CD19-targeted Chimeric Antigen Receptor T Research Articles

Early evaluation of CAR-T cell therapy response in R/R DLBCL patients using 18F-FDG PET/CT.

CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy provides a durable response in patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). The role of fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) for early evaluation of response in patients with that immunotherapy was evaluated. Three separate 18F-FDG PET/CT examinations of 53 patients (29 males, 24 females; median 62 years old) with R/R DLBCL were conducted; before bridging therapy [time of decision (TD)], before CAR-T (tisagenlecleucel, n=37; lisocabtagenemaraleucel, n=16) infusion [time of CAR-T infusion (IT)], and one month (M1) after CAR-T infusion. Response was evaluated based on the Deauville 5-point scale and Lugano criteria. Among 21 patients (39.6%) with complete metabolic response (CMR) at IT-PET, 20 were able to continue CMR, while one showed progression at M1-PET. Among 32 patients (60.4%) with non-CMR at IT-PET, 12, 8, 4, and 8 showed CMR, partial metabolic response (PMR), (non-metabolic response (NMR), and progressive metabolic disease (PMD), respectively, at M1-PET as compared with IT-PET. Evaluations of M1-PET as compared with baseline TD-PET indicated 32, 7, 5, and 9 patients with CMR, PMR, NMR, and PMD, respectively. After a median 10.1 months, 26 patients showed progression and 13 had died from DLBCL. The 32 who achieved CMR showed significantly longer progression-free (P<0.0001) and overall survival (P<0.0001) periods as compared to the 21 non-CMR patients. Fluorine-18-FDG PET/CT findings obtained one month after CAR-T cell therapy showed accuracy for early response evaluation and prediction of progression in patients with R/R DLBCL.

Efficacy and Safety of a Third Generation CD20 CAR-T (MB-106) for Treatment of Relapsed/Refractory Indolent B-Cell Non-Hodgkin Lymphoma: Phase-1 Results from a Multicenter Trial

Background: Indolent B-cell non-Hodgkin lymphomas (NHL) remain incurable despite multiple therapeutic options, including chemoimmunotherapy, targeted therapies, bispecific antibodies, and CD19-targeted chimeric antigen receptor T (CAR-T) cells. MB-106, a fully human 3 rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains, had high overall response rate (100%) and complete response (CR) rate (91%) in follicular lymphoma (FL) patients at doses ≥ 3.3×10 6 cells/kg in a single institution trial (Shadman et al., 17-ICML, 2023). We seek to replicate the favorable efficacy and safety profile of MB-106 in a multicenter trial with centralized manufacturing. Methods: MB-106 is being evaluated in a Phase 1/2, open label, multicenter study utilizing a 3+3 dose-escalation design in R/R B-cell NHL patients with confirmed CD20 expression. Prior treatment with CD19-targeted CAR-T therapy is permitted. Following lymphodepletion (cyclophosphamide 300 mg/m 2/day and fludarabine 30 mg/m 2/day for 3 days), MB-106 is administered to patients with indolent B-cell NHL at one of two dose levels (DL): DL1, 3.3×10 6; and DL2, 1.0×10 7 cells/kg. MB-106 is infused in the outpatient setting if allowed by the institution, except for the first patient in each dose level cohort (overnight observation). Dose-limiting toxicities (DLT) are monitored through day 28. The treatment response is assessed on day 28 and on subsequent visits, and best overall response is reported here. CAR-T persistence is assessed by both flow cytometry and qPCR in peripheral blood mononuclear cells. Results: Three patients with indolent NHL (2 FL, 1 Waldenström macroglobulinemia [WM]) received MB-106 at DL1. In addition, one patient with hairy cell leukemia variant (HCL-v) received non-conforming material (drug product that failed to meet release criteria) at DL1, following FDA authorization. These four patients were heavily pre-treated with a median of 5.5 prior lines of treatment. High-risk features included progression of disease within 24 months of first-line treatment (POD24, n=1), prior autologous stem cell transplant (n=1) and prior CD19-targeted CAR-T therapy (n=1). No DLTs were observed at DL1. Three patients experienced Grade 1 cytokine release syndrome. There have been no occurrences of immune effector cell-associated neurotoxicity syndrome (ICANS). The 2 FL patients achieved CR by both PET-CT and bone marrow biopsy, one of whom had been previously treated with CD19-targeted CAR-T. The WM patient, who had nine prior treatments, including autologous stem cell transplant, and high disease burden, achieved very good partial response, with complete metabolic response by PET-CT, morphologic clearance of lymphoma in bone marrow, and resolution of the IgM paraprotein. The patient with HCL-v, who had been transfusion dependent, continues to have stable disease with decreased disease in the bone marrow, and achieved complete transfusion independence which is ongoing at 6 months. All patients displayed MB-106 expansion with peak levels between 7-14 days post-infusion. Persistence of CAR T-cells is ongoing at 6 months. Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in responses, including CRs, and CAR-T persistence in patients with R/R indolent NHL and was associated with favorable safety profile with no occurrence of Grade 3 or Grade 4 cytokine release syndrome and no ICANS of any grade. CRs have been observed in patients previously treated with CD19-targeted CAR-T in both this multicenter trial and the original single institution trial. Dosing is ongoing in the final dosing level (1.0×10 7 cells/kg) to establish the recommended Phase 2 dose for a planned pivotal trial in WM. It is anticipated that the complete data set from the Phase 1 indolent NHL arm will be presented.

