFF-R CAR T cells from small blood samples collected from CLL patients and then demonstrated the cytotoxic effects of these patient-derived CAR T cells against autologous tumor cells. Given these promising results, BAFF-R CAR T-cell therapy has the potential to meet the long-standing need for an effective treatment on CLL patients.

Case study of CD19 CAR T therapy in a subject with immune-mediate necrotizing myopathy treated in the RESET-Myositis phase I/II trial

Under compassionate use, chimeric antigen receptor (CAR) T-cells have elicited durable remissions in patients with refractory idiopathic inflammatory myopathies (IIM)1. Here, we report on the safety, efficacy, and correlative data of the first subject with the immune-mediated necrotizing myopathy (IMNM) subtype of IIM who received a fully human, 4-1BBz anti-CD19-CAR T-cell therapy (CABA-201) in the RESET-Myositis™ phase I/II trial (NCT06154252). CABA-201 was well-tolerated following infusion. Notably, no evidence of cytokine release syndrome (CRS) or immune effector-cell associated neurotoxicity syndrome (ICANS) was observed. CK levels decreased, and muscular strength improved post-infusion. Peripheral B-cells were depleted rapidly following infusion, and the subject achieved peripheral B-cell aplasia by day 15 post-infusion. Peripheral B-cells returned at 2 months post-infusion and were almost entirely transitional. Autoantibodies to SRP-9, SRP-72, SRP-54, and Ro-52, decreased relative to baseline whereas antibodies associated with pathogens and vaccinations remained stable. The infusion product consisted of predominantly CD4+ effector memory T-cells & exhibited in vitro cytolytic activity. Post-infusion, CABA-201 expansion peaked at day 15 and was preceded by a serum IFN-γ peak on day 8 with peaks in serum IL-12p40 and IP-10 on day 15. These data detail the safety, efficacy, and pharmacodynamics of CABA-201 in the first IMNM subject.

Autologous CD7 CAR-T cells generated without T cell pre-selection in pediatric patients with relapsed/refractory T-ALL: A phase I trial

Chimeric antigen receptor (CAR) T-cell therapy showed preliminary activity in patients with refractory or relapsed T-cell acute lymphoblastic leukemia (r/r T-ALL). However, many obstacles remain, including manufacturing difficulties and risk of infections. This phase I study (NCT04840875) evaluated autologous CD7 CAR T cells manufactured without pre-selection of healthy T cells in r/r T-ALL. Thirty patients (29 children and one adult) with a median of two lines of prior therapy but without detectable peripheral leukemia were enrolled. Excluding three cases of manufacturing failures, a total of 27 (90%) patients received infusions after products were confirmed free of leukemia contamination, including 16 (59%) meeting planned target doses. Common adverse events within 30 days included grade 3-4 cytopenias (100%), grade 1-2 (70%) and 3-4 (7%; including one dose-limiting toxicity) cytokine release syndrome, grade 1 neurotoxicity (7%), grade 2 infection (4%), and grade 2 graft-versus-host disease (4%). Two patients developed grade 2 infections after day 30. At day 30, 96% responded and 85% achieved CR or CRi. 74% underwent transplantation. Twelve-month progression-free survival with and without censoring transplantation was 22% (95%CI 4-100) and 57% (41-81), respectively. These results support that autologous CD7 CAR T-cell therapy without T-cell pre-selection is feasible in patients with r/r T-ALL.

CD28 hinge used in chimeric antigen receptor (CAR) T-cells exhibits local structure and conformational exchange amidst global disorder

T-cell therapies based on chimeric antigen receptor (CAR) targeting of a tumor-specific antigen offer hope for patients with relapsed or refractory cancers. CAR hinge and transmembrane regions link antigen recognition domains to intracellular signal transduction domains. Here, we apply biophysical methods to characterize the structure and dynamic properties of the CD28 CAR hinge (CD28H) used in an FDA-approved CD19 CAR for the treatment of B-lineage leukemia/lymphoma. By using nuclear Overhauser effect spectroscopy (NOESY), which detects even transiently occupied structural motifs, we observed otherwise elusive local structural elements amidst overall disorder in CD28H, including a conformational switch from a native β-strand to a 310-helix and polyproline II helix-like structure. These local structural motifs contribute to an overall loosely formed extended geometry that could be captured by NOESY data. All FDA-approved CARs use prolines in the hinge region, which we find in CD28, and previously in CD8α, isomerize to promote structural plasticity and dynamics. These local structural elements may function in recognition and signaling events and constrain the spacing between the transmembrane and antigen recognition domains. Our study thus demonstrates a method for detecting local and transient structure within intrinsically disordered systems and moreover, our CD28H findings may inform future CAR design.

