Bispecific Chimeric Antigen Receptor Research Articles

Simultaneous targeting of B-cell malignancies and human immunodeficiency virus with bispecific chimeric antigen receptor T cells.

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State of the Art in CAR-T Cell Therapy for Solid Tumors: Is There a Sweeter Future?

Chimeric antigen receptor (CAR)-T cell therapy has proven to be a powerful treatment for hematological malignancies. The situation is very different in the case of solid tumors, for which no CAR-T-based therapy has yet been approved. There are many factors contributing to the absence of response in solid tumors to CAR-T cells, such as the immunosuppressive tumor microenvironment (TME), T cell exhaustion, or the lack of suitable antigen targets, which should have a stable and specific expression on tumor cells. Strategies being developed to improve CAR-T-based therapy for solid tumors include the use of new-generation CARs such as TRUCKs or bi-specific CARs, the combination of CAR therapy with chemo- or radiotherapy, the use of checkpoint inhibitors, and the use of oncolytic viruses. Furthermore, despite the scarcity of targets, a growing number of phase I/II clinical trials are exploring new solid-tumor-associated antigens. Most of these antigens are of a protein nature; however, there is a clear potential in identifying carbohydrate-type antigens associated with tumors, or carbohydrate and proteoglycan antigens that emerge because of aberrant glycosylations occurring in the context of tumor transformation.

Bispecific CAR T cell therapy targeting BCMA and CD19 in relapsed/refractory multiple myeloma: a phase I/II trial

Despite the high therapeutic response achieved with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapy in relapsed and refractory multiple myeloma (R/R MM), primary resistance and relapse exist with single-target immunotherapy. Here, we design bispecific BC19 CAR T cells targeting BCMA/CD19 and evaluate antimyeloma activity in vitro and in vivo. Preclinical results indicate that BC19 CAR specifically recognize target antigens, and BC19 CAR T cells mediate selective killing of BCMA or CD19-positive cancer cells. BC19 CAR T cells also exhibit potent antigen-specific anti-tumor activity in xenograft mouse models. We conduct an open-label, single-arm, phase I/II study of BC19 CAR T cells in 50 patients with R/R MM (ChiCTR2000033567). The primary endpoint was safety. BC19 CAR T cells are well tolerated with grade 3 or higher cytokine release syndrome in 8% of patients and grade 1 neurotoxic events in 4% of patients, which meet the pre-specified primary endpoint. Secondary endpoints include overall response rate (92%), median progression-free survival (19.7 months), median overall survival (19.7 months) and median duration of response (not reached). Our study demonstrates that bispecific BC19 CAR T cells are feasible, safe and effective in treating patients with R/R MM.

Abstract 1091: Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma

Abstract Goal: Evaluate αvβ3 and HER2 as antigens for a bispecific chimeric antigen receptor T (CAR T) cell therapy in pediatric rhabdomyosarcoma. Background: Current treatments are ineffective against relapsed/refractory or metastatic rhabdomyosarcoma (RMS). Antigen loss or modulation on tumor cells commonly leads to tumor escape across diverse cancer types after CAR T therapy targeting different antigens. We hypothesize that simultaneously targeting two RMS antigens with a bispecific CAR T will reduce the likelihood of tumor escape. The integrin alphavbeta3 (αvβ3) plays a role in cancer progression, metastasis and neovascularization in several malignancies. HER2 also contributes to cancer progression and metastasis. Methods: Control of intramuscular RMS tumors by αvβ3 or HER2 CAR Ts was assessed in NSG mice, with non-specific CD19 CAR Ts as controls. Tumor burden was assessed weekly by bioluminescence imaging. Persistence and phenotype of CAR Ts were evaluated by flow cytometry. CAR T cell infiltration, tumor antigen expression and residual disease were determined by immunohistochemistry. Results: Six of six pediatric patient-derived RMS cell lines express αvβ3 and/or HER2. αvβ3 and HER2 CAR Ts kill RMS cells in vitro at low effector-to-target ratios. αvβ3 and HER2 CAR Ts independently control—and nearly eliminate—orthotopic RMS tumors. Fewer HER2 CAR Ts (5 × 106) are needed to control tumors than αvβ3 CAR Ts (10 × 106), but this is not due to lack of persistence or exhaustion of αvβ3 CAR Ts. Tumor control by αvβ3 CAR Ts is delayed (>21 days) compared to that by HER2 CAR Ts (7 days), and is robust, despite initial tumor progression following treatment. Importantly, despite strong tumor control by both CAR Ts, at 7 weeks post-treatment residual tumor nests are evident by histology. At this time, CAR Ts are still abundant, are not exhausted and express memory markers, suggesting that the remaining tumor cells are no longer susceptible to CAR T killing. We are currently evaluating the hypothesis that the remaining tumor cells have down-regulated antigen expression. Delayed tumor control by αvβ3 CAR Ts was ameliorated by engineering αvβ3 overexpression in RMS tumors. Conclusions: αvβ3 and HER2 CAR Ts control orthotopic RMS tumors by distinct mechanisms with changes in αvβ3 antigen expression, likely due to tumor progression, one prevalent factor. This is consistent with our prior reports of αvβ3 CAR Ts preventing breast cancer metastases, which naturally upregulate αvβ3 compared to the primary tumor. Despite near elimination of tumors, residual nests of tumor cells are likely to rebound as CAR T resistant tumors, similar to what is seen in patients treated with monospecific CAR Ts. These data support the incorporation of αvβ3 and HER2 CAR Ts into a bispecific CAR T approach to mitigate antigen escape. Our data also support investigation into the role of αvβ3 in RMS. Citation Format: Amanda M. Lulu, Nagyeong Park, Joseph J. Caruso, Dustin A. Cobb, Daniel W. Lee. Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1091.

Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) Tcell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR Tcells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

CAR-T cell therapy: a game-changer in cancer treatment and beyond.

In recent years, cancer has become one of the primary causes of mortality, approximately 10 million deaths worldwide each year. The most advanced, chimeric antigen receptor (CAR) T cell immunotherapy has turned out as a promising treatment for cancer. CAR-T cell therapy involves the genetic modification of T cells obtained from the patient's blood, and infusion back to the patients. CAR-T cell immunotherapy has led to a significant improvement in the remission rates of hematological cancers. CAR-T cell therapy presently limited to hematological cancers, there are ongoing efforts to develop additional CAR constructs such as bispecific CAR, tandem CAR, inhibitory CAR, combined antigens, CRISPR gene-editing, and nanoparticle delivery. With these advancements, CAR-T cell therapy holds promise concerning potential to improve upon traditional cancer treatments such as chemotherapy and radiation while reducing associated toxicities. This review covers recent advances and advantages of CAR-T cell immunotherapy.

Current Clinical Application of the CAR-T Cell Therapy

In recent years, the U.S. Food and Drug Administration (FDA) has approved Chimeric Antigen Receptor (CAR)-T cell therapy as a novel approach to treating hematological diseases, including multiple myeloma, large B-cell lymphoma, and B-cell acute lymphoblastic leukemia. Using the patient's genetically altered T-cells, this novel strategy precisely targets antigens linked to cancer. Even with the encouraging outcomes of using bi-specific chimeric antigen receptors to stop tumor antigen evasion, difficulties still arise, especially when dealing with solid tumors. There are several challenges because of the intricate interactions among many elements in the tumor microenvironment, such as immunosuppression, hypoxia, decreased T-cell infiltration, elevated reactive oxygen species, and the erratic nature of tumor-associated antigens. This study examines current efforts to find reliable tumor-associated antigens in an attempt to address issues impeding the efficacy of CAR-T treatment. The fundamental objective is to create CAR-T cells that are economical while being adapted to the distinct characteristics of the tumor microenvironment. By means of a comprehensive analysis of the course of the therapy, the study explores its potential therapeutic uses for a range of tumor types, including solid and hematological malignancies. This work not only sheds light on the enormous obstacles to obtaining precision medicine in cancer treatment, but it also advances our knowledge of the promise that the specific therapy has. The presented results open the door for future developments in the field and provide a nuanced viewpoint on the revolutionary role that the treatment has played in the contemporary cancer scene.

ATA3431: Allogeneic CD19/CD20 Bispecific CAR EBV T Cells for the Treatment of B-Cell Malignancies