Multiplex Genome Editing of Human T Cells with Innovative Transformer Base Editor (tBE) for Construction of Next Generation CAR-T Therapies

Autologous chimeric antigen receptor T (CAR-T) cell therapy has achieved great success for antitumor treatments especially against hematological malignancies, and several autologous CAR-T therapies have been approved around the world. However, high manufacturing cost and long manufacturing process hinder the broader application of autologous CAR-T cell therapy. As a result, off-the-shelf universal CAR-T cell therapy with donor derived healthy T cells is of great interest and under development by many companies globally. Gene editing technologies including TALENs, CRISPR/Cas nucleases and base editors are of great potential in T cell engineering for allogeneic T cell therapy. The innovative transformer base editor (tBE) is a base editing system that avoids to cause DNA double strand breaks (DSBs) and exhibits higher editing efficiency, no detected off-target mutations and lower cytotoxicity. Due to its capability to avoid off-target mutations and DSB-caused chromosomal translocations, tBE is potentially one of the best gene editing tools to induce multiplex editing in cells. Here we show that the tBE was used to induce highly efficient single or multiplex gene editing in primary human T cells. With optimized electroporation conditions for tBE, TRAC, CD52 and PDCD1 could be knocked out simultaneously with editing efficiency up to ~90% for each target gene. tBE triggered no detected off-target mutation or chromosomal abnormalities, which were reported in the cases of CRISPR/Cas- or TALEN-mediated multiplex editing. CD19-targeted CAR-T cells with triplex base editing exhibits enhanced antitumor efficacy in vitro and in mouse models compared with unedited CAR-T cells. Our results demonstrated the application of tBE for construction of next generation universal or enhanced CAR-T therapies.

Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B cell non-Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR-T cell kinetics is fundamental to understand the correlation between CAR-T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR-T cell detection using flow cytometry. We set up and compared with each other three assays for CD19 CAR-T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single-chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC-conjugated antihistidine antibody and CD19bio + APC-conjugated antibiotin antibody) showed better sensitivity and specificity compared with the direct staining with CD19-FITC, and CD19his had a better cost-effective profile. We validated CAR detection with CD19his with parallel quantitative real-time polymerase chain reaction data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR-T cell positive subpopulations. Finally, this method can be used for different anti-CD19 CAR-T cell products and for different sample sources. These data demonstrate that detection of CAR-T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR-T cells.

The nutritional impact of CD19-targeted CAR-T therapy versus BEAM chemotherapy for adult patients with lymphoma.

