Dual Chimeric Antigen Receptor Research Articles

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

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

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

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

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

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

Successful 24-Hours Manufacture of Anti-CD19/CD22 Dual Chimeric Antigen Receptor (CAR) T Cell Therapy for B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Introduction Multiple issues arise for a wider application of chimeric antigen receptor (CAR) T cell therapy including manufacturing time and antigen escape. Here we report data on an anti-CD19/CD22 dual CAR-T (GC022F) therapy based on a novel manufacturing platform, from a phase I clinical study (NCT04129099) in treating patients with B-cell acute lymphoblastic leukemia (B-ALL). Methods Peripheral blood (PB) mononuclear cells were obtained by leukapheresis. T-cells were separated and transduced with lentivirus that encodes a CD19/CD22 directed 4-1BB: ζ CAR. GC022F cells were manufactured using a novel FasTCARTM platform which takes 24 hours, while the conventional CD19/CD22 dual CAR-T (GC022C) cells used as parallel control in the preclinical study were manufactured by conventional process which typically takes 9-14 days. The phase I dose escalation study was initiated to explore the safety and efficacy of GC022F in patients with B-ALL. All patients received a conditioning regimen of IV fludarabine (25mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days prior to GC022F infusion. Results When compared with the GC022C, GC022F cells showed 1) less exhaustion as indicated by lower percentage of PD-1+LAG3+ cells following co-culturing with tumor cells, 2) younger phenotypes as demonstrated by more abundant T central memory cells (Tcm; CCR7+CD45RA+ or CD45RO+CD62L+), 3) higher expansion fold at in vitro culture, and 4) high anti-leukemia efficacy in mice model (Fig.1). Comparing in vivo efficacy of GC022F with GC022C cells at lower doses, GC022F treatment were more potent and could reduce tumor burden earlier and faster, and led to significantly prolonged overall survival of the experimental animals. From Nov. 2019 to Jun. 2020, 9 children and 1 adult with B-ALL were enrolled and infused with GC022F, 2 in low-dose (6.0×104/kg), 7 in medium dose (1.0-1.5×105/kg), 1 in high-dose (2.25×105/kg). Patients' median observation time was 99 (14-210) days on the day of cut-off. Characteristics of enrolled patients are shown in Table 1. The median age was 10 (3-48) years, and the median bone marrow (BM) blasts were 21.0 (0.1-63.5) % at enrollment. Three patients had prior CD19 CAR-T cell therapy history and one of whom had prior allogeneic hematopoietic stem cell transplantation (allo-HSCT). After infusion, the median peak of circulating CAR-T cell copy number was 2.29 ×105 copies/µg genomic DNA (0.0014-5.66), which occurred around day 14 (day10 - day 28). Importantly, GC022F persisted well in PB with a median of 2.40×105 copies/µg genomic DNA (0.75-3.98) on day 28 in 5 of 9 patients with available 4 weeks of cellular kinetics data. GC022F exerted a superior safety profile with no observed grade ≥ 3 cytokine release syndrome (CRS) and neurotoxicity in all patients. Among those 6 patients with CRS, only 1 at high dose level had grade 2 CRS; only 1 developed grade 1 neurotoxicity. After GC022F infusion, 6/6 patients with BM blasts > 5% at enrollment achieved complete remission (CR) by day 28, 5/6 with minimal residual disease (MRD)-negative CR. For those 4 patients with MRD positive disease at enrollment, 3 became MRD-negative CR by day 28, 1 had persist MRD positive disease and withdrew from the study by 2 weeks. Five of 8 MRD-negative CR patients subsequently made a choice to pursue consolidation allo-HSCT with a median time interval of 57 (48-71) days post GC022F infusion and all have remained in MRD-negative CR except 1 died from graft-versus-host disease (GVHD) and infection 143 days post GC022F infusion. Of the other 3 patients without allo-HSCT, 2 relapsed with CD19+/CD22+ disease at 12-16 weeks follow-up, including the patient with prior history of CD19 CAR-T treatment and transplant. Conclusion This study demonstrated that anti-CD19/CD22 dual CAR T-cells could be successfully manufactured by FasTCARTM technology in 24 hours, with younger and less exhausted phenotypes. Moreover, the Dual FasTCAR-T cells showed more potent efficacy in xenograft mouse model compared to the conventional dual CAR-T cells. Our clinical data demonstrate that GC022F is safe and efficacious in treating patients with CD19+CD22+ B-ALL. More data on additional patients and longer observation time are needed to further evaluate CD19/CD22 dual FasTCAR-T cell product. Disclosures Cai: Gracell Biotechnologies Ltd: Current Employment. Wang:Gracell Biotechnologies Ltd: Current Employment. Chen:Gracell Biotechnologies Ltd: Current Employment. Ye:Gracell Biotechnologies Co., Ltd.: Current Employment. He:Gracell Biotechnologies Co., Ltd.: Current Employment. Cao:Gracell Biotechnologies Ltd: Current Employment. Sersch:Gracell Biotechnologies Co., Ltd.: Current Employment.

Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis in vivo.

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4+ T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4+ T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

Abstract 6606: Develop dual-targeted CAR-T cells to achieve RCC cures

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. ccRCC is characterized by the loss of chromosome 3p, where the von-Hippel-Lindau (VHL) gene is found. Its gene product, pVHL suppresses the hypoxia inducible factor (HIF) transcription factors and the deletion of VHL leads to HIF hyperactivation, upregulating the expression of many downstream genes involved in angiogenesis, metabolism, and cell-cycle regulation. Two gene products, carbonic anhydrase IX (CAIX) and CD70 which we are pursuing as therapeutic targets are downstream genes of the HIF-1α pathway and thus commonly overexpressed in ccRCC. Despite the development of checkpoint blockade inhibitors (CBIs) for the treatment of this disease, curative therapies are rare. Chimeric Antigen Receptor (CAR)-T cells are a new type of “living drug”, in which T cells are engineered to express a single chain variable antibody fragment (scFv) linked to an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB (second generation), or both (third generation) costimulatory domains. CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment (TME), and on-target off-tumor toxicities resulting from the sharing of CAR-T target epitopes on healthy tissues. To translate CAR-T cell therapy to ccRCC, we developed dual-targeted CAR-T cells against both CAIX and CD70, in which dual CAR can target double positive (CAIX+ CD70+) and single positive (CAIX+ CD70-, CAIX- CD70+) tumor cell populations to address this heterogeneity. By IHC staining, we demonstrated that these two target antigens are overexpressed in ccRCC and represent circa 90% of the tumor cell population regardless of disease stages. Here we build on our previous findings and demonstrate that 41BB CAR and CD4/CD8 CAR-T cell mixtures exhibit the superior efficacy and persistence in our ccRCC orthotopic NSG-SGM3 mouse model compared to other CAR constructs and CD8 alone. A panel of dual-targeted CARs have been encoded in our 41BB CAR construct and assessed in vitro and in vivo by using Celigo imaging cytometry assay and humanized ccRCC orthotopic NSG-SGM3 mouse model respectively. Dual-targeted anti-CAIX/CD70 CD4/8 CAR-T cells outperformed singly-targeted CAR-T cells with elevated efficacy. In summary, anti-CAIX/CD70 CAR-T cells hold a great promise to achieve ccRCC cures. Citation Format: Yufei Wang, Marion Grimaud, Alicia Buck, Atef Fayed, Matthew Chang, Rebecca Jennings, Maura Sticco-Ivins, Miriam Ficial, Leo L. Chan, Sabina Signoretti, Quan Zhu, Wayne A. Marasco. Develop dual-targeted CAR-T cells to achieve RCC cures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6606.

Bispecific CAR T cells for multiple myeloma: natural ligand compared to tandem scFv design