BACKGROUND: Chimeric antigen receptor (CAR) therapy targeted against CD19 has yielded remarkable clinical responses in the treatment of relapsed/refractory B-cell malignancies, but about a third of patients ultimately relapse due to the loss or downregulation of CD19. Furthermore, technical and logistical challenges associated with autologous CAR T therapy limits access for a majority of eligible patients. To help address these challenges, we have developed an allogeneic bispecific tandem CAR directed against both CD19 and CD20 (ATA3431) built on our Epstein-Barr virus (EBV) T-cell platform. EBV T cells maintain expression of endogenous T-cell receptors that have inherently low alloreactivity due to their recognition of defined viral antigens. CAR EBV T cells have demonstrated a favorable safety profile with no associated evidence for graft-versus-host disease or high-grade cytokine release syndrome (Curran KJ, 2020). The bispecific tandem CAR design allows either CD19 or CD20 target antigen recognition to activate the CAR EBV T cell, with the goal of extending durability of response and limiting antigen escape. In clinical trials, autologous bispecific CAR T cells were shown to be efficacious and safe (Larson 2023, Shah 2020). Additionally, a 1XX mutated CD3ζ signaling domain and our optimized CAR EBV T-cell platform preserve a central memory phenotype, robust expansion, functional persistence, and potent antitumor activity of ATA3431. METHODS: We generated CD20/CD19 bispecific CAR EBV T cells containing a modified CD3ζ signaling domain 1XX using our EBV T-cell platform. As a benchmark comparator, we generated autologous CD20/CD19 bispecific CAR T cells using a 12-day process derived from OKT3/CD28-activated T cells. The final products were characterized via immunophenotyping and evaluated for in vitro and in vivo response to antigen expressing targets. RESULTS: CD20/CD19 bispecific CAR EBV T cells generated from multiple healthy donors demonstrated stable CAR expression, a high proportion of T-cell memory populations, and consistent HLA-independent killing of CD19 + and/or CD20 + tumor cells over repeated in vitrochallenges. ATA3431 mediated highly potent antitumor activity in an established disseminated tumor model of Burkitt's lymphoma and correlated with long-term in vivo persistence of CAR T cells without additional cytokine support. Moreover, ATA3431-treated animals showed superior tumor growth inhibition compared to animals treated with autologous CD20/CD19 bispecific benchmark CAR T cells. No treatment-related toxicities were observed in animals treated with ATA3431. CONCLUSIONS: Preclinical evaluations of ATA3431 show B-cell tumor growth inhibition without any treatment-related toxicities and thereby support advancing ATA3431 for clinical evaluation. Curran KJ, Sauter CS, Kernan CS, et al. Durable remission following “off-the-shelf” chimeric antigen receptor (CAR) T-cells in patients with relapse/refractory (R/R) B-cell malignancies. 2020 Transplantation & Cellular Therapy Meetings. Orlando. Larson SM, Walthers CM, Ji B, Ghafouri SN, et al. CD19/CD20 bispecific chimeric antigen receptor (CAR) in naive/memory T cells for the treatment of relapsed or refractory non-Hodgkin lymphoma. Cancer Discov. 2023 Mar 1;13(3):580-597. doi: 10.1158/2159-8290.CD-22-0964. PMID: 36416874; PMCID: PMC9992104. Shah NN, Johnson BD, Schneider D, et al. Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial. Nat Med. 2020 Oct;26(10):1569-1575. doi: 10.1038/s41591-020-1081-3. Epub 2020 Oct 5. PMID: 33020647.

Generation of Bi-Specific Chimeric Antigen Receptor (CAR) T Cell Therapy for the Treatment of AIDS-Related B Cell Malignancies from Healthy Donors and HIV Patients

Introduction: Individuals with human immunodeficiency virus (HIV) face a substantially greater likelihood of developing cancers compared to the general population. The prevalence of cancer diagnosis among HIV-positive individuals ranges from 25% to 40%. Non-Hodgkin lymphoma (NHL) stands out as the most prevalent cancer type associated with people living with HIV/AIDS (PLWHA). NHL primarily manifests in two forms in PLWHA: Burkitt lymphoma, which poses an 18-fold higher risk, and diffuse large B cell lymphoma (DLBCL), accounting for 75% of cases. The advent of chimeric antigen receptor (CAR) T cell therapies, notably CD19-targeting CAR T cells, has emerged as a groundbreaking treatment for B cell NHL. Unfortunately, PLWHA has been excluded from participating in all of these clinical trials for years due to safety concerns and the challenges associated with manufacturing CAR T cell products from HIV-positive donors. Recent researches explored the feasibility of developing CD19-targeting CAR T cell products utilizing cells from HIV-positive donors with lymphoma. Although these studies have provided promising evidence that alleviates concerns regarding the safety profile and limited effectiveness of CAR T cell therapy in this particular population, they do not address the underlying HIV condition. We previously generated CAR T cells targeting either lymphoma (CD19-CAR) or HIVgp120 (N6-CAR) that show efficacy against their respective diseases in clinical and preclinical testing, respectively. We hypothesized that a dual construct capable of targeting both antigens could simultaneously target lymphoma cells and HIV-infected cells in a same individual. We report our experience developing a bi-specific N6-huCD19 CAR T cell platform that can target both antigens in a single therapeutic product. Methods: We engineered 3 bi-specific CAR candidate designs including 2 tandem and 1 loop constructs. These constructs were developed by integrating a humanized (hu) CD19 single-chain variable fragment (scFv) and an N6 scFv from an anti-HIV broadly neutralizing antibody (bNAb) into a backbone of a 2 nd-generation CAR construct. The bi-specific tandem CAR constructs consisted of N6 and huCD19 scFvs fused with a G4S linker in either huCD19-N6 or N6-huCD19 orientation. The loop-CAR was generated by fusing huCD19(VL):N6(VH):N6(VL):huCD19(VH) with a whitlow linker. All constructs included the CD4 transmembrane domain, a double-mutated IgG4 Fc spacer, 4-1BB co-stimulatory and CD3-zetta signaling domains, and EGFRt separated by a T2A ribosomal skip sequence (Fig.1). We tested the 3 bi-specific constructs in healthy donor-derived T cells using cytotoxicity co-culture assay against either Raji (CD19+) or 8E5 (gp120+) target cells. We generated N6-huCD19 CAR T cells from HIV-positive patients and confirmed the functionality of these CAR T cells against tumor cells. Results: The functional assessment of all 3 bi-specific CAR constructs revealed their efficacy against CD19 and HIVgp120 antigens. However, the N6-huCD19 tandem CAR T cells demonstrated comparable and better efficacy against both antigens. Notably, the N6-huCD19 tandem CAR T cells exhibited the capability to sequentially target either a single antigen or alternate between the 2 antigens (Fig.2). We successfully generated N6-huCD19 tandem CAR T cells using HIV-positive donors, signifying the practicality of this approach within the context of PLWHA. Remarkably these HIV donor-derived N6-huCD19 CAR T cells effectively killed tumor cells expressing CD19 or HIVgp120 antigens, thus confirming their dual functionality against these 2 antigens. Conclusion: Here we demonstrate the development of the novel N6-huCD19 bi-specific CAR T cells with functionality against both CD19 and HIVgp120 antigens. We also showed the feasibility of generating these CAR T cells from HIV-donors. This strategy could potentially provide life-saving approach tailored for treatment of HIV-patients who develop CD19+ lymphomas.