Chimeric antigen receptor T (CAR-T) cell therapy is a novel therapy demonstrating durable remissions in patients with refractory or relapsing non-Hodgkin's B-cell lymphoma. Maintaining a patient's nutritional status has been demonstrated to improve outcomes in cancer treatment. However, no studies have investigated how CAR-T therapy affects nutritional status, nor compared its impact with other cancer treatments for this patient group. The primary aim of the present study was to investigate the effect of CAR-T therapy on the prevalence of nutrition impact symptoms (NIS) and nutritional status within 30 days post-treatment of patients with lymphoma compared to a conditioning regimen for autologous haematopoetic stem cell transplant (carmustine/BCNU, Etoposide, cytarabine/Ara-C, Melphalan [BEAM] auto-haematopoetic stem cell transplant [HSCT]). Clinical notes of patients with lymphoma who underwent either CAR-T therapy or BEAM auto-HSCT between 2018 and 2021 were reviewed. Data extracted included body weight measurements and NIS, including decreased appetite, nausea, vomiting, diarrhoea, constipation, mucositis, cytokine release syndrome (CRS) and neurotoxicity at baseline and 30 ± 7 days post-treatment. In total, 129 adults with lymphoma (n = 88 CAR-T vs. n = 41 BEAM) were included. Nutritional status was assessed in both groups at baseline prior to treatment. Mean absolute weight change was significantly different between groups (3.05 kg in CAR-T, -5.9 kg in BEAM, p ≤ 0.001). This was also significant when weight loss was categorised into percentage weight loss (p = 0.01). CAR-T patients experienced a significantly lower prevalence of decreased appetite (52.3% vs. 97.6%) nausea (25% vs. 78%,) vomiting (10.2% vs. 53.7%), diarrhoea (43.2% vs. 96.7%) and mucositis (5.7% vs. 75.6%) combined across all levels of severity compared to BEAM chemotherapy (all p ≤ 0.01). CRS and neurotoxicity, which are specific side effects of CAR-T therapy, were moderately positively associated with weight loss. Weight loss, percentage weight loss and NIS were significantly reduced in CAR-T compared to BEAM treatment. However, patients who experienced neurotoxicity during treatment did have significant weight loss.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Safety and efficacy of autologous and allogeneic humanized CD19-targeted CAR-T cell therapy for patients with relapsed/refractory B-ALL

BackgroundMurine chimeric antigen receptor T (CAR-T) cell therapy has demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). However, the potential immunogenicity of the murine single-chain...

Timing of PD-L1 Blockade with Durvalumab May Affect Outcomes of CD19 CAR-T Cell Therapy for Relapsed/Refractory Large B-Cell Lymphoma

Introduction CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown high overall response rates (ORRs) in patients (pts) with relapsed/refractory (R/R) large B-cell lymphoma (LBCL); however, CD19 CAR-T cells fail to elicit a complete response (CR) in a considerable proportion of pts. PD-L1 expression in the tumor microenvironment could impair the efficacy of CAR-T cell therapy. PD-1/PD-L1 pathway blockade may enhance the function and antitumor activity of CAR-T cells. Here, we report the analysis of a phase I dose-finding study (NCT02706405) of combination therapy with CD19-targeted 4-1BB-costimulated CAR-T cells (JCAR014) and escalating doses of durvalumab (durva), an anti-PD-L1 monoclonal antibody, in adults with R/R LBCL. Methods Pts were treated in 1 of 2 groups. All pts received lymphodepletion (LD) with cyclophosphamide (Cy) and fludarabine (Flu) followed by JCAR014 infusion. Pts in group 1 (Grp 1) received the first dose of durva (225 mg or 750 mg) after JCAR014 infusion. Pts in group 2 (Grp 2) received the first dose of durva (7.5 mg, 22.5 mg, 75 mg, 225 mg, or 750 mg) 1 day prior to JCAR014 infusion. Up to 10 doses of durva were administered at 4-week intervals after JCAR014 at the highest identified safe dose until toxicity or disease progression. Adverse events (AEs) were graded by CTCAE 4.03, except for cytokine release syndrome (CRS), which was graded according to Lee 2014 consensus criteria. Response was reported according to PET/CT 2014 Lugano criteria. Results Between 01/23/2017 and 07/02/2020, 34 pts were screened, 32 underwent leukapheresis, and 29 were enrolled and received LD and JCAR014. Eleven pts were treated in Grp 1 and 18 in Grp 2 (Table 1). All pts received 2 x 106 JCAR014 cells/kg, except for the first 2 pts treated on the study who received 7 x 105 cells/kg. The most common AEs of any grade related to LD and JCAR014 with or without durva were CRS (41%), neutropenia (21%), neurotoxicity (17%), and hypogammaglobulinemia (17%). The incidences of any grade and grade ≥ 3 CRS and neurotoxicity were similar between Grp 1 and Grp 2 (Table 2A). Patients in Grp 2 had later onset of CRS after JCAR014 infusion compared with Grp 1 (median of 6 vs. 4 days, P = .05) and shorter duration of CRS (median of 3 vs. 8 days, P = .08). Among the 27 pts evaluable for dose-limiting toxicity (DLT), 2 (7%) had DLT: 1 pt with grade 4 CRS in Grp 2 (durva 225 mg) and 1 pt with prolonged grade 3 neurotoxicity in Grp 2 (durva 750 mg). No maximum-tolerated dose of durva was identified. In pts evaluable for response (n = 26), the ORR and the CR rate at 3 months were 35% (95% CI, 17-56) and 27% (95% CI, 14-46). We retrospectively compared the response rates of LBCL pts treated with JCAR014 in combination with durva (NCT02706405) with LBCL pts who received the same Cy/Flu lymphodepletion regimen and JCAR014 dose (2 x 106 cells/kg) without durva (JCAR014 alone cohort) on our previous phase I/II clinical trial (NCT01865617). There was a trend towards lower ORR (P = .08) and CR rate (P = .09) in pts treated with JCAR014 in combination with durva compared with those treated in the JCAR014 alone cohort (Table 2B). There were no significant differences in ORR and CR rate between patients in Grp 1 and those who received JCAR014 alone. However, despite a trend towards lower tumor burden, pts in Grp 2 (first durva before JCAR014) had lower ORR (P = .07) and CR rate (P = .03) compared with those treated in the JCAR014 alone cohort, and a trend towards lower CR rate compared with those treated in Grp 1 (first durva after JCAR014; P = .16). Patients treated with JCAR014 alone had significantly shorter time to CAR-T cell peak counts in blood compared with patients treated with JCAR014 in combination with durva (median of 8 vs. 14 days, P = .01). We did not observe significant differences in peak CAR-T cell expansion, area under the curve (AUC) from day 0 to 28 after JCAR014 infusion, and day 28 CAR-T cell counts by qPCR between the cohorts. Conclusion The combination of JCAR014 with durva for the treatment of adult pts with LBCL was safe. Our findings suggest that the timing of initiation of durva therapy is a key variable that may affect outcomes. To our knowledge, this is the first report suggesting that PD-L1 blockade may impact toxicity and antitumor response in pts with LBCL undergoing CD19 CAR-T cell therapy. Additional studies will be required to determine the optimal approach for combining CD19 CAR-T cell therapy with PD-1/PD-L1 pathway blockade in LBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1440: BTK INHIBITORS IMPROVE CART19 CELL THERAPY BY MODULATING IMMUNE SYSTEM