Abstract One of the most promising candidates for Chimeric Antigen Receptor (CAR) T cell therapy beyond CAR19 is treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen (BCMA). However, current reports of BCMA CAR in the clinic have a median progression free survival of 11.8 months, suggesting that targeting BCMA alone may not be sufficient. Evidence suggests patients treated with BCMA-targeted therapies may be thwarted by BCMA negative relapse. We have developed a novel second generation CAR T cell against another multiple myeloma target: transmembrane activator and CAML interactor (TACI). Mice were immunized against TACI and we designed a new scFv targeting TACI based on the resulting antibodies. Anti-TACI CAR T cells are cytotoxic in vitro and in vivo against multiple myeloma. To overcome single antigen loss, we designed bispecific tandem scFv CAR T cells targeting BCMA and TACI. These two antigens share a natural ligand, A Proliferation-Inducing Ligand (APRIL), which has also been used to design a dual-targeting CAR called TriPRIL. We show that these dual targeting CARs, based on tandem scFv or natural ligand design, have similar efficacy against wild type multiple myeloma models. However, this changes in the context of single antigen loss. We have characterized the proliferative (population doublings) and activation capability (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotype (PD-1, TIM-3, LAG-3), with long term exposure to single antigen. Our studies show that sensitivity to antigen density differs between the tandem bispecifics and the natural ligand CAR. These data provide insight into how structural differences between dual-targeting CAR T cells affects their function.

The Evolving Protein Engineering in the Design of Chimeric Antigen Receptor T Cells

The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the treatment of haematological cancers has encouraged the extensive development of CAR design to improve their function and increase their applicability. Advancements in protein engineering have seen modifications to both the ecto- and endo-domains of the CAR, with recent designs targeting multiple antigens and including inducible elements. These developments are likely to play an important role in inducing effective CAR T cell responses in a solid tumour context, where clinical responses have not been effective to date. This review highlights the spectrum of novel strategies being employed in CAR design, including for example variations in targeting tumour antigens by utilising different ectodomain designs such as dual chain CARs, natural receptor or ligand-based CARs, and T cell receptor fusion constructs, and also reviews some of the innovative approaches to a “universal” CAR and various multi-antigen targeting CAR strategies. We also explore how choices in the endodomain impact CAR function and how these need to be considered in the overall CAR design.

Resistance Mechanisms to CAR T-Cell Therapy and Overcoming Strategy in B-Cell Hematologic Malignancies.

Chimeric antigen receptor (CAR) T-cell therapy has shown promising clinical impact against hematologic malignancies. CD19 is a marker on the surface of normal B cells as well as most B-cell malignancies, and thus has a role as an effective target for CAR T-cell therapy. In numerous clinical data, successes with cell therapy have provided anticancer therapy as a potential therapeutic option for patients who are resistant to standard chemotherapies. However, recent growing evidence showed the limitations of the treatment such as antigen-positive relapse due to poor CAR T-cell persistence and antigen-negative relapses associated with CAR-driven mutations, alternative splicing, epitope masking, low antigen density, and lineage switching. The understanding of the resistance mechanisms to the cell therapy has developed novel potential treatment strategies, including dual-targeting therapy (dual and tandem CAR), and armored and universal CAR T-cell therapies. In this review, we provide an overview of resistance mechanisms to CD19 CAR T-cell therapy in B-cell malignancies and also review therapeutic strategies to overcome these resistances.

Abstract 3179: Design and activity of 2nd generation, dual-targeted CAR T cell factories against ccRCC

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC which is among the 10 most common cancers in both men and women. Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR T cells, the suppressive tumor microenvironment, and on-target off-tumor toxicities resulted from the sharing of CAR T targeting epitopes on healthy tissues. To combat the suppressive microenvironment, immune checkpoint blockade has shown promising effect on antitumor response by restoring the local antitumor immunity. CAR T cell factories were designed to empower CAR T cells through the secretion of human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site. Our results show a dramatic improvement in CAR T killing of ccRCC in vitro and in vivoby reversing CAR T cell and tumor infiltrating lymphocyte (TIL) exhaustion. CAIX is an ideal target for ccRCC therapy and used as a CAR target for the first clinical trial. However, it led to adverse side effects (ADEs) due to CAIX expression on the bile duct. Therefore, it is crucial to develop a CAR with elevated efficacy and safety (limited on-target off-tumor effect). To achieve that, the 2nd targeting scFv was introduced in the CAR T cell factory together with anti-CAIX scFv to enable the CAR to target two unique antigens simultaneously. By IHC staining of ccRCC patient samples, we found that target B is an ideal target to be utilized as the 2nd target since it is highly expressed on ccRCC and co-expressed with CAIX. Our 27 billion-member human scFv-phage display library was panned against the antigen expressing skrc-59 ccRCC cells to identify novel scFvs. Their binding kinetics (Kon/Koff) were then measured and scFvs with desirable kinetics were evaluated for their ability to bind with antigen expressing cells. Ideal candidates were cloned into vectors where anti-target B and anti-CAIX scFvs were combined in different permutations by changing the order of the two targeting scFvs with various linkers connected to a costimulatory domain (CD28, 41BB) and an activating domain (CD3). Primary T cells isolated from PBMCs were transduced to express dual CAR and were tested against different cell lines in vitro. For further evaluation in vivo, a humanized orthotopic ccRCC mouse model was established by injecting luciferized ccRCC cells under the kidney capsule of NSG-SGM3 mice with a reconstituted human immune system. In summary, utilizing a dual CAR T discovery platform, we generated a series of CARs with different scFvs, linkers, and hinges. By combining the best dual CAR with immune checkpoint blockade as payload, we propose that lead 2nd generation CAR T cell factory candidates will be discovered that should mitigate against ADEs on normal tissues and hold great promise for the treatment of ccRCC. Citation Format: Yufei Wang, Eloah Rabello Suarez, Matthew Chang, Rebecca Jennings, Sabina Signoretti, Quan Zhu, Wayne Marasco. Design and activity of 2nd generation, dual-targeted CAR T cell factories against ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3179.

