Dual CAR-T Research Articles

Updated results of a multicenter first-in-human study of BCMA/CD19 dual-targeting fast CAR-T GC012F for patients with relapsed/refractory multiple myeloma (RRMM).

8005 Background: GC012F is a B cell maturation antigen (BCMA)/CD19 dual-targeting CAR-T developed on the novel FasT CAR-T platform with overnight manufacturing and designed to improve depth of response and efficacy. Data was presented at ASCO and EHA 2021 for initial 19 pts. We present updated data for study (NCT04236011; NCT04182581) with longer follow up and 9 additional pts treated (n = 28) in 3 different dose levels. Methods: From October 2019 to November 2021, 28 heavily pretreated RRMM pts (age 27-76) median of 5 prior lines (range 2-9) were treated on a single-arm, open label, multicenter Investigator Initiated Trial receiving a single infusion of GC012F. 89.3% (25/28) were high risk (HR- mSMART), 8 pts had EM disease, 3 had never achieved a CR including after transplant, 1 pts presented with plasma cell leukemia, 24/28 pts were refractory to last therapy, 3 pts primary refractory. 9/28 pts had received prior anti-CD38, 27/28 pts prior IMiDs. 26/28 pts were refractory to PI, 26/28 pts to IMiDs. After lymphodepletion over 2-3 days (30 mg/m2/d, 300mg/ m2/d Flu/Cy) GC012F was administered as single infusion at 3 dose levels: 1x105/kg (DL1) n = 2, 2x105/kg (DL2) n = 10 and 3x105/kg (DL3) n = 16. Results: As of Jan 26th 2022, 28 pts - median follow-up (f/u) 6.3 mths (1.8-29.9) - had been evaluated for response. Overall response rate (ORR) in DL1 was 100% (2/2)- DL 2 -80% (8/10) DL 3 -93.8% (15/16) with 27 pts MRD negative by flow cytometry (sensitivity 10-4-10-6). 100% of MRD assessable pts (27/27) achieved MRD negativity. One patient out of 28 could not get assessed. At d28, 21/24 assessable patients were MRD negative (81.5%), 4/28 pts could not get d28 MRD assessment f/u due to COVID-19 restrictions however were assessed at a later timepoint. To date best response is MRD- sCR in 21/28 patients(75.0%) across all dose levels. Some pts after short f/u show responses that are still deepening. Cytokine Release Syndrome (CRS) was mostly low grade: gr 0 n = 3 (10.7%), gr 1-2 n = 23 (82.1%), gr 3 n = 2 (7.1%) – no gr 4/5 CRS and no ICANs were observed (Graded by ASBMT criteria). Median duration of CRS was 3 d (1-8 d). PK results showed no difference amongst dose levels DL1 to DL3. Overall, CAR-T median Tmax was 10 d (range 8-14 d), median peak copy number (Cmax) was 97009 (16,011-374,346) copies /μg DNA with long duration of persistence of up to d793 (data cut-off). CAR-T geometric mean AUC0-28 for DL1, DL2 and DL3 were 468863, 631540 and 581620 copies/μg DNA×day, respectively. Pts continue to be monitored for safety and efficacy including DOR. Conclusions: BCMA-CD19 dual FasT CAR-T GC012F continues to provide deep and durable responses with a favorable safety profile in additional RRMM pts across all dose levels demonstrating a very high MRD negativity rate including in pts refractory to anti-CD38, PI and IMIDs. GC012F is currently being studied in earlier lines of therapy as well as additional indications. Clinical trial information: NCT04236011; NCT04182581.

CD19/BAFF-R Dual-Targeted CAR T Cells for the Treatment of Mixed Antigen-Negative Variants of Acute Lymphoblastic Leukemia

The results of clinical trials evaluating CD19-targeting chimeric antigen receptor (CAR) T cells are impressive, with overall response rates of up to 90% in B cell acute lymphoblastic leukemia (ALL) and 50-80% in lymphoma. Despite initial responses, antigen-negative relapse is common following treatment with CD19-targeted therapies and is estimated to occur in up to 39% of patients. One approach of addressing this problem is to utilize dual-targeting CAR T cells, a strategy that has recently been applied to CD19/CD22 for ALL, CS1/BCMA, and BCMA/GPRC5D for multiple myeloma. Dual-targeting CARs can simultaneously target two tumor antigens and, therefore, potentially eradicate heterogeneous tumors. The initial response to dual-targeted CAR T cells is expected to provide greater tumor coverage compared to single-targeted therapy, and can potentially circumvent antigen escape. Moreover, if one tumor antigen becomes downregulated during treatment, the second targeting domain will continue to be reactive to tumors. Therefore, it is critical to identify novel targets that can be combined with CD19 in a dual-targeted immunotherapeutic platform.To expand the potential for dual targeting of ALL, we developed a CAR T cell therapy against a novel target, B-cell activating factor receptor (BAFF-R), based on the remarkable specificity of anti-BAFF-R antibodies that we previously generated. BAFF-R is expressed almost exclusively on B cells, including in patients with CD19-negative relapse, making it an ideal immunotherapeutic target. Studies demonstrate that the role of BAFF-R in B cell function and survival is conclusive, an important feature that may mitigate the tumor's ability to escape therapy through antigen loss, particularly if a non-redundant role for BAFF-R is confirmed. BAFF-R-CAR T cells demonstrate in vitro effector function and in vivo therapeutic efficacy in CD19-negative models, including patient-derived xenograft models, and are currently being evaluated clinically for the treatment of ALL (NCT04690595). We hypothesized that simultaneous targeting of CD19 and BAFF-R in a bispecific CAR platform could confer a therapeutic advantage and avoid the challenges of sequential administration of CD19 and BAFF-R monospecific CAR T cells.We leveraged our experience with CD19- and BAFF-R-CAR T cells to develop a dual-targeting, bispecific CAR with a 41BB costimulatory domain (CD19/BAFF-R dual CAR). Here, we identified the optimal orientation of the single-chain variable fragment (loop scFv) domains within the dual construct and tested the CD19/BAFF-R dual CAR T cells for their in vitro effector function and in vivo anti-leukemia activity. To evaluate the specific targeting to CD19 and BAFFR, we developed Nalm-6-BAFF-R-knockout (KO) and Nalm-6-CD19-KO cell lines. CD19/BAFF-R dual CAR T cells specifically released IFN-g following incubation with Nalm-6 CD19-/- or BAFF-R-/- cells (P<0.001) compared with un-transduced mock T cells. Both CD4+ and CD8+ CAR T cell populations exhibited effector function. To evaluate the antigen-dependent targeting of the CD19/BAFF-R dual CAR T cells in vivo, we utilized a mixed B-cell leukemia model that simulates clinical tumor heterogeneity. NOD-scid IL2Rgammanull (NSG) mice were inoculated with 2-3x10 5 of mixed Nalm-6 BAFF-R-/- and CD19-/- cells at a 1:1 ratio with a single injection. 1x10 6 CD19/BAFF-R dual CAR, CD19, or BAFF-R single CAR-T cells were administered intravenously 9-10 days later. Tumor growth was monitored by bioluminescent imaging weekly. We observed superior tumor eradication (P<0.01) and survival (P<0.01) (Figure 1) by CD19/BAFF-R dual CAR T cells compared to either single-targeting CAR and mock T cells. The adoptively transferred CD19/BAFF-R dual CAR T cells were able to persist in vivo.Our unique CD19/BAFF-R dual-targeting CAR T cells will be the first to target this combination of tumor-associated antigens. Our study demonstrated the reliability of bispecific CD19/BAFF-R dual CAR T cell therapy in inducing remission in ALL consisting of CD19-/- and BAFF-R-/- tumors. We hypothesize that simultaneous immunotherapy targeting of heterogeneous leukemic cell populations may diminish the likelihood of antigen escape and may have a significant impact on leukemia treatment by improving the therapeutic benefits of CAR T cell therapy. [Display omitted] DisclosuresWang: Pepromene Bio, Inc.: Consultancy. Forman: Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company. Kwak: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company. Qin: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company.