Background: CD19-targeted chimeric antigen receptor-T (CART19) cell therapy has achieved extraordinary success in B-cell lymphoma and leukemia. However, recurrence and adverse effects, including cytokine release syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS), remain its obstacles. Recurrence, even resistance to CART19 cell therapy, might be attributed to CAR-T cell dysfunction and suppressor cells in the microenvironment. Cytokines required for CRS and ICANS are predominantly derived from monocytes and macrophages. Given that Bruton’s tyrosine kinase (BTK) is expressed on myeloid cells, we considered that BTK inhibitors (BTKi) could improve CAR-T cell therapy by modulating immune system. Aims: To systematically evaluate the effects of three BTKi, ibrutinib (IB), zanubrutinib (ZB) and orelabrutinib (OB), on T cells, CART19 cells and tumor microenvironment. Methods: T and CART19 cells were cultured with the three BTKi for 4 days, and the immune and differentiated phenotypes (PD1, TIM3, CTLA4, CD25, CD69 and CD28, CD45RA and CD62L) were analyzed by flow cytometry (FCM). The effects of BTKi on CART19 cytotoxicity against NALM-6 were assessed for 24-hour coculture. CART19, NALM-6 and macrophages differentiated from THP-1 upon PMA stimulation were cocultured and IL-6 in the supernatants was detected by cytometry bead assays. Lymphoma mouse models were established with BALB/c mice by subcutaneous injection of 5x106 A20 cells. IB, ZB and OB were administered to the BALB/c mice at 25, 10 and 10mg/kg respectively by oral gavage once daily for 28 days. Vehicle treatment was performed using 0.5% carboxymethylcellulose sodium. Mice in healthy controls were fed with no additional treatment. Splenocytes, bone marrow (BM) samples and tumor specimens were harvested, and single-cell suspensions were prepared to analyze immune phenotypes by FCM. Results: Compared with control T cells, IB-supplemented T cells significantly reduced the expression of PD-1 (62.28% vs 14.95%, P = 0.0024), TIM-3 (71.38% vs 7.88%, P <0.0001) and CTLA-4 (92.65% vs 15.5%, P <0.0001) following CD3/CD28 stimulation (Figure 1A). The downregulation of the suppressive markers was also observed in T cells treated with ZB, but not with OB (Figure 1A). In parallel, IB and ZB decreased activation markers expression and increased effector memory cell subsets, while decreased naïve T cells (Figure 1A and B). BTKi decreased IL-6 level in the supernatants of CART19, NALM-6 cells and macrophages (Figure 1C), and decreased CTLA-4 expression on CART19 (Figure 1D). The effects of BTKi on CART19 differentiated phenotypes were not observed (Figure E). Considering strong CD3/CD28 stimulation and tonic signaling could drive terminal differentiation and even apoptosis of CAR-T cells, we supposed BTKi could protect CART19 from excessive activation in long-term cultivation and stimulation (not shown). The three BTKi did not show synergistic effects on cytotoxicity of CART19 against NALM-6 cells (not shown). In lymphoma mice models, the three BTKi all increased the ratio of type 1 macrophages (M1) to type 2 macrophages (M2) and Th1 to Th2 in tumor, and reduced tumor-infiltrating macrophages (Figure 1F-H). BTKi induced significant decreases in exhaustion markers expression on T cells in BM tissues, but not in tumor specimens (Figure I). Image:Summary/Conclusion: Differentiation and exhaustion of CAR-T cells highly activated upon CD3/CD28 stimulation and tonic signaling can be prevented by BTKi. Moreover, BTKi can alter the tumor microenvironment and downregulate IL-6 level to improve the efficacy and safety of CART19 cell therapy.