C-CAR039, a Novel Anti-CD20/CD19 Bi-Specific CAR T-Cell Therapy Shows Deep and Durable Clinical Benefits in Patients with Relapsed or Refractory (r/r) B-Cell Non-Hodgkin Lymphoma (B-NHL) in Long Term Follow up

Background: We previously reported that C-CAR039, an autologous anti-CD20/CD19 bispecific chimeric antigen receptor (CAR) T-cell therapy, demonstrated a favorable safety profile and promising efficacy in patients (pts) with r/r B-NHL. Of the initial 28 pts treated with C-CAR039 with a median of 3 prior lines of therapy. Only 1 patient experienced grade ≥3 cytokine release syndrome (CRS), and no patient reported grade ≥2 immune effector cell-associated neurotoxicity syndrome (ICANS). The overall response rate (ORR) was at 92.6%, with 85.2% complete response (CR). After a median follow-up of 7 months, 74.1% of pts remained in CR, and the Kaplan-Meier (KM) estimation of progression-free survival (PFS) at 6 months was 83.2%. (Liang et al. ASCO 2021. #2507). Here we present the updated results to include more pts (48 pts) and a longer duration of follow-up (median 23.9 months). Methods: This is an open-label, dose escalation and expansion investigator-initiated trial (IIT) of C-CAR039. This trial was conducted at 4 sites to determine the safety and efficacy of C-CAR039 in pts with r/r B-NHL. Pts with r/r diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL) were enrolled and received a single C-CAR039 infusion at a dose of 1.0-5.0x10 6 CAR-T cells/kg after a 3-day conditioning chemotherapy. The primary objective was to assess the safety and tolerability. CRS and ICANS were graded according to ASTCT 2019 criteria. The secondary objectives were to evaluate C-CAR039 efficacy and pharmacokinetics. Response was assessed per Lugano 2014 criteria. Results: Between Nov 5, 2019, and Jan 11, 2022, 48 pts received C-CAR039 manufactured using Miltenyi Biotec CliniMACS Prodigy® System. Of the 48 pts, 44 pts had large B-Cell Lymphoma (LBCL) (DLBCL, n = 37; PMBCL, n = 3; tFL, n=4), 3 pts had FL and 1 patient had MCL. The median age was 55 (range, 25-71) years, and 11 (22.9%) pts were ≥ 65 years. Thirty-six (75%) pts were in Ann Arbor Stage III/IV with 3 median prior lines of therapy (range, 1-7). All pts received anti-CD20 antibody and alkylating agents. Eight (16.7%) pts had prior ASCT, 32 (66.7%) pts were refractory to their last treatment, and 22 (45.8%) pts never achieved CR to their prior therapies. Prior to C-CAR039 infusion, 12 (25%) pts received bridging therapy. As of Jan 15, 2023, 45 pts (93.8%) experienced CRS, of which 44 (91.7%) pts reported grade 1 or 2, only 1 (2.1%) patient experienced grade 3 CRS. Median time to CRS onset was 3 days (range, 1-12), with median duration of 5 days (range, 2-78). Three pts had ICANS at a dose of 5.0x10 6 CAR-T cells/kg, of which 2 were grade 1 and 1 was grade 2. The median time to onset of ICANS was 6 days (range, 5-29), with a median duration of 12 days (range, 3-53). All CRS and ICANS were resolved. Grade ≥3 neutropenia, anemia, thrombocytopenia and infection were reported in 83.3%, 14.6%, 27.1% and 10.4% of pts, respectively. Second primary malignancy after C-CAR039 infusion were observed in 3 pts; 2 acute myeloid leukemia (AML) occurred at 2 and 10 months, and 1 Epstein-Barr virus-positive cytotoxic T-cell lymphoma was at 8 months, the tumor biopsy was tested with qPCR and CAR transgene was negative and none were related to C-CAR039. Twelve deaths occurred, 9 due to disease progression, 2 due to AE of AML, and 1 due to unknown cause. Of the 48 pts, 47 pts were evaluable for efficacy (1 patient with DLBCL had no measurable disease at baseline). The ORR and CR rate among evaluable pts were 91.5% and 85.1% respectively. Median time to first response and CR was 1.0 month (range, 0.9-1.9) and 1.1 month (range, 0.9-8.9), respectively. Of the 43 LBCL pts, the ORR was 90.7%, with 86.0% CR. Median DOR, PFS and OS were not reached. The KM estimation of PFS and OS rate at 24 months was 66.0% (95%CI 53.2%-81.9%) and 77.9% (95% CI, 66.6%-91.1%) respectively. C-CAR039 showed cellular kinetic profile consistent with commercial CAR-T therapies. In 48 evaluable pts, the median T max was 11.5 day (range, 7-31), the median C max was 139,416 copies/g gDNA (range, 708-962,445), and the median AUC 0-28day was 1,513,442 copies/ug∙day (range, 3,818-11,594,912.5). Conclusions: At a longer median follow-up of 23.9 months, C-CAR039 demonstrated a favorable safety profile with deep and durable response in pts with r/r B-NHL, especially in LBCL pts.