How to Sequence Therapies in Diffuse Large B-Cell Lymphoma Post-CAR-T Cell Failure.

Post CAR-T failures represent a new unmet need in R/R LBCL. The prognosis is usually very poor and standard treatment options that can guide clinicians are, unfortunately, not available. While polatuzumab, tafasitamab, selinexor, and loncastuximab tesirine are available as SOC since they are FDA approved, data is lacking in the post CAR-T setting. However, they could be used in the absence of other treatment options (clinical trials). A selected group of patients may be treated with checkpoint inhibitors, likely low tumor burden or low proliferative lymphomas or those with PD-L1 expression. For localized relapses, radiation therapy could be considered. A main consideration should be given to clinical trials. So far, it appears that bi-specific antibodies have the best encouraging data (high response rates) with manageable toxicities and logistics; thus, we recommend clinicians to enroll patients in clinical trials utilizing these agents. Other cell therapies (such as dual CAR-T or allogeneic products) should also be considered; however, challenges with logistics and further immunosuppression (especially if patients had prolonged cytopenias from prior CAR-T therapy) may affect its applicability right after CAR-T relapse. It is unclear whether these options will lead to long-term remissions; thus, consolidation with stem cell transplantation (either auto or allogeneic SCT) could be considered in eligible patients.

Efficacy and Safety of Triple versus Dolutegravir-based Dual Therapy in Patients with HIV-1 Infection: A Meta-analysis of Randomized Controlled Trials.

A new strategy of simplification therapy shown the unique benefits in clinical treatment, by reducing pill burden and avoid drug exposure. To provide more evidence for the strategy, we compared the efficacy and safety of dolutegravir (DTG)-containing simplified dual combination antiretroviral therapy (cART) and traditional triple cART for people living with HIV/AIDS. The meta-analysis of randomized controlled trials compared DTG-containing dual therapy with triple cART. The primary outcome was virologic suppression. The secondary outcomes included CD4T cell recovery, lipids change from baseline, and adverse events (AEs). A total of 7 studies, 4852 patients were eligible, 2423 (49.9%) received DTG-based simplified dual cART, and 2429 (50.1%) received triple cART. The viral suppression rate was 94.7% at 24 weeks, 93.0% at 48 weeks, and 96.6% at 96 weeks in dual cART. The viral suppression rate of dual cART was non-inferior to triple cART at 24 weeks (risk difference [RD], -0.00; 95% confidence interval [CI] -0.02-0.01), at 48 weeks (RD, -0.01; 95% CI -0.02-0.01), and at 96 weeks (RD, -0.01; 95% CI -0.02-0.00). Sub-analysis results were consistent with the overall results. With regard to other outcomes (CD4T counts, lipids, any AEs, and AEs grade ≥ 3), there was no significant statistical difference between the two regimens. DTG-based simplified dual cART was non-inferior to triple cART in terms of efficacy and safety. This finding provides strong support for current consensus guidelines recommended the dual regimen as first-line treatment.

Long-term follow-up results of a multicenter first-in-human study of the dual BCMA/CD19 Targeted FasT CAR-T GC012F for patients with relapsed/refractory multiple myeloma.