Relma-cel (JWCAR029) in relapsed and refractory B-cell non-Hodgkin lymphoma: A two-year survival update of a phase I study.

e19555 Background: B-cell non-Hodgkin lymphoma (B-NHL) has a very poor prognosis. Relma-cel (JWCAR029) is a CD19-targeted chimeric antigen receptor T (CAR-T) cell suspension. In a phase I trial performed in November 2017 in Beijing Cancer Hospital, China, JWCAR029 was used to treat relapsed/refractory B-NHL (R/R B-NHL) in adult patients, showing a high response rate and persistent response. This report aimed to update 2-year survival data generated in the above trial. Methods: This was an open-label, dose-escalating phase I study (JWCAR029-001, NCT03344367) involving patients with R/R B-NHL. The patients received a 3-day treatment with the conditioning chemotherapy regimen, consisting of fludarabine (25 mg/m2/day) and cyclophosphamide (250 mg/m2/day). Then, they received a single dose of intravenous JWCAR029 (25×106, 50×106, 100×106, or 150×106 CAR T cells). Two-year PFS and OS after JWCAR029 treatment were evaluated. The data were updated till the cut-off date of January 18, 2021. Results: A total of 23 patients with R/R B-NHL were enrolled, of whom 22 completed single-dose JWCAR029 cell infusion. All patients completed the 2-year follow-up, with a median time of 24.2 (95%CI 24.0-24.3) months. Based on the efficacy analysis set comprising 20 patients, the best ORR and CRR were 85.00% and 70.00%, respectively. In subgroup analysis, 1-year ORR and CRR in patients administered 100×106 cells were both 3/5 (60%); they were both 5/8 (62.5%) in those administered 150×106 cells. The 1-year and 2-year PFS rates (11 patients censored) were both 55.0%. Median PFS was not reached (95%CI 3.0 months-NA). The 1-year and 2-year PFS rates in patients administered 100×106 cells were both 60.0%; these values were both 62.5% in individuals treated with 150×106 cells. The 1-year and 2-year OS rates (3 patients censored) were both 68.6%. Median OS was not reached (95%CI 10.4 months-NA). The 1-year and 2-year OS rates in patients administered 100×106 cells were both 60.0%; these values were both 75.0% in those treated with 150×106 cells. A total of 6 patients (30%) died. No grade 3 and above CRS or NT were found. Conclusions: JWCAR029 was approved in China in September 2021 after the RELIANCE study (NCT04089215) reported its excellent efficacy and tolerable toxicity in the treatment of relapsed/refractory B-cell lymphoma. This report indicated that R/R B-NHL cases could benefit from JWCAR029. R/R B-NHL cases administered JWCAR029 at 100×106 or 150×106 cells had a promising response rate, with durable PFS and sustained OS benefits after 12 months. The effectiveness and safety of JWCAR029 need to be confirmed in the real-world setting. Clinical trial information: NCT03344367.