Tandem bispecific CD123/CLL-1 CAR-T cells exhibit specific cytolytic effector functions against human acute myeloid leukaemia.

The treatment of refractory and recurrent acute myeloid leukaemia (AML) is still a challenge with poor response rates and short survival times. In an attempt to solve this problem, we constructed a tandem bispecific chimeric antigen receptor (CAR) targeting CD123 and C-type lectin-like molecule 1 (CLL-1), two different AML antigens, and verified its cytotoxic effects in vitro. We established and cultured K562 cell lines expressing both CD123 and CLL1 antigens. Single-target CAR-T cells specific to CD123 and CLL1 were engineered, alongside tandem CD123/CLL1 bispecific CAR-T cells. Flow cytometry was used to determine cell phenotypes, transfection efficiencies, cytokine release, and CAR-T-cell proliferation, and an lactate dehydrogenase assay was used to detect the cytotoxicity of CD123/CLL-1 bispecific tandem CAR-T cells in vitro. Two types of tandem CAR-T cells exhibited significant killing effects on CLL-1 + CD123+ leukaemia cell lines and primary AML tumour cells. The killing efficiency of tandem CAR-T cells in the case of single antigen expression is comparable to that of single target CAR-T cells. When faced with dual target tumour cells, dual target CAR-T cells significantly surpass single target CAR-T cells. CD123/CLL-1 CAR-T cells in tandem targeted and killed CD123- and CLL-1-positive leukaemia cell lines and released a large number of cytokines. CD123/CLL-1 CAR-T cells in tandem can simultaneously target CD123 and CLL-1 on AML cells, demonstrating a significant ability to kill single antigens and multi-target tumour cells. This suggests that CD123/CLL-1 CAR-T cells exhibit significant advantages in the expression of multiple antigens in a wide range of target cells, which may help overcome the challenges posed by tumour heterogeneity and evasion mechanisms.

AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells.

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.

Impact of fludarabine exposure on the depth and duration of lymphopenia and CAR T-cell outcomes.