8014 Background: The dual CAR-T GC012F developed on the novel FasT CAR-T platform targeting B cell maturation antigen (BCMA), and CD19 was designed to improve depth of response and overall efficacy for CAR-T as therapy for Multiple Myeloma. Here, we present updated data for study (NCT04236011; NCT04182581) including additional pts treated. Methods: From October 2019 to July 2020, 19 heavily pretreated Relapsed/Refractory Multiple Myeloma (RRMM) pts (age 27-71) with a median of 5 prior lines (range 2-9) received a single infusion of GC012F. 94.7% (18/19) were high risk (HR- defined by mSMART), 5 pts had extramedullary disease, 1 pts presented with plasma cell leukemia, and 15/19 were refractory to last therapy. 4/19 pts had received prior anti- CD38, 18/19 pts prior IMiD. 18/19 pts were refractory to PI, 17 pts to IMiD, 3 pts being primary refractory. After lymphodepletion over 2-3 days (30 mg/m2/d, 300mg/ m2/d Flu/Cy) CAR-T cells were administered as single infusion at 3 dose levels: 1x105/kg (DL1) n=1, 2x105/kg (DL2) n=9 and 3x105/kg (DL3) n=9. Results: As of Jan 12, 2021 cut-off, 19 pts were evaluated for response. Overall response rate (ORR) was 94.7% - all responses VGPR or better (94.7% - 16/18 sCR, 2/18 VGPR) with all pts MRD- by flow cytometry (10-4-10-6) - earliest response d 28 post infusion. 100% of pts achieved a reduction of paraprotein, 18/19 a 100% reduction. Best response was MRD- sCR in 16/19 patients (84.2%). In DL3 (n=9) 4 additional pts were response evaluable for 6 mth follow-up: 100% (9/9) of pts achieved MRD-sCR as best response, 87.5% (7/8) of response evaluable pts maintained MRD-sCR at landmark analysis of 6 mths. At data cut off, the median time to follow up was 13.8 mths (6.1-16.4) – median duration of response not yet reached. Cytokine Release Syndrome (CRS) and ICANs were graded by ASBMT criteria: grade 1-2 n=16 (84.2%), grade 3 n=2 (10.5%). Median duration of CRS was 4 d (1-8 d). No grade 4/5 CRS or ICANS were observed. 2 deaths occurred on study and were assessed as not related to therapy – one reported previously; one patient was diagnosed with pseudomonas pneumonia on Day 52, refused life-saving treatment and passed. CAR-T median Tmax was 10 d (range 8-14d), median peak copy number (Cmax) was 127548 (16,011-374,346) copies /ug DNA with long duration of persistence of up to 60 weeks at time of data cut off. Patients continue to be monitored for safety and efficacy. Conclusions: BCMA-CD19 dual FasT CAR-T GC012F showed early, deep and durable responses with a high ORR (94.7% - VGPR and better) including a high MRD- sCR rate (DL3=100%, n=9) in high risk RRMM pts including those refractory to anti-CD38, PI and IMIDs with a favorable safety profile consistent with previous findings. BCMA-CD19 dual FasT CAR-T GC012F may present a new treatment approach for patients with RRMM including those with high-risk features refractory to standard of care. Clinical trial information: NCT04236011; NCT04182581.

The efficacy and safety of anti-CLL1 based CAR-T cells in children with relapsed or refractory acute myeloid leukemia: A multicenter interim analysis.

10000 Background: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable success in treating a variety of blood cancers, such as CD19 CAR-T for B-cell malignancies and BCMA CAR-T for myeloid myeloma (MM). However, similar achievement has yet to be replicated in patients with relapsed and refractory acute myeloid leukemia (R/R AML), primarily due to the AML heterogeneity, making it difficult to find an ideal CAR-T target. Previous efforts have targeted single CD33, CD123, LeY, NKG2D, or CD70 receptors, but the overall response rate is very disappointed. To address these challenges, we aim to find an effective target for AML without the need for the hematopoietic stem cells transplant (HSCT). In our study, CLL1 is chosen as a promising target as it is not expressed on normal HSCs, but highly expressed on AML blasts cells and leukemia stem cells (LSCs). Here we report the interim analysis from a Phase I clinical trial using anti-CLL1 based CAR-T cells to treat children with R/R AML. The primary and secondary objectives were to evaluate the safety and anti-AML responses, respectively, with long-term prognosis within those patients who did not receive allogeneic HSCT (allo-HSCT) as an additional objective. Methods: We have generated a 2nd generation of CLL1 CAR-T, the extracellular scFv was derived from a murine CLL1 monoclonal antibody, which was generated by hybridoma technology. Autologous CAR-T cells were manufactured in a cGMP facility. Between Oct 2019 and Jan 2021, 11 pediatric R/R AML patients were infused. CLL1 or CLL1-CD33 dual CAR-T cells were given by a dose at 0.3-1x106/kg with a single dose after lymphodepleting conditioning with cyclophosphamide/fludarabine(Cy/Flu). Results: Of the 11 patients infused, Grade 3-4 hematologic adverse events were observed before and during CAR-T cell infusion, and no dose-limiting toxicities were observed. Meanwhile, grade 1-3 cytokine release syndrome was observed but without any lethal events. All the adverse effects were resolved after guideline-directed intervention. Anti-CLL1 CAR-T cells efficiently expanded in vivo, the median expansion peaking time was at Day 8. For these 11 R/R-AML patients, 10 patients completely responded to anti-CLL1 based CAR-T cell therapy, with CLL1 positive AML blast eliminated within one month. Among the responded 10 patients, 5 patients reached CR/MRD-, 3 patients reached CR/MRD+, 1 patient reached PR and 1 patient showed SD, with only CLL1 negative AML cells. Conclusions: Our study demonstrated that 10/11 patients responded to CLL1 CAR-T cell therapy within one month. For patients showing MRD+ with CLL1 negative AML blast, chemotherapy like Azacitidine, and combined with HSCT may help those patients to reach complete response. These initial results suggested that anti-CLL1 base CAR-T cells can be a well-tolerated and candidate option for treating children with R/R-AML. Clinical trial information: ChiCTR1900027684.

Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer

Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage “don’t eat me” signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas.