Five-Year Follow-Up of Standard-of-Care Axicabtagene Ciloleucel for Large B-Cell Lymphoma: Results From the US Lymphoma CAR T Consortium.

Axicabtagene ciloleucel (axi-cel) is an autologous CD19 chimeric antigen receptor (CAR) T-cell therapy that is approved for the treatment of relapsed or refractory large B-cell lymphoma. Little is known about the long-term survivorship after CAR T-cell therapy. We previously reported the results of 298 patients who were leukapheresed with the intent to receive standard-of-care axi-cel (n = 275 infused) after two or more previous lines of therapy at a median follow-up of 12.9 months. Here, we report extended follow-up of this cohort to a median of 58 months, with a focus on late survivorship events. Among axi-cel-infused patients, progression-free survival at 5 years was 29% and overall survival (OS) at 5 years was 40%. The 5-year lymphoma-specific survival was 53% with infrequent late relapses. However, the 5-year nonrelapse mortality (NRM) was 16.2%, with over half of NRM events occurring beyond 2 years. Patients who were 60 years and older had a lower risk of relapse (P = .02), but a higher risk of NRM compared with patients younger than 60 years (NRM odds ratio, 4.5 [95% CI, 2.1 to 10.8]; P < .001). Late NRM was mainly due to infections and subsequent malignant neoplasms (SMNs). In total, SMNs occurred in 24 patients (9%), including therapy-related myeloid neoplasms (n = 15), solid tumors (n = 7), and unrelated lymphoid malignancies (n = 2). In the standard-of-care setting, axi-cel exhibits outcomes consistent with those reported in clinical trials, with sustained, durable responses observed at the 5-year time point. However, late infections and the development of SMN are key survivorship issues that reduce long-term survival after CAR T-cell therapy, particularly in the elderly.

Expression of concern: “Combined 4-1BB and ICOS co-stimulation improves anti-tumor efficacy and persistence of dual anti-CD19/CD20 chimeric antigen receptor T cells” [Cytotherapy, Volume 23, Issue 8, 2021, 715-723]

Expression of concern: “Combined 4-1BB and ICOS co-stimulation improves anti-tumor efficacy and persistence of dual anti-CD19/CD20 chimeric antigen receptor T cells” [Cytotherapy, Volume 23, Issue 8, 2021, 715-723]

Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between Tcell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) Tcells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR Tcells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR Tcells, CD33PAN CAR Tcells showed greater invitro and invivo efficacy against several human AML cell lines withdiffering levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR Tcells had greater invitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR Tcells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR Tcells further toward possible clinical application.

Tethered IL15-IL15Rα augments antitumor activity of CD19 CAR-T cells but displays long-term toxicity in an immunocompetent lymphoma mouse model

BackgroundAdoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy...

CD19 CAR-T treatment shows limited efficacy in r/r DLBCL with double expression and TP53 alterations