Blocking CD30 on T Cells by a Dual Specific CAR for CD30 and Colon Cancer Antigens Improves the CAR T Cell Response against CD30- Tumors.

Chimeric antigen receptor (CAR)-engineered Tcells are efficacious in controlling advanced leukemia and lymphoma, however, they fail in the treatment of solid cancer, which is thought to be due to insufficient Tcell activation. We revealed that the immune response of CAR Tcells with specificity for carcinoembryonic antigen (CEA) was more efficacious against CEA+ cancer cells when simultaneously incubated with an anti-CD30 immunotoxin or anti-CD30 CAR Tcells, although the targeted cancer cells lack CD30. The same effect was achieved when the anti-CD30 single-chain variable fragment (scFv) was integrated into the extracellular domain of the anti-CEA CAR. Improvement in Tcell activation was due to interfering with the Tcell CD30-CD30L interaction by the antagonistic anti-CD30 scFv HRS3; an agonistic anti-CD30 scFv or targeting the high-affinity interleukin-2 (IL-2) receptor was not effective. Tcells with the anti-CD30/CEA CAR showed superior immunity against established CEA+ CD30- tumors in a mouse model. The concept is broadly applicable since anti-CD30/TAG72 CAR Tcells also showed improved elimination of TAG72+ CD30- cancer cells. Taken together, targeting CD30 on CAR Tcells by the HRS3 scFv within the anti-tumor CAR improves the redirected immune response against solid tumors.

5th Annual Immuno-Oncology 360° Conference: Spanning Science and Business to Bring New Therapies to Patients

5th Annual Immuno-Oncology 360° Conference: Spanning Science and Business to Bring New Therapies to Patients

CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment.

Chimeric antigen receptor (CAR) T cells, T cells that have been genetically engineered to express a receptor that recognizes a specific antigen, have given rise to breakthroughs in treating hematological malignancies. However, their success in treating solid tumors has been limited. The unique challenges posed to CAR T cell therapy by solid tumors can be described in three steps: finding, entering, and surviving in the tumor. The use of dual CAR designs that recognize multiple antigens at once and local administration of CAR T cells are both strategies that have been used to overcome the hurdle of localization to the tumor. Additionally, the immunosuppressive tumor microenvironment has implications for T cell function in terms of differentiation and exhaustion, and combining CARs with checkpoint blockade or depletion of other suppressive factors in the microenvironment has shown very promising results to mitigate the phenomenon of T cell exhaustion. Finally, identifying and overcoming mechanisms associated with dysfunction in CAR T cells is of vital importance to generating CAR T cells that can proliferate and successfully eliminate tumor cells. The structure and costimulatory domains chosen for the CAR may play an important role in the overall function of CAR T cells in the TME, and “armored” CARs that secrete cytokines and third- and fourth-generation CARs with multiple costimulatory domains offer ways to enhance CAR T cell function.