Clinical Results of a Multicenter Study of the First-in-Human Dual BCMA and CD19 Targeted Novel Platform Fast CAR-T Cell Therapy for Patients with Relapsed/Refractory Multiple Myeloma

Introduction To further improve efficacy and duration of response of CAR-T therapy for Relapsed/Refractory Multiple Myeloma (R/R MM), we have designed a dual FasT CAR-T targeting both B cell maturation antigen (BCMA), a well-established MM target, and CD19, which is expressed on MM cells and their progenitors. Here we report early results from the first-in-human multicenter clinical study (NCT04236011; NCT04182581) to determine safety, pharmacokinetics (PK) and efficacy of BCMA-CD19-directed FasT CAR-T (GC012F) in patients with R/R MM. Methods The BCMA-CD19 dual CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3z. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis, T cells were isolated and CAR-T cells were manufactured (FasT CAR platform). From September 2019 to April 2020, we enrolled 16 heavily pretreated R/R MM patients (Age range 27-71), with a median of 5 prior lines of therapies (range 2-7), 93.8% (15/16) of these patients were high risk as defined by mSMART criteria, 5 had extramedullary disease. 4 out of 16 patients had received prior anti CD38 therapy, 93.8% (15/16) patients had received prior IMiD, all patients received at least 1 prior PI and corticosteroids with 3 patients being primary refractory to last therapy. Prior to CAR-T infusion patients received a conditioning regimen over 3 days of 30 mg/m2/d fludarabine and 300 mg/m2/d cyclophosphamide. CAR-T cells were administered in a single infusion at 3 dose levels 1x105/Kg (DL1) (1 patients), 2x105/Kg (DL2) (9 patients) and 3x105/Kg (DL3) (6 patients). Results As of July 17th 2020, all 16 patients were evaluable for response assessment, 15 out of 16 patients responded to treatment (ORR 93.8%) in all dose levels with the earliest response observed at day 28. Best response to date is MRD- CR/sCR in 9/16 patients (56.3%). In DL3 100% (6/6) of patients achieved sCR, 3 at data cut off had been confirmed by PET-CT. In all response evaluable patients, 78.6% (11/14) were MRD- by flow at month 1, and 100% at month 3 (11/11) and 6 (10/10) (sensitivity by flow cytometry measured at 10-4 in 7 patients, and at 10-6 in 9 patients tested by EuroFlow with at least 1.08x107 cells analyzed). At data cut off, the median follow up time was 7.3 months, the longest follow up was 10 months post infusion. CAR-T PK in PB was monitored by qPCR and flow cytometry. The CAR-T median proliferation peak was reached on Day10 (Day8-Day14), and the median peak copy number was 140,982 (16,011-374,346) copies /ug DNA. GC012F showed an acceptable safety profile with 14 out of 16 patients experiencing a cytokine release syndrome (CRS) grade 1-2 (n=14, 87.5%) and 2 grade 3 (n=2, 12.5%). The median duration of CRS was 4 days (1-8 days). No neurotoxicity of any grade was observed. One patient (DL2) presented with fever and died shortly after Day 78 of unknown cause during the COVID-19 Pandemic. Two patients had progression of extramedullary disease while achieving MRD negativity at month 1 and 3, respectively. At landmark analysis at 6 months, all patients in DL3 had achieved and maintained MRD- sCR including patients heavily pretreated including Daratumumab - among them 83.3% (5/6) patients in DL3 had high risk features according to mSMART criteria, and 5 out of 6 patients in DL3 were assessed by 10-6 Euroflow for MRD. The study is still enrolling patients and we will continue to be monitoring safety and efficacy including duration of response. Conclusion The data of BCMA-CD19 dual FasT CAR-T showed an early and high response rate with 93.8% ORR to date with a promising early high MRD-sCR rate in the highest dose level DL3 (100%) which was sustained with a median duration of follow up of 7.3 months at cut off. The data shows very promising activity of the BCMA-CD19 dual FasT CAR-T with a favorable safety profile in R/R MM patients. 93.8% (15/16) of the treated patients exhibited high risk features - a specifically difficult to treat patient population which remains a high unmet medical need in Multiple Myeloma. This data indicates that BCMA-CD19 dual FasT CAR-T (GC012F) may present an effective new treatment option for patients with R/R MM including those with high-risk features who failed multiple prior therapies including anti-CD38. The study is still ongoing and enrolling patients, we will update the results as they become available. Disclosures Zhao: Gracell Biotechnologies Ltd: Current Employment. Han:Gracell Biotechnologies Co., Ltd.: Current Employment. Chen:Gracell Biotechnologies Ltd: Current Employment. Xu:Gracell Biotechnologies Ltd: Current Employment. Zhang:Gracell Biotechnologies Ltd: Current Employment. He:Gracell Biotechnologies Co., Ltd.: Current Employment. Shi:Gracell Biotechnologies Ltd: Current Employment. Han:Gracell Biotechnologies Co., Ltd.: Current Employment. Ye:Gracell Biotechnologies Co., Ltd.: Current Employment. Wang:Gracell Biotechnologies Ltd: Current Employment. Liu:Gracell Biotechnologies Co., Ltd.: Current Employment. Shen:Gracell Biotechnologies Ltd: Current Employment. Cao:Gracell Biotechnologies Ltd: Current Employment. Sersch:Gracell Biotechnologies Co., Ltd.: Current Employment.

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 &amp;gt; 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.

Improved Safety and Efficacy of a Multi-Target Chimeric Antigen Receptor Modified T Cell Therapy (4SCAR2.0) Against Relapsed or Refractory Lymphomas