10033 Background: Chimeric antigen receptor (CAR) T-cells are highly effective for patients with relapsed or refractory B-ALL. Lymphodepletion has been paramount to CAR T-cell expansion and efficacy. Recent data identified an association of fludarabine exposure with reduced relapse rates, longer leukemia free survival, and more prolonged B-cell aplasia following tisagenlecleucel. However, the relationship of fludarabine exposure to lymphopenia and the mechanism by which dosing impacts outcomes following CAR T-cells remains unknown. Methods: We conducted a retrospective review of children and young adults with B-ALL who received fludarabine for lymphodepletion (LD) prior to infusion of anti-CD19, anti-CD22, or anti-CD19/22 bispecific CAR T-cells. The primary objective of this study was to investigate the impact of fludarabine on the depth and duration of lymphopenia. Secondary objectives included examining the impact of fludarabine exposure on: 1) overall clinical response, and 2) CAR T-cell expansion. All patients in the analysis cohort received fludarabine 25 mg/m2/day x 3 days ( = 75 mg/m2 cumulative dosing). Fludarabine exposure (AUC per dose) for each patient was predicted using a validated population PK model and analyzed with various responses/endpoints using the 3-day cumulative exposure/patient. Results: Of 137 children and young adults, the median cumulative AUC was 8.3 mg*hr/mL (range, 3.00-13.83 mg*hr /mL). The overall complete response (CR) rate was: 67.9% (93/137). The median duration of lymphopenia (defined as an absolute lymphocyte count (ALC) £ 150 K/uL) was 5 days (range, 0-15); and the median ALC on Day 0 was 80 K/uL (range, 0-1250 K/uL). When stratified by the median fludarabine exposure, there was no difference in depth or duration of lymphopenia or CAR T-cell expansion collectively or per trial. The median fludarabine exposure also did not differ between responders and non-responders. However, depth and duration of lymphopenia did differ in the CD22 CAR T-cell trial. Specifically, in those achieving CR, the duration of lymphopenia was longer than in non-responders (7 days versus 4 days, p = 0.030). Similarly, CAR T-cell expansion was higher if lymphopenia duration was greater than 5 days (p = 0.019). These findings were not seen across our less persistent CD19 or CD19/22 CAR T-cell trials. Conclusions: Although lymphodepletion impacts CAR T-cell response, we did not find an association between CR rate, depth of lymphopenia, or duration of lymphopenia with varying fludarabine exposures. This raises additional questions regarding the impact of LD therapy on CAR T-cell outcomes—especially since a more prolonged lymphopenia was associated with high CR rates and greater peak expansion in at least one of our studies (CD22 CAR T-cells). Future analysis will evaluate the impact of fludarabine on Day 0 cytokines and other immune parameters. Clinical trial information: NCT03448393 .

CD19/CD22 bispecific chimeric antigen receptor‑NK‑92 cells are developed and evaluated.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have improved the outcomes of patients with B cell leukemia and lymphoma. However, their applications and positive outcomes remain limited. CAR-T cells are currently restricted to autologous blood as their source and their use can lead to downregulation of CD19 expression along with complications such as graft-versus-host disease and cytokine release syndrome. The present study aimed to develop anti-CD19/CD22 bispecific CAR structures using an anti-CD22 monoclonal antibody clone from chickens and analyze them in natural killer (NK)-92 cells, a human NK cell line, in vitro and in vivo. Anti-CD19/CD22 CAR-NK-92 cell cytotoxicity was assessed by the survival of target cells and counted using flow cytometry. Anti-CD22/CD19 and loop-structured anti-CD19/CD22 bi-specific CAR-NK-92 cells showed improved efficacy against OCI-Ly7 cells, a human B cell lymphoma cell line, compared with other CAR structures. These results demonstrate the potential of anti-CD19/CD22 bispecific CAR-NK cells and suggested that optimizing CAR structures in NK cells can improve the efficacy of CAR therapy.

Abstract CT023: Phase 1 trial of CD19/CD20 bispecific chimeric antigen receptor-engineered naïve/memory T cells for relapsed or refractory non-Hodgkin lymphoma