Early Clinical Experience of CD19 x CD22 Dual Specific CAR T Cells for Enhanced Anti-Leukemic Targeting of Acute Lymphoblastic Leukemia

Introduction:Advances in chimeric antigen receptor (CAR) T cell therapy have yielded complete remission (CR) rates in relapsed/refractory B-ALL (rrB-ALL) of 70-95%. However, disease recurrence after CD19 or CD22 CAR therapy is greater than 50% at 1 year, and approximately half of recurrences are due to antigen escape. To reduce antigen escape and optimize the durability of remission, we sought to design a CAR T cell product with dual specificity that is capable of simultaneously targeting both CD19 and CD22. Preclinical testing of our bi-specific CAR showed a preference for signaling through CD22 over the CD19 CAR. In contrast, dual transduced T cells signaled through both the CD19 and CD22 CAR with lytic activity and cytokine production similar to single transduced CAR T cells of the same specificity. Therefore, we opted to move forward with dual transduced T cells for clinical use. We are currently testing SCRI-CAR19x22v1 in PLAT-05 (NCT03330691), a phase 1 clinical trial for pediatric and young adult patients with CD19+CD22+ rrB-ALL, with the primary objectives to determine the feasibility of manufacturing products with dual specificity, to assess the safety of the cryopreserved product infusion, and to describe the full toxicity profile.Methods:Subjects undergo apheresis, after which the CD4 and CD8 T cell subsets are immunomagnetically selected and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two separate SIN lentiviral vectors that direct the expression of a CD19-specific FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR with an Her2tG tag and expression of a CD22-specific m971scFv:IgG4hinge:CH2(L235D)-CH3:CD28tm:4-1BB:ζ CAR with an EGFRt tag, creating three distinct populations of CAR T cells (anti-CD19, anti-CD22, and anti-CD19x 22). Transduced cells are expanded in serum free media formulation with IL-7, IL-15, and IL-21. Following lymphodepleting chemotherapy, cryopreserved products are thawed and infused at the protocol-prescribed dose level. Cytokine release syndrome (CRS) is graded according to Lee et al. (Blood 2014) and is treated according to our early intervention strategy of tocilizumab and dexamethasone for persistent, mild CRS.Results:Seven subjects (ages 1-26 yr) with rrB-ALL have been enrolled with 4 treated at dose level 1 (1 x 106 CAR T cells/kg) and 3 treated at dose level 2 (3 x 106 CAR T cells/kg). The mean culture time was 7.9 days (range 7-11) and subjects received infusions with a mean CD8:CD4 ratio of 1.7 (range 0.2 - 3.1). CD8 CAR composition, on average, consisted of 21.6 % CD19 CAR, 37.8 % CD22 CAR, and 40.6 % CD22xCD19 CAR T cells. CD4 CAR composition, on average, consisted of 25.8 % CD19 CAR, 30.6 % CD22 CAR, and 43.6 % CD22xCD19 CAR T cells (Figure). Peak engraftment occurred between days 7 and 14 for all patients and was predominantly composed of the CD19 CAR population with median peak values for CD19 CAR, CD22 CAR, and CD19xCD22 CAR T cell populations of 9.1%, 1.2%, and 2.4%, respectively. A CR was achieved in 5/7 (71%) subjects by day 21, 4 of which were minimal residual disease negative. The two subjects without a CR did not exhibit evidence of CAR T cell engraftment; one had previously received CD19 CAR T cells, and the other had progressive disease and pursued alternative therapy at day 10. Therapy was well tolerated with no dose limiting toxicities. CRS occurred in 5 subjects (Grade 1) with 2 of these subjects experiencing mild neurotoxicity (Grade 1). Four subjects received tocilizumab +/- dexamethasone, and two of these received multiple doses of dexamethasone.Conclusions:Preclinical testing showed superior efficacy against both CD19 and CD22 when using two separate CARs and dual transduction, compared to a single bi-specific CAR. Preliminary analysis of PLAT-05 supports feasibility of product manufacturing, and toxicity and response rates that are consistent with the reported CD19 CAR T cell experience. While the infused SCRI-CAR19x22v1 products consist of a near-uniform distribution of the 3 distinct populations, we observed selective in vivo expansion of the CD19 CAR T cell population. Further investigation is required to understand the mechanism of CD19 CAR dominance in vivo. Continued accrual of subjects is ongoing to further assess the impact of dual antigen targeting on the prevention of antigen escape and the potential to provide a more durable remission. [Display omitted] DisclosuresPark:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Chimeric Antigen Receptor T Cells: Extending Translation from Liquid to Solid Tumors