BACKGROUND: Chimeric antigen receptor-modified T cell (CART) therapy holds great promise as a novel cancer therapy approach. Although CART cell therapy has revolutionized the treatment of B-cell non-Hodgkin lymphomas (NHLs), relapsed/refractory NHLs including diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), and follicular lymphoma (FL) have not responded well to the currently approved CART cell products. The existing limitations include antigen escape, toxicities of currently approved CART cells, and the high cost of manufacture of a product which is only used for a single patient (pt). The development of an innovative multi-CART therapy using a safety improved CAR design may overcome the above limitations. METHODS: Lymphoma pts who have exhausted all available treatments with progressive or stable disease and life expectancy &amp;gt;2 months were enrolled in the study. Lymphoma biopsies were immunostained for various target antigens including CD19, CD20, CD22, CD30, CD38, CD70 and PSMA. An advanced CART therapy regimen, CAR 2.0, has been developed, which involves a primary and a booster CAR-T cell infusions targeting multiple antigens identified on each pt's tumor. Autologous T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). Pts received cyclophosphamide/fludarabine chemotherapy conditioning 1-2 days before infusion of 1.21- 4.87x106 CART cells/kg per infusion. The quality of apheresis cells, efficiencies of gene transfer and T cell proliferation, CAR T infusion dose and blood CAR copies were quantitatively documented. RESULTS: Total 60 pts were evaluated at three months follow-up time, including 42 pts with DLBCL, 10 with PMBCL, 7 with FL and 1 with gastric mucosal associated lymphoid tissue lymphoma (MALT). The median age was 47 years (range, 2-77), including 31 males and 29 females. Pt characteristics include 16 pts with stage IV disease (27%), 2 after allo-transplantation (3.3%), 4 with bone marrow involvement (6.7%), 8 with CNS involvement (13%), and 7 received prior PD-1 antibody treatment (12%). The performance status of 52 of the 60 patients (87%) was Eastern Co-operative Oncology Group score 0 at time of infusion. The study included two treatment cohorts: 30 pts received single 4SCAR19 cells, and 30 pts received CAR2.0 targeting CD19 plus an additional target, depending on the individual pt's tumor antigen staining results. In the DLBCL cohort, 25 pts received single target 4SCAR19 cell infusion, at an average dose of 1.42x106 CART cells/kg, and resulted in 7 complete response (CR), 9 partial response (PR), 3 stable disease (SD) and 6 progressive disease (PD). The other 17 DLBCL pts received dual target CART infusions (CD19 plus CD20, CD22, CD38 or CD70) at an average dose of 2.32x106 CART cells/kg, and resulted in 7 CR, 8 PR ,1 SD and 1 PD. The 5 FL pts received dual target CD19+CD22 CART infusions, at an average dose of 4.64x106 CAR-T cells/kg, and resulted in 4 CR, and 1 PR. The 10 PMBCL pts, 3 received single target 4SCAR19 cell infusions, at an average dose of 2.85x106 CART cells/kg, and resulted in 1 PR and 2 PD; the other 7 PMBCL pts received dual target CART infusions (CD19+CD22, CD30, or CD20), at an average dose of 4.87x106 CART cells/kg, and resulted in 4 CR, 2 PR and 1 PD. The MALT pt received double CART cell infusions, 2.8x106 4SCAR19 cells/kg and 2x106 4SCAR-PSMA cells/kg, and achieved PR. None of these pts developed greater than grade 2 CRS response, except for 1 DLBCL pt, who achieved CR following a grade 3 CRS. The toxicity profile of the 4SCART is consistent with our previous experience with B cell acute lymphoblastic leukemia. In summary, the comparison between single versus double CART cell infusions clearly illustrates an increased response rate for the double CART cohorts, either in DLBCL pts or in all lymphoma pts, as illustrated in the summary pie graphs below. CONCLUSIONS: The results of the multi-target 4SCAR2.0 therapy for the treatment of highly resistant lymphomas have demonstrated increased safety and improved response rate with this novel approach. There is clear overall clinical benefit with the multi-target CART regimen as compared with the single CD19 CART treatment. Continued follow-up will verify whether the 4SCAR2.0 therapy regimen can achieve long term overall survival. Figure Disclosures No relevant conflicts of interest to declare.

CD19/CD22 Dual-Targeted Chimeric Antigen Receptor T-Cell Therapy for Relapsed/Refractory Aggressive B-Cell Lymphoma: a Safety and Efficacy Study

Introduction Chimeric antigen receptor (CAR)-T-cell therapy has revolutionized the treatment of relapsed/refractory (R/R) B-cell hematological malignancies, primarily acute lymphoblastic leukemia (ALL), and B-cell non-Hodgkin lymphoma (NHL). CD19 CAR-T cells have been extensively studied and have been shown to yield complete remission (CR) rates of about 90% in R/R ALL, but substantially lower (50%) rates in R/R NHL. Moreover, persistence is usually limited, and antigen escape-mediated relapse is a major limitation. Dual CAR-T cells targeting both CD19 and CD22 may address these limitations. Patients and methods We developed a bispecific CAR-T cells that could concomitantly recognize CD19- and CD22-expressing targets by incorporating both CD19 and CD22 single-chain variables in a single CAR construct (Figure 1A). We designed a prospective study to assess the safety and efficacy profiles of the dual CAR-T therapy in patients with R/R aggressive B-cell lymphoma. Results The preclinical cytotoxicity evaluation of the CD19/CD22 dual-targeted CAR-T cells was performed in comparison with mono-specific CD19-BB-002 and CD22-BB-002 CAR-T cells in HeLa cells that were engineered to express CD19, CD22, or both antigens. The dual-antigen specific CAR-T cells performed equally well when compared with the mono-specific CAR-T cells when there was only a single antigen present on the target cells; better performance was observed when both antigens were present on target cells (Figure 1B). In addition, the dual-antigen specific CAR-T cells induced equal amounts of interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-γ, when compared with the two mono-specific CAR-T cells (Figure 1C). Furthermore, the CD19 CAR-T cells induced more IL-2 and tumor necrosis factor (TNF)-α than the CD22 CAR-T cells and dual-antigen CAR-T cells. However, in the presence of both CD19 and CD22 antigens, the dual-specific CAR-T cell tended to produce more granzyme B, which may explain the higher degree of cytotoxicity when compared with the two mono-specific CAR-T cells (Figure 1D). Twenty-four patients were screened. Of the 16 eligible patients 14 (87.5%) achieved objective response (RR), with 10 (62.5%) achieving complete response (CR). The 2-year overall survival (OS) and progression-free survival (PFS) rates were 77.3% and 40.2%, respectively (Figure 2A). Achieving CR (HR: 0.017, 95% CI: 0.000-0.935; P=0.046) and number of prior lines of chemotherapy (n=2) (HR:135.784, 95% CI: 1.069-17248.110, P=0.047) were found as independent prognostic factors associated with favorable PFS. The 2-year OS and PFS of the CR patients were higher than those of the non-CR patients (100% versus 41.7%, P=0.015; 66.7% versus 0%, P &amp;lt; 0.001), respectively (Figure 2B). The 2-year PFS in patients received 2 prior lines of chemotherapy was higher as compared to those that received more than 2 lines of chemotherapy (68.6% versus 16.7%, P=0.049) whereas the OS in the 2 groups did not differ significantly (83.3% and 71.1%, P=0.613) (Figure 2C). Severe grade 3 cytokine release syndrome (CRS) was observed in only one patient, while 4 had grade one and 11 had grade 2, respectively. No patient developed neurotoxicity. Conclusions Immunotherapy with a novel CD19/CD22 dual targeted CAR-T cells yields a potent and durable anti-lymphoma response with no neurotoxicity or severe CRS. Bispecific CD19/CD22 CAR-T cells represent a safe and potent anti-lymphoma cellular based targeted immunotherapy. Figure 1 Disclosures No relevant conflicts of interest to declare.