Abstract Despite excellent responses to anti-CD19 chimeric antigen receptor (CAR)-T cell therapy in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL), more than half of patients will relapse due to poor CAR-T persistence or CD19 antigen escape. Engineering naïve/memory T (TN/MEM) cells with a bispecific anti-CD19/CD20 CAR could improve outcomes by mitigating these limitations. Here we report an update of the first-in-human phase 1 clinical trial with TN/MEM cells expressing a bispecific anti-CD19/CD20 CAR (CART19/20) for patients with R/R NHL (NCT04007029). Eligible patients were ≥18 years old with R/R diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma (PMBCL) after ≥2 prior lines of therapy, or with mantle-cell lymphoma (MCL), follicular lymphoma (FL), or CLL/SLL after ≥3 prior lines of therapy. Prior CAR-T cell therapy was excluded, but other forms of CD19- or CD20-targeted therapies were allowed. Autologous leukocytes were obtained by leukapheresis and sorted for CD14-/CD25-/CD62L+ TN/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Bridging therapy after leukapheresis was allowed. Lymphodepletion chemotherapy with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day was administered for 3 days from Day −5 to Day −3 prior to CAR-T cell infusion. The primary endpoint was safety and the secondary endpoints were ORR, progression free survival (PFS), overall survival (OS), and CART19/20 transgene persistence. As of December 19, 2022, 11 patients have been treated with CART19/20 cells (7 DLBCL, including 4 transformed FL and 1 PMBCL; 3 FL; and 1 MCL). Patients were enrolled at two dose levels, including 50 × 106 CAR+ cells (8 patients) and 200 × 106 CAR+ cells (3 patients). The median age was 58 (range: 34 to 70). All patients had stage IV disease and 9 of 11 patients received bridging therapy. Six patients had grade-1 cytokine release syndrome (CRS), without any occurrence of higher-grade CRS. Neurotoxicity was not observed. Ten of the 11 patients achieved an objective response (91% ORR), with 8 patients (73%) achieving complete response (CR). One patient with FL relapsed at 18 months, received a second CART19/20 cell infusion (112 × 106 CAR+ cells), and re-achieved a CR that has persisted for >6 months at last follow-up. With a median follow-up of 20.9 months (range: 3.4 - 37 months), 7 patients remain in CR with a median PFS of 18.2 months (95% CI 3.4 months - not estimable) and the median OS was not reached. This phase 1 study demonstrates robust safety and tolerability of CART19/20 T-cells in patients with R/R NHL, without occurrence of severe CRS or neurotoxicity. CART19/20 cells, utilizing a bispecific CAR and TN/MEM cells, may be an effective strategy to overcome the challenges of poor CAR T-cell persistence and antigen escape. Citation Format: Benjamin R. Puliafito, Christopher Walthers, Brenda Ji, Sanaz N. Ghafouri, Jacob Naparstek, Jacqueline Trent, Jia M. Chen, Mobina Roshandell, Caitlin Harris, Mobina Khericha, Thomas Schweppe, Beata Berent-Maoz, Stanley B. Gosliner, Amr Almaktari, Melanie Ayala Ceja, Martin S. Allen-Auerbach, Jonathan Said, Karla Nawaly, Monica Mead, Sven de Vos, Patricia A. Young, Caspian Oliai, Gary J. Schiller, John M. Timmerman, Antoni Ribas, Yvonne Y. Chen, Sarah M. Larson. Phase 1 trial of CD19/CD20 bispecific chimeric antigen receptor-engineered naïve/memory T cells for relapsed or refractory non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT023.

Effect of Special Structure Bi-Specific Chimeric Antigen Receptor T Cell on Tumor Cells

To explore and design a novel bi-specific chimeric antigen receptor (CAR) structure. To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo. Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79b scFv, CD8 hinge & TM-4-1BB-CD3ζ and/or CD3ε chain intracellular regions were constructed and prepared. CAR-19-79b cells were obtained. Five kinds of CAR-T cells were co-incubated with the 3M-CD19-CD79b-Luc target cells. Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared. The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells. And the treatment efficacy was evaluated. At the same time, other targets were used in this structure. With the same methods, the stability and effectiveness of the structure were verified. CAR-19-79b-T cells were cultured for 7 days, the expression rates of CAR-19 and CAR-79b were 21.6%-36.3% and 21.7%-37.8%, respectively. The killing rates of 5 kinds of CAR-19-79b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1. Among them, the killing rates of CAR-19-79b-T cells with No. III and No. IV structures were the strongest. After co-incubation with 3M-CD19-CD79b-Luc target cells, the amount of IFN-γ and TNF-α secreted by CAR-T cells with CAR IV and CARV structures was the lowest. And there was no significance between the two groups (P>0.05). CAR IV cells with remarkable killing effect and low secretion factor had obvious therapeutic effect on Daudi-Luc leukemia mice, extending the survival period of mice to 64 days. And all mice in the T cell control group died at 41.0±2.4 days. The CAR-19-BCMA-T and CAR-19-22-T with the same structure showed significant killing ability and low cytokine expression levels. A novel bi-specific CAR structures was successfully designed, which could efficiently kill the corresponding tumor cells and secrete less cytokines (such as TNF-α, IFN-γ). Moreover, it shows obvious therapeutic effect on Daudi lymphoma mouse model. The bi-specific CAR structure shows good killing specificity and safety.

LigandCD70.CAR As a Platform for Dual-Targeting CAR T Cells for Acute Myeloid Leukemia