Successful translation of chimeric antigen receptor (CAR) T cells designed to target and eradicate CD19+ lymphomas has emboldened scientists and physicians worldwide to explore the possibility of applying CAR T-cell technology to other tumor entities, including solid tumors. Next-generation strategies such as fourth-generation CARs (CAR T cells redirected for universal cytokine killing, also known as TRUCKs) designed to deliver immunomodulatory cytokines to the tumor microenvironment, dual CAR designs to improve tumor control, inclusion of suicide genes as safety switches, and precision genome editing are currently being investigated. One major ongoing goal is to determine how best to generate CAR T cells that modulate the tumor microenvironment, overcome tumor survival mechanisms, and thus allow broader applicability as universal allogeneic T-cell therapeutics. Development of state-of-the-art and beyond viral vector systems to deliver designer CARs coupled with targeted genome editing is expected to generate more effective off-the-shelf CAR T cells with activity against a greater number of cancer types and importantly solid tumors.

Driving CARs on the uneven road of antigen heterogeneity in solid tumors

Uniform and strong expression of CD19, a cell surface antigen, on cells of B-cell lineage is unique to hematologic malignancies. Tumor-associated antigen (TAA) targets in solid tumors exhibit heterogeneity with regards to intensity and distribution, posing a challenge for chimeric antigen receptor (CAR) T-cell therapy. Novel CAR designs, such as dual TAA-targeted CARs, tandem CARs, and switchable CARs, in conjunction with inhibitory CARs, are being investigated as means to overcome antigen heterogeneity. In addition to heterogeneity in cancer-cell antigen expression, the key determinants for antitumor responses are CAR expression levels and affinity in T cells. Herein, we review CAR T-cell therapy clinical trials for patients with lung or pancreatic cancers, and provide detailed translational strategies to overcome antigen heterogeneity.

Nanobody Based Dual Specific CARs.

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

Abstract A193: Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection

Abstract Myelosuppressive and ablative regimens are used to deplete lymphocytes in some patients before adoptive cell therapy. CD19-specific engineered T-cells have been used successfully to eliminate B-cells in patients with B-cell leukemias and lymphomas. Prolonged immunosuppression, including B-cell aplasia is associated with increased risk of invasive fungal infection (IFI). Thus, patients will benefit if bio-engineered T cells can control IFI as well as kill tumor cells. The Sleeping Beauty gene transfer system was used to render T cells to express a chimeric antigen receptor (CAR) on the cell surface to redirect specificity via Dectin-1. Upon binding with the β-1,3-glucan sugar moiety found in IFI, Dectin-1 CAR+ T cells signal via chimeric CD28 and CD3-ζ signaling domain. The activated T cells secrete perforin, granzyme and granulysin to damage hyphae and thus inhibit the hyphal growth of Aspergillus and Candida. The T cells also secrete IFN-γ to activate other immune cells to target IFI. To ready the Dectin-1 CAR-modified T cells for the human application, we have modified the Fc domain of the extracellular stalk to alleviate deleterious uptake by Fcγ receptors and thus in vivo loss of infused T cells. We have shown that the Dectin-1 CAR+ T cells do not kill the yeast form of Candida so normal commensals in the gut microbiota should not be affected. We have also demonstrated that Dectin-1 CAR+ T cells can control Aspergillus infection in the presence of immunosuppressive drugs at physiological concentrations. Finally, we have generated dual CAR+ T cells by co-expressing a CD19-specific CAR with Dectin-1 CAR rendering the resultant genetically modified T cells able to target both B-cell malignant cells as well as fungal hyphae. These dual CAR-T cells can thus control both leukemia and invasive fungal infections. These data provide a path forward for clinical trials testing Dectin-1 CAR+ T cells. Citation Format: Pappanaicken R. Kumaresan, Nathaniel Albert, Harjeet Singh, Simon Olivares, Sourindra N. Maiti, Tiejuan Mi, Helen Huls, Richard E. Champlin, Dimitrios P. Kontoyiannis, Laurence J.N. Cooper. Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection . [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A193.