The Safety and Efficacy of a CRISPR/Cas9-Engineered Universal CAR-T Cell Product (CTA101) in Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Introduction Although autologous CAR-T therapy targeting CD19+ B-cell has proven efficacy in hematological malignancies. The challenges such as undesirable waiting time, possible manufacturing failures and high cost, remain to be solved. In addition, antigen-loss/downregulation leads to treatment failure after single-target CAR-T therapy. To tackle these issues, we developed a universal CD19/CD22-targeting CAR-T cell with the TRAC region and CD52 gene disrupted using CRISPR/Cas9 technology. Pre-clinical data demonstrated the safety of CRISPR gene editing and the anti-leukemia ability of CTA101. Method Pts with CD19+ or CD22+ r/r B-ALL received a single dose of CTA101 with traditional 3+3 dose escalation (dose level: 1, 3 and 6×106 CAR+ T cells/kg). Prior to CTA101 infusion, pts received pre-conditioning chemotherapy regimen consisting of cyclophosphamide, fludarabine and alemtuzumab. The primary endpoint is the frequency of DLTs and AEs and secondary endpoints including ORR, OS, cellular PK, etc. (NCT04227015) Results Characteristics of CTA101 CTA101 was manufactured by electroporation of ribonucleoprotein complexes (RNP) comprising Cas9 loaded with sgRNA targeting TRAC and CD52 followed by lentiviral transduction of the CAR transgene. All products were expanded above 100-folds after 10-12 days manufacturing (Fig.1a). CAR expression was detected by FCM staining, ranging from 40~80% (Fig.1b). The frequency of editing as determined by flow cytometry was consistently above 85% (88%-97%) for TRAC and above 65% (68%-83%) for CD52 (Fig.1c, 1d). The CD19/CD22 dual targeting universal CAR-T cells showed potent antigen specific cytotoxicity, and mitigated tumor antigen escape (Fig.1e). Whole genome sequencing was carried out to assess the on-target and off-target editing events. While most mutations were on target, a few off-target mutations were identified, which needed further investigation (Fig.2). QPCR assays identified no detectable rearrangements occurred with the simultaneous editing of four loci: TRAC and CD52 (TRAC-CD52) during the manufacture process. Residual Cas9 protein was quantified during the manufacturing process, showing declining levels that were 0.195 fg/cell (detection limit of sensitive ELISA) in the final product. Clinical result As of August 5th 2020, 8 adult pts were screened, and 6 adult pts were enrolled. All of 6 patients received one infusion of CTA101 (DL1: 3 pts; DL2: 3 pts), with no more than 8 days between enrollment and infusion. The median age of 6 pts was 49 years (range, 26 to 56), the median prior lines of therapies were 5 (range, 2 to 8), and the median marrow blast percentage was 52% (range, 1 to 82). 3 pts had hyperleukocytosis prior to enrollment with difficulties in apheresis for autologous CAR-T, 1 relapsed after autologous CD22 CAR-T, and 3 had high-risk genomic lesions (Table 1). All 6 pts were available for evaluation of safety and efficacy. No DLTs, GvHD, ICANS and death occurred to date. All pts experienced CRS (3 G1, 2 G2, 1 G3), and the G3 CRS recovered within 7 days with one dose of tocilizumab and glucocorticoids. Other common AEs were prolonged cytopenia (3 G3), virus reactivation/infections (CMV 1 G2, 2 G3; ADV 1 G1), bacterial pneumonia (1 G3), and fungal sepsis (1 G3). The majority of events recovered to date. No replication competent lentivirus (RCL) has been detected. On D28 after CTA101 infusion, 5/6 (83.3%) pts achieved CR/CRi, and 5/5 (100%) pts achieved MRD-. With a median follow-up of 85 days (range, 53 to 202), 4/5 pts remained MRD-, 1/5 had MRD+ CR (Fig. 3). Only 1 pt received HSCT in remission on D60. Expansion had been observed in all pts and peaked from D10 to D14 (Fig.4). The correlation among dose level, donor difference, tumor burden and degree of expansion could not be identified. The median duration of persistence was 42 days. The lowest peak expansion was detected by qPCR in the pt without response and whose T lymphocyte recovered within D7, while FCM did not detect CTA101 expansion in the pt. Conclusions CTA101 demonstrates manageable safety profile and deep response of high MRD- CR/CRi rate. No GvHD and CRISPR/Cas9 genome editing associated AEs occurred. Early data of cellular PK and efficacy illustrate that CRISPR gene editing does not curtail the expansion and anti-leukemia capacity of CAR-T. Updated data, including long term gene editing-related AEs, will be presented after further follow-up. Disclosures Zhou: Nanjing Bioheng Biotech Co., Ltd: Current Employment. Ge:Nanjing Bioheng Biotech Co., Ltd: Current Employment. Han:Nanjing Bioheng Biotech Co., Ltd: Current equity holder in private company. Wang:Nanjing Bioheng Biotech Co., Ltd: Current Employment. Zhang:Nanjing Bioheng Biotech Co., Ltd: Current Employment. Ren:Nanjing Bioheng Biotech Co., Ltd: Current equity holder in private company.