Special issue: CAR‐T cell therapy for hematological malignancies

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized the treatment of relapsed/refractory (R/R) B-cell derived hematological malignancies, including acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM). CD19-targeted CAR-T cells yield complete remission (CR) rates of about 90% in R/R ALL and about 50% in R/R NHL, respectively, while BCMA-targeted CAR-T cells yield CR rate of about 50%−80% in R/R MM. Notably, the US Food and Drug Administration have approved six CAR-T cell products (tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, and ciltacabtagene autoleucel) for the treatment of R/R B-NHL, R/R ALL, and R/R MM. However, some patients might not respond to such therapy or quickly relapse after CAR-T cell infusion, which is likely due to the dysfunction and poor persistence of CAR-T cells or the modulation of specific antigen. Thus, alternative treatment strategies must be investigated. In addition, accumulating research have been conducted to discover novel target of antigens with therapeutic efficacy and utility for generating CAR-T cells against acute myeloid leukemia (AML). Safety during CAR-T cell treatment is another important issue, concerning complications such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS), pancytopenia, and infection. Despite the fact that the majority of those adverse effects are minimal, the risk of a life-threatening situation still exists. Cautious treatment for severe adverse events requires a multidisciplinary approach with involvement of not only oncologists but also other internal medicine doctors to guarantee diagnosis and treatment in time. Therefore, it is of vital importance to understand the novel advances of CAR-T cell therapy. This special issue of ImmunoMedicine focuses on novel developments of CAR-T cell therapy for hematological malignancies. The review from Xiangmin Wang (https://doi.org/10.1002/imed.1030) summarized the non-BCMA targeted CAR-T cell therapies for MM. Novel targets including CS1, CD38, CD138, NKG2D, CD70, TACI, and etc. might have the potential to prevent the recurrence and enhance treatment efficiency. The review from Elaine Tan Su Yin (https://doi.org/10.1002/imed.1039) summarized the current breakthrough and future perspectives of CD20-targeted CAR-T cell therapy for hematologic malignancies, especially for R/R B-NHL. The review from Yue Huang (https://doi.org/10.1002/imed.1031) summarized the current achievement and potential AML-associated cell markers of CAR-T cell therapy in AML preclinical studies and clinical trials, and discusses the future directions of CAR-T cell therapy in patients with AML. The review from Yuanyuan Hao (https://doi.org/10.1002/imed.1029) summarized the effects of tumor-derived exosomes (TEXs) on the survival and functions of T cell subsets, as well as their clinical applications. This special issue also contains three original research articles reporting clinical trials of CAR-T cell therapy. Linghui Zhou et al. (https://doi.org/10.1002/imed.1036) reported the data of infections post CAR-T cell therapy, and found that it was a common complication in patients with hematological malignancies treated by CD19 CAR-T cells, which means clinicians should pay more attention to such situations. Kaiting Tang et al. (https://doi.org/10.1002/imed.1037) presented three cases of compassionate CAR-T cell therapy in advanced B-ALL, and shared their experiences on treating patients with high disease burden. Yue Huang et al. (https://doi.org/10.1002/imed.1032) reported an R/R ALL patient with high leukemia burden and central nervous system involvement, who responded to donor-derived HLA-matched allogeneic CAR-T treatment and achieved quick CR. We thank all the authors for their insightful contributions. This special issue provides an overview of the current knowledge regarding the most recent advances of CAR-T cell therapy, including CAR-T cell function, diverse applications, and complications with coping strategies. We hope that this issue will appeal to researchers as they explore fundamental and clinical aspects of CAR-T cell therapy.

Safety and efficacy of a humanized CD19 chimeric antigen receptor T cells for relapsed/refractory acute lymphoblastic leukemia.

CD19-targeted chimeric antigen receptor T (CAR-T) cells using murine single-chain variable fragment (scFv) has shown substantial clinical efficacy in treating relapsed/refractory acute lymphoblastic leukemia (R/R ALL). However, potential immunogenicity of the murine scFv domain may limit the persistence of CAR-T cells. In this study, we treated 52 consecutive subjects with R/R ALL with humanized CD19-specific CAR-T cells (hCART19s). Forty-six subjects achieved complete remission (CR) (N=43) or CR with incomplete count recovery (CRi) (N=3) within 1month post infusion. During the follow-up with a median time of 20 months, the 1-year cumulative incidence of relapse was 25% (95% confidence interval [CI] 13-46), and 1-year event-free survival was 45% (95% CI 29-60). To the cutoff date, 20 patients presented CD19+ relapse and 2 had CD19- relapse. Among the 22 relapsed patients, 14 had treatment-mediated and treatment-boosted antidrug antibodies (ADA) as detected in a sensitive and specific cell-based assay. ADA positivity was correlated with the disease relapse risk. ADA-positive patients had a significantly lower CAR copy number than ADA-negative patients at the time of recurrence (p < .001). In conclusion, hCART19s therapy is safe and highly active in R/R ALL patients, and the hCART19s treatment could induce the emergence of ADA, which is related to the recurrence of the primary disease.