Chimeric antigen receptor (CAR) T cells have demonstrated remarkable potency in acute lymphoblastic leukemia (ALL), but similar success has yet to be seen in acute myeloid leukemia (AML). Due to the variable expression of targetable antigens on AML blasts and leukemic stem cells, treatment with single-antigen targeting immunotherapy may select for antigen-negative clones that ultimately cause disease progression or relapse. An appropriate combination of two different CAR-binders and co-stimulatory domains could both increase tumor control and reduce antigen escape. We utilized a binder derived from human CD27 protein, the only known ligand of CD70, to generate the LigandCD70.CAR, which demonstrated superior in vivo activity against AML compared to conventional antibody-derived CARs (Sauer et al 2021). As the LigandCD70.CAR consists of a full length CD27 fused with CD3-zeta, this design takes advantage of the physiologic receptor-ligand interaction of CD70/CD27 to bind with high affinity to CD70+ cells and provides CD27-mediated costimulation to the T cells. These characteristics combined with the high expression of CD70 on more than 80% of AML make the LigandCD70.CAR a promising candidate for combination with other CARs to target AML. We evaluated the LigandCD70.CAR in combination with a CLL-1.CAR and a CD123.CAR, as both have shown preclinical efficacy and are currently being tested in clinical trials. We co-transduced activated T cells with LigandD70.CAR and CLL-1 CAR to generate LigandCD70/CLL-1 bispecific CAR (CLL-1.BiCAR) T cells. CLL-1.BiCAR T cells had comparable in vitro expansion to single-CAR expressing T cells but with enhanced cytotoxicity against CLL-1+ AML cell lines compared to single CLL-1.CARs, even if CD70 expression on tumor cells was low. In xenograft AML models of CD70-low CLL-1-high tumors (HL60), CLL-1.BiCAR T cells enhanced tumor control, survival and T cell persistence compared to single CLL-1.CAR T Cells (Figure 1). In order to determine whether the LigandC70.CAR can enhance other CARs, we co-transduced ligandCD70.CAR with CD123.CAR (Riberdy et al 2020) to generate a Ligand.CD70/CD123 bispecific CAR (CD123.BiCAR). The in vitro expansion and cytotoxicity was comparable to single-CAR expressing T cells. Treatment with CD123.BiCAR T cells significantly improved in vivo survival and tumor control in a xenograft model of AML with CD70-low/CD123-high target cells. In summary, LigandCD70.CAR co-expressed with either CLL-1.CAR or CD123.CAR demonstrate enhanced anti-tumor efficacy compared to single-targeting CLL-1.CAR and CD123.CAR, even in CD70-low tumors. The simultaneous targeting of two AML antigens may thus mitigate the risk of antigen-negative relapse. These data indicate that the Ligand70.CAR combined with other AML targeting CARs could be an effective therapeutic option for AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multispecific CAR T Cells Deprive Lymphomas of Escape via Antigen Loss.

Chimeric antigen receptor (CAR) modified T cell therapy has transformed the management of relapsed/refractory B cell malignancies. Despite high overall response rates, relapse post CAR T treatment remains a clinical challenge. Loss of target antigen, specifically CD19, is one well-defined mechanism of disease relapse. The mechanism of CD19 loss and which patients are at higher risk of CD19 loss remain poorly understood. To overcome CD19 loss, CARs targeting multiple antigens are being tested in clinical trials. CD19/20 and CD19/22 bispecific CARs demonstrate cytotoxicity against CD19-negative cells in preclinical studies. These CARs have also shown efficacy, safety, and a relatively low rate of CD19-negative relapse in phase I trials. These small studies suggest that multispecific CAR T cells can deprive lymphomas of escape via antigen loss. However, the selection of an ideal target, the right CAR construct, and whether these multispecific CARs can induce long-term remissions are still under investigation.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “off-target” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.MethodsThe peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups.ResultsThe mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal “off-target” combination existed on CoG133-CAR T cells and major organs.ConclusionOur study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

Current Status and Perspectives of Dual-Targeting Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Hematological Malignancies.

Simple SummaryApproved chimeric antigen receptor (CAR) T cells recognize and bind to only one tumor target (single-targeted CAR T cells, Si-CART) on cancer cells by the special receptor and followed with activation, thus removing cancers from patients. However, cancer cells can resist the treatment of Si-CART by hiding the single target to prevent the recognition and survive, causing recurrence of cancers in patients. Dual-targeting CAR T-cell therapy contains CAR T cells recognizing two targets on cancer cells and can overcome the resistence in cancers to Si-CART. We summarize the latest preclinical and clinical development of dual-targeting CAR T-cell therapies to provide perspectives for optimization and shed light on new hope for patients after the treatment of Si-CART.Single-targeted chimeric antigen receptor (CAR) T cells tremendously improve outcomes for patients with relapsed/refractory hematological malignancies and are considered a breakthrough therapy. However, over half of treated patients experience relapse or refractory disease, with antigen escape being one of the main contributing mechanisms. Dual-targeting CAR T-cell therapy is being developed to minimize the risk of relapse or refractory disease. Preclinical and clinical data on five categories of dual-targeting CAR T-cell therapies and approximately fifty studies were summarized to offer insights and support the development of dual-targeting CAR T-cell therapy for hematological malignancies. The clinical efficacy (durability and survival) is validated and the safety profiles of dual-targeting CAR T-cell therapy are acceptable, although there is still room for improvement in the bispecific CAR structure. It is one of the best approaches to optimize the bispecific CAR structure by boosting T-cell transduction efficiency and leveraging evidence from preclinical activity and clinical efficacy.