Tandem CD19/CD22 Dual Targets CAR-T Cells Therapy Acquires Superior CR Rate Than CD19 CAR-T Cells: A Case Controlled Study

Background: CD19 chimeric antigen receptor (CAR) T-cells therapy has shown unprecedented success in relapsed/refractory (r/r) ALL. Now it is recommended for the treatment of patients up to 25 years with r/r precursor B-ALL. Despite its high response rate, approximately 50% of r/r ALL patients relapsed after CD19 CAR T-cell therapy, which was mainly due to CD19 antigen loss or short persistence of CAR T-cells in vivo. CD19 and CD22 dual target CAR T-cells have demonstrated to reduce relapse post CD19 CAR T-cell therapy in limited cases. This study was designed to investigate the efficacy and safety of CD19/CD22 dual targets CAR-T cells and the correlation between that and CD19 CAR-T cells. Methods: A novel tandem CD19/CD22 CAR-T construct with CD28 and OX40 co-stimulatory domains were administered. All patients received FC (fludarabine, 30 mg/m2, days 1-3 and cyclophosphamide, 300 mg/m2, days 1-3) based regimen chemotherapy pre-infusion. Median infusion dose of CAR-T cells was 1(0.5-2.5) *107 cells/kg. All the patients accepted the bone marrow examination 28 days post CAR-T infusion. We conducted a retrospective, case controlled study analysis at the First Hospital of Soochow University. Every patient with active disease treated with CD19/CD22 CAR-T cells therapy was paired with one control subject who received CD19 CAR-T cells from January 2017 to September 2019. We matched the control group to prevent bias according to: (1) bone marrow blast before CAR-T infusion; (2) extramedullary involvement; (3) cytogenetic risk groups according to NCCN guideline; (4) transplantation status pre-infusion. Results: From 2017 Oct to 2020 Jul, 36 patients were enrolled into the clinical trial (NCT: 03614858). Patients who accepted more than 4 previous treatments account for 58.3% and 8 patients received stem cell transplantation pre-infusion. Bone marrow blasts 40.75 (0~94.5)%. After tandem CD19/CD22 CAR-T infusion, all patients achieved completely remission (CR) and minimal residual disease negative (MRD-) CR rate is 77.8%. The 6-month OS rate is 88.36% and 12-month OS rate is 70.6%. The 6-month LFS rate is 88.072% and the 12-month LFS rate is 69.216%. CAR-T bridging HSCT (25/36) obviously improves the 1-year LFS rate compared with non-transplantation group (11/36) (82.309% vs 31.169%, p=0.0135). All the side effects were mild and can be relieved through support treatments. 8 out of 36 patients developed with grade 3~4 CRS. 2 patients had HLH but soon relieved through low dose steroids. 28 out of 36 patients who accepted dual targets CAR-T cells didn't get complete remission before infusion. Therefore, 28 patients were enrolled in the CD19 CAR-T group. There are no significant differences between two groups at baseline. The CR rate was higher in CD19/CD22 group than that in CD19 group(100% vs 53.57%, p=0.000), so was MRD-CR rate(71.43% vs 42.86%,p=0.000). In survival analysis, 6 month overall survival rate of CD19/CD22 group was 69.276% while CD19 group was 53.571%. Of 28 patients who received dual target treatment, 7 patents relapsed while 4 relapsed out of 15 CR patients in CD19 group. Conclusions:The tandem CD19/22 dual CAR-T cell therapy is a safe and high efficacy treatment for R/R ALL patients. Our study demonstrated that dual targets CAR-T cells acquires higher CR rate than CD19 CAR-T cells. It is possible that multi-targeted CAR-T cell therapy may overcome this resistance mechanism and improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

Enhanced Safety and Antitumor Efficacy of Switchable Dual Chimeric Antigen Receptor-Engineered T Cells against Solid Tumors through a Synthetic Bifunctional PD-L1-Blocking Peptide.

Chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) therapy has an excellent efficacy in cancer treatment, especially its impressive results in hematological malignancies. Unfortunately, its application on solid tumors is challenged by the off-target effects caused by lacking of tumor specific antigens and the immunosuppression caused by the tumor microenvironment. We constructed a switchable dual receptor CAR-T cell (sdCAR-T) whose activity relied upon double antigens (mesothelin and fluorescein isothiocyanate) and was strictly controlled by a "switch" (FPBM) consisting of a PD-L1 blocking peptide conjugated to fluorescein isothiocyanate. SdCAR-T cells were activated only when FPBM and cognate tumor cells expressing both PD-L1 and mesothelin coexist. Importantly, long-term proliferation experiments in vitro and the pharmacodynamic study in vivo showed a stronger antitumor activity of this system compared to the second generation mesothelin CAR-T cells. In view of this novel treatment paradigm being safer and more effective than traditional CAR-T cells, it may become a new strategy for the treatment of solid tumors.

A brief history of CAR-T cells: from laboratory to the bedside

AbstractChimeric antigen receptors (CARs) are genetically engineered receptors that provide specific properties to an immune effector cell and these receptors gain the specificity of a monoclonal antibody targeted against specific tumor cells. T cells with engineered CARs acquire potent immunological properties and redirect the immune system in order to eliminate malignant cells. First-engineered T cells with chimeric molecule (CAR-T cells) were developed in 1989–1993 by Israeli immunologists Zelig Eshhar and Gideon Gross. The first clinical application of CAR-T cells was done in the University of Pennsylvania and Children’s Hospital in Philadelphia by the immunologist Carl June and hematologist David Porter to patients with chronic lymphocytic leukemia in 2011 and together with the pediatrician Stephan Grupp to patients with acute lymphoblastic leukemia (ALL) in 2012. The US Food and Drug Administration Agency (FDA) in 2017 and the European Medicines Agency (EMA) in 2018 have licensed two products of CAR-T cells: tisagenlecleucel for the use in children and young adults up to 25 years of age with B-cell ALL who do not respond to treatment or have relapsed two or more times and tisagenlecleucel and axicabtagene ciloleucel for the use in adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Current progress in CAR technology includes the use in other hematological malignancies, solid tumors, the use of dual CAR-T cells and chimeric antigen receptor natural killer cells (CAR-NK cells).