ObjectAutologous CD19 chimeric antigen receptor T-cell therapy (CAR-T) significantly modifies the natural course of chemorefractory diffuse large B-cell lymphoma (DLBCL). However, 25% to 50% of patients with relapsed/refractory DLBCL still do not achieve remission. Therefore, investigating new molecular prognostic indicators that affect the effectiveness of CAR-T for DLBCL and developing novel combination therapies are crucial. MethodsData from 73 DLBCL patients who received CD19 CAR-T (Axi-cel or Relma-cel) were retrospectively collected from Shanghai Tongji Hospital of Tongji University, The Second Affiliated Hospital Zhejiang University School of Medicine, and The Affiliated People's Hospital of Ningbo University. Prior to CD19 CAR-T-cell transfusions, the patients received fludarabine and cyclophosphamide chemotherapy regimen. ResultsOur study revealed that relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) patients with both Double-expression (MYC > 40% and BCL2 > 50%) and TP53 alterations tend to have a poorer clinical prognosis after CAR-T therapy, even when CAR-T therapy is used in combination with other therapies. However, CAR-T therapy was found to be effective in patients with only TP53 alterations or DE status, suggesting that their prognosis is in line with that of patients without TP53 alterations or DE status. ConclusionsOur study suggests that r/r DLBCL patients with both DE status and TP53 alterations treated with CAR-T therapy are more likely to have a poorer clinical prognosis. However, CAR-T therapy has the potential to improve the prognosis of patients with only TP53 alterations or DE status to be similar to that of patients without these abnormalities.

Efficacy and safety of bendamustine-containing bridging therapy in R/R LBCL patients receiving CD19 CAR T-cells.

Bridging therapy (BT) after leukapheresis is required in most relapsed/refractory (R/R) large B-cell lymphoma (LBCL) patients receiving chimeric antigen receptor (CAR) T cells. Bendamustine-containing regimens are a potential BT option. We aimed to assess if this agent had a negative impact on CAR-T outcomes when it was administered as BT. We included R/R LBCL patients from six centers who received systemic BT after leukapheresis from February 2019 to September 2022; patients who only received steroids or had pre-apheresis bendamustine exposure were excluded. Patients were dividedinto two BT groups, with and without bendamustine. Separate safety and efficacy analyses were carried out for axi-cel and tisa-cel. Of 243 patients who received BT, bendamustine (benda) was included in 62 (26%). There was a higher rate of BT progressors in the non-benda group (62% vs. 45%, p = 0.02). Concerning CAR-T efficacy, complete responses were comparable for benda versus non-benda BT cohorts with axi-cel (70% vs. 53%, p = 0.12) and tisa-cel (44% vs. 36%, p = 0.70). Also, 12-month progression-free and overall survival were not significantly different between BT groups with axi-cel (56% vs. 43% and 71% vs. 63%) and tisa-cel (25% vs. 26% and 52% vs. 48%); there were no differences when BT response was considered. CAR T-cell expansion for each construct was similar between BT groups. Regarding safety, CRS G ≥3 (6% vs. 6%, p = 0.79), ICANS G ≥3 (15% vs. 17%, p = 0.68), severe infections, and neutropenia post-infusion were comparable among BT regimens. BT with bendamustine-containing regimens is safe for patients requiring disease control during CAR T-cell manufacturing.

Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

Chimeric antigen receptor (CAR) T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen. scFv selection should involve safety profiling with evaluation of the efficacy/toxicity balance, especially when the target antigen also is expressed on healthy cells. Here, to assess differences in terms of efficacy and on-target/off-tumor effects, we generated five different CARs targeting CD123 by substituting only the scFv. In in vitro models, T cells engineered to express three of these five CD123 CARs were effectively cytotoxic on leukemic cells without increasing lysis of monocytes or endothelial cells. Using the IncuCyte system, we confirmed the low cytotoxicity of CD123 CAR T cells on endothelial cells. Hematotoxicity evaluation using progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR T cells were less cytotoxic on hematopoietic stem cells. Using a humanized mouse model, we confirmed that CD123- cells were not eliminated by the CD123 CAR T cells. Two CD123 CAR T cells reduced tumor infiltration and increased the overall survival of mice in three in vivo models of blastic plasmacytoid dendritic cell neoplasm. In an aggressive version of this model, bulk RNA sequencing analysis showed that these CD123 CAR T cells upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Together, these results emphasize the importance of screening different scFvs for the development of CAR constructs to support selection of cells with the optimal risk-benefit ratio for clinical development.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B-cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B-cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T-cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared to conventional CARs and demonstrated the up-regulated genes associated with T-cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T-cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B-ALL and B-NHL xenograft models while demonstrating persistent T-cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B-cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.