Single-Cell Analyses Identify Transcriptional Characterizations of Subsets Associated with Efficacy and Toxicity for CAR-T Immunotherapy in B-ALL Patients

BackgroundThe development of mRNA sequencing has contributed greatly to the mechanism exploration in hematologic malignancies disease. With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize every subset of expression programs and functional states in a comprehensive and unbiased manner. Here, we present a systematic evaluation of engineered chimeric antigen receptor T (CAR-T) products and patient bone marrow profiles in terms of primary resistance and severe cytokine release syndrome (CRS) at the single-cell level.MethodsUsing single-cell mRNA sequencing in conjunction with flow cytometry (FCM), we performed characterization of CD19-targeted CAR-T and mononuclear bone marrow cells from 4 on-trial B acute lymphoblastic leukemia (B-ALL) patients (NCT02965092). Bioinformatics analysis was utilized to explore diversity between patients with different grades of response or CRS. Basing on marker genes, CAR-T products were divided into four groups, which were double-positive T (DPT), CD4 positive T (CD4), CD8 positive T (CD8), and double-negative T (DNT) cells. Meanwhile, both the mononuclear bone marrow cells before and after CAR-T infusion were grouped into six clusters, which were B-ALL, stem, progenitor, B, T, and myeloid cells. The expression and enrichment analyses results were calculated by R (version 3.6.3) and then verified in a 22-sample conventional transcription sequencing cohort of the same clinical trial. Patient efficacy was assessed by the national comprehension cancer network guidelines version 2.2020 for acute lymphoblastic leukemia, and CRS was graded by CTCAE 5.0.ResultsBy FCM detection, the variances of CAR-T infusion products between patients with different clinical outcomes were limited, and nor did mononuclear bone marrow cells. The scRNA sequencing results showed that distinct CAR-T and bone marrow cell subsets indicated differentiated expression in proliferation, cytotoxicity, and intercellular signaling pathways. Expression differentiation variances in CAR-T infusion products were minor than in mononuclear bone marrow cells. CD8+ CAR-T products of complete response (CR) patients were still significantly enriched in pathways such as cell killing (p adjust=0.0012), antigen processing and presentation (p adjust=0.0027), and cell cycle (p adjust=0.0231), exhibiting greater immune function when compared with no response patients. Also, DPT CAR-T products of the non-CRS patients were meaningfully enriched in negative regulation of cytokine production pathway (p adjust=0.0127) when compared with CRS ones. In mononuclear bone marrow cells, B-ALL cells before CAR-T treatment of CR patients presented negatively in cell-cycle (p adjust=0.0019), leading to a low malignant cell proliferation level; and stem-progenitor cells after CAR-T treatment of CR patients showed a stronger ability of neutrophil activation (p adjust<0.0001). As with comparisons between CRS and non-CRS, B-ALL cells before infusion manifested a cell cycle arrest profile (p adjust<0.0006) in non-CRS patients, whereas the immune cells at the same time point were enriched in positive regulation of cell cycle process (p adjust=0.0002).ConclusionsThrough single-cell RNA-seq profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the cell cycle, immune phenotype, and metabolic profiles of subsets during CAR-T therapy, providing a mechanistic basis for ameliorating clinical outcomes and individualized management. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Serial surveillance by circulating tumor DNA profiling after chimeric antigen receptor T therapy for the guidance of r/r diffuse large B cell lymphoma precise treatment.