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Introduction Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated a high response rate, the durable response rate is around 30%. Most patients lose their CART cells, and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in this field. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and immunosuppression. Objective CAFs from MM patients lead BCMA CART cell dysfunction and targeting both MM cells and CAFs can overcome this resistance. Methods CAFs were isolated from the bone marrow (BM) of MM patients and purified using anti fibroblast beads. MM-TME mouse model was established by injecting a 1:1 ratio of BCMA+ OPM2 (MM cell line) to CAFs. Five different CART cells were tested in this study. For single transduced CAR: BCMA, CS1, and fibroblast activation protein (FAP). For dual transduced CAR: BCMA-CS1 and BCMA-FAP CART. These were generated through the double transduction of two lentiviral vectors. Results BCMA CART cell proliferation assay showed inhibition of CART expansion in the presence of CAFs, and this was mediated by TGF-β (Fig A). MM-TME mouse model revealed a significant acceleration of MM progression (Fig B). To identify targets, we verified FAP expression on CAFs. Interestingly, there was a low level of CS1, but not BCMA expressed on CAFs (Fig C). Then, we evaluated the impact of CAFs on functions of BCMA CART cells compared to dual CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of CAFs, the proliferation of BCMA CART cells, but not the dual CART cells was significantly inhibited (Fig A). Similarly, in the presence of CAFs, the production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, we studied the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells was able to eradicate MM (Fig E). Then, the MM-TME model was used to assess the effect of targeting CAFs. Mice were treated with 1) control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A low dose (1 × 106 iv) of CART cell was used to induce relapse. BCMA CART cells led to a transient antitumor activity in this model (mice died within 2 weeks), while dual CART cells resulted in durable remissions, a higher number of circulating T cells, and long term survival (Fig F). Conclusion We demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in MM.

BCMA and CD19 Targeted Fast Dual CAR-T for BCMA+ Refractory/Relapsed Multiple Myeloma

BCMA and CD19 Targeted Fast Dual CAR-T for BCMA+ Refractory/Relapsed Multiple Myeloma

Safety of Autologous Humanized Anti-CD19 and Anti-CD20 Dual Specific CAR-T Cells in Adult Patients With Diffuse Large B-cell Lymphoma

Safety of Autologous Humanized Anti-CD19 and Anti-CD20 Dual Specific CAR-T Cells in Adult Patients With Diffuse Large B-cell Lymphoma

Anti-CD19/CD22 Dual CAR-T Therapy for Refractory and Relapsed B-Cell Acute Lymphoblastic Leukemia

Introduction Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has demonstrated high success for B-cell acute lymphoblastic leukemia (B-ALL). Despite the initial high complete remission (CR) rate, about half of patients (pts) relapse at 1 year. CD19 antigen loss was observed in a significant number of relapsed patients. CD22 is another leukemic marker that often expressed on the surface of CD19- relapsed B-ALL blasts. We have developed a bispecific CAR construct targeting CD19 and CD22. Here we report results from a phase Ⅰ clinical trial of CD19/CD22 (GC022) dual CAR-T to evaluate the safety and feasibility of treating patients with relapsed/refractory B-ALL. Methods The CD19/CD22 dual CAR-T cells were manufactured in a cGMP facility. Patients' peripheral blood (PB) mononuclear cells were first collected, and CD3+ T cells were separated. The cells were then transfected by lentivirus encoded with CD19 CD22 bispecific scFv sequences. The CAR-T cells produced in this way contained a 4-1BB co-stimulatory signal domain. The CAR-T cells were then cultured for 8-14 days until sufficient cells were harvested for infusion. All pts received conditioning regimen of fludarabine and cyclophosphamide intravenously for 3 consecutive days with doses of 30 mg/m2/day and 250 mg/m2/day, respectively before a single infusion of CAR-T cells. The level of infused CAR-T cell proliferation in PB was analyzed by qPCR and flow cytometry. The primary end points were to evaluate feasibility and toxicity, and the secondary end points included disease response and engraftment/persistence of infused CD19/CD22 dual CAR-T cells. Results From Feb. 2019 to 23 July. 2019, 17 patients (pts) with relapsed/refractory B-ALL including 4 pts who previously treated with CD19 CAR-T cells were enrolled and pts were treated with CD19/CD22 dual CAR-T GC022 (US NIH Clinical#: NCT03825731). Four were adults, 13 pediatrics (age 1-45, Table 1). The median bone marrow (BM) blasts was 19.09 (0.36-87.82) %. Four patients received a low-dose (2.5-5×105/kg) dual CAR-T, 7 received a medium-dose (1-2.5×106/kg) and 5, a high-dose (3-5×106/kg). One patient withdrew immediately before CAR-T infusion due to his personal issue. Anti-leukemic efficacy was evaluated in 11/16 pts (5 pts have not yet reached D15). The 3/4 pts received low dose of GC022 had no response to treatment and 1 had MRD-positive CR. Seven patients who received medium dose achieved 100% CR on D15, highlighting the dose-dependent anti-leukemic activity. Six out of seven pts had MRD negative CR in this medium dose group. Five pts in high dose group have not reached the time for evaluation. No one relapsed with a median observation time of 60 (7-139) days. Cellular kinetic data was analyzed. Median peak of CAR-T copies was 1.09 (0.0022-4.98) x105 copy number/µg PB genomic DNA (Fig.1). The proliferation of medium or high dose groups was significantly better than the low dose group 3.47(0.43-4.98) x105 vs. 0.023(0.0022-0.81) x105(P=0.02) and 2.02(1.89-2.16) x105 vs. 0.023(0.0022-0.81) x105(P=0.004), (Fig.2). The peaks of IL-6, IFN-γ, IL-10, and CD25 were observed around day 7-10. Sixteen out of seventeen pts had grade 0-1 cytokine release syndrome (CRS) and only 1 patient experienced grade 2 CRS. None developed neurotoxicity. Conclusion Our study demonstrates safety and technical feasibility of CD19 and CD22 dual CAR-T in treating patients with CD19+CD22+ relapsed/refractory B-ALL. A low toxicity with dose-dependent high CR rate including pts who previously treated with CD19 CAR-T cells were observed. Longer observation time and more patients are needed to evaluate a beneficial advantage of the CD19/CD22 dual CAR-T over CD19 CAR-T product. Disclosures No relevant conflicts of interest to declare.

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents &amp; Royalties. Cox:Humanigen: Patents &amp; Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents &amp; Royalties, Research Funding; Morphosys: Research Funding.