Abstract 2797: Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) system

Abstract Introduction: The use of adoptive transfer of T cells for the treatment of cancer has been hampered by the low persistence of the infused cells and the difficulty to isolate and expand tumor-specific lymphocytes. The use of chimeric antigen receptors (CARs) allows the circumvention of these problems. CARs consist of an antibody-derived scFv, a transmembrane region and an intracellular activating signaling domain, recognizing the target antigen with high affinity and in a MHC-independent fashion. Nonetheless, off-target responses owing to the recognition of the target antigen in healthy cells limit the application of this therapy. The antigen CD19 represents a good target for the elimination of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and anti-CD19 CAR+ lymphocytes are being used clinically. However, the expression of CD19 as a pan B-lineage marker leads to mature B cell depletion. Objective: Since mature B cells express both CD19 and CD20 antigens, we propose the creation of an inhibitory CAR directed to CD20, allowing the lymphocytes to discriminate between BCP-ALL and mature B cells. Methods: Jurkat cells expressing the plasmid pGL4.30 (luciferase controlled by a NFAT responsive element) were generated and used as a reporter cell line. As target cells we used the K562 cell line modified to express CD19 (K5-19), CD20 (K5-20) or CD19 and CD20 (K5-19/20). The expression of the activation marker CD69 was evaluated by flow cytometry. The Sleeping Beauty transposon system was used to modify primary human T lymphocytes and the effector activity was evaluated by cytotoxicity and IFNg production assays. Results: Three different inhibitory αCD20-CARs containing signaling domains CTLA-4, PD-1 or BTLA were constructed. Jurkat cells expressing the αCD19- activating CAR 19BBz showed high activity of luciferase when cultured with K5-19 or K5-19/20. However, Jurkat cells expressing both the activating and inhibitory CARs showed a potent inhibition of luciferase activity when incubated with K5-19/20, while maintaining a high activity when cultured with K5-19 cells. Moreover, the three inhibitory CARs were able to inhibit the de novo expression of CD69 induced by 19BBz in Jurkat cells. In primary T cells, the CAR 20PD1 was not able to inhibit the production of IFNg or the cytotoxic activity induced by 19BBz. Surprisingly, T cells expressing both 19BBz and 20PD1 showed preferential lysis of CD19+ CD20+ targets, suggesting that addition of PD1 signaling domain might augment T cell function in particular contexts. Conclusions: The results in Jurkat cells suggest that αCD20-CARs can inhibit the activation of lymphocytes mediated by αCD19-CAR. However, additional experiments are required to evaluate the inhibitory activity of 20PD1 in primary human T cells. Experiments with CARs 20CTLA4 and 20BTLA are in progress and might allow the validation of the dual CAR hypothesis in vitro and in vivo. Citation Format: Leonardo Chicaybam, Martin H. Bonamino. Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2797. doi:10.1158/1538-7445.AM2014-2797

Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28‐OX40 signalling

The therapeutic success of adoptive therapy with chimeric antigen receptor (CAR) engineered T cells depends on the appropriate costimulation of CD3ζ to induce full T cell activation. Costimulatory endodomains of the CD28 family are therefore fused with CD3ζ in a dual signalling CAR. Serious adverse events in two most recent trials; however, highlight the need to analyse in more detail the impact of each costimulatory endodomain on individual effector functions of redirected T cells. We therefore performed a thoroughly controlled side-by-side comparison of the most frequently used endodomains with respect to their impact on CD4(+) and CD8(+) T cell effector functions. CD28 reinforced T cell proliferation and is mandatory to induce IL-2. In the absence of added IL-2, CD28 and OX40 (CD137) but not 4-1BB (CD134) enhanced specific cytolysis. While CD28, 4-1BB and OX40 similarly improved pro-inflammatory cytokine secretion, OX40 most efficiently prevented activation induced cell death of CD62L(-) effector memory T cells. CD28 was superior to initiate the T cell response, OX40 and 4-1BB sustained the response in long term with OX40 being most effective. We consequently combined the beneficial functions in a 3rd generation CD28-OX40 CAR which substantially improved the antitumor response without loosing specificity.