Background: Circulating tumor DNA (ctDNA) released from tumor cells carries the tumor-associated genetic and epigenetic characteristics of cancer patients. Next-generation sequencing (NGS) facilitates the application of ctDNA profiling for identification and monitoring of minimal residual disease (MRD) in cancer, and can serve as the guidance for precise treatment.Methods: In this study, we profiled genomic alterations in the baseline, relapsed, and progressive tumor samples of eight diffuse large B cell lymphoma (DLBCL) patients (NCT03118180) after chimeric antigen receptor T (CAR-T) cell therapy.Results: The median follow-up was 41 months. 4 (50%) patients achieved complete remission (CR), 1 (12.5%) patient achieved partial remission (PR), and the other 3 (37.5%) patients showed no response. 3 of 5 patients who achieved remission relapsed within 4 months after CAR-T therapy, while the rest 2 patients remained CR for more than 3 years. Based on the positron emission tomography-computed tomography (PET-CT) scan, the current gold standard for evaluating response to therapy in lymphoma, the sensitivity and specificity of our ctDNA profiling in detecting tumor-related ctDNA mutations were 94.7% and 83.3%, respectively. The median numbers of baseline plasma ctDNA mutations in patients who remained long-term CR and patients who relapsed or became refractory to CAR-T therapy were 3 and 14.3, respectively. GNA13, SOCS1, TNFAIP3 and XPO1 mutations appeared to be associated with poor prognosis after CAR-T cell therapy. Our results also suggested that lenalidomide might relieve relapsed lymphoma with mutations in NFKBIA 202C>T (p.Q68*) and NFKBIE 433A>T (p.K145*) by targeting NF-Kappa B signaling. In addition, the inhibitor selinexor may be another choice for refractory or relapse (r/r) DLBCL patients after CAR-T cell treatment.Conclusion: Serial ctDNA monitoring is an emerging technology for the surveillance of disease status and prognosis prediction. In this work, we demonstrated the use of serial ctDNA monitoring in r/r DLBCL patients after CD19-targeted CAR-T cell therapy. Our longitudinal NGS profiling revealed the changes of ctDNA mutation in accordance with prognosis, and shed some light on exploring more targeted treatment options together with CAR-T cell therapy.

Efficient Gene Editing of CART Cells with CRISPR-Cas12a for Enhanced Antitumor Efficacy

Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirects T cells to recognize cancer cells expressing a particular surface antigen. CART cell therapy has been astonishingly successful at eradicating certain B cell malignancies, but relapse is common, and efficacy is lacking in many cancers. Gene editing of CART cells is being investigated to enhance efficacy and safety and to develop off-the-shelf products. Currently, genome engineering tools used to modify CART cells include zinc finger nucleases, transposons, TALENs, and CRISPR-Cas9. CRISPR-Cas9 uses a trans-activating (tracrRNA): CRISPR RNA (crRNA) duplex to trigger imprecise DNA repair through targeted double stranded breaks, causing indels and often resulting in loss of protein function. Gene-edited CART cells have entered the clinic to provide an allogeneic cell source (TALEN TCRα knockout), safer treatment (CRISPR-Cas9 GM-CSF knockout), and resistance to exhaustion (CRISPR-Cas9 PD-1 knockout). CRISPR-Cas9 PD-1 knockout (PD-1k/o) CART cells were well-tolerated in a first-in-human clinical trial. However, clinically tested CRISPR-Cas9-edited CART cells showed only modest loss of function (~25%) of PD-1 upon infusion. Additionally, off-target editing has been observed in the clinic and remains a concern. We hypothesized that using next-generation CRISPR-Cas12a systems will result in enhanced editing efficiency and precision. CRISPR-Cas12a has a smaller protein component than CRISPR-Cas9, uses a single crRNA without a tracrRNA for simplified delivery and leaves staggered 5' overhangs. These properties, along with lower intrinsic off-target activity than Cas9, render Cas12a a powerful gene editing tool. First, we compared the knockout efficiency of Cas9 and Cas12a in three therapeutically relevant genetic targets in T cells by delivering ribonucleoprotein complexes containing the crRNA and Cas protein of interest. We showed that Cas12a more effectively knocked out CD3, GM-CSF, and PD-1 expression compared to Cas9 (Figure 1A), demonstrating the potential of Cas12a in further genetically editing T cell therapies. We then used electroporation with Cas9 and Cas12a to generate PD-1k/o in lentivirally transduced CD19-targeted CART (CART19) cells with the aim of making exhaustion-resistant CART19 cells through CRISPR gene editing. CART19 and PD-1k/o CART19 cells were repeatedly stimulated with CD19+ NALM6 target cells for one week, and exhaustion marker expression was measured over time with flow cytometry. The expression of CTLA4, TIM3, and LAG3 were similar between CART19 groups, but PD-1 expression was lower in Cas9 PD-1k/o CART19 cells and almost completely eradicated in Cas12a PD-1k/o CART19 cells compared to wildtype or mock shocked CART19 cells (Figure 1B). We then compared the functionality of wildtype, mock shocked, and Cas9 or Cas12a PD-1k/o CART19 cells in vitro to ensure that neither the electroporation process nor PD-1 knockout impaired CART19 cell antitumor activity. Over a range of effector-to-target ratios and with repeated stimulation with target cells, cytotoxicity was comparable across all CART19 cell groups (Figure 1C). All CART19 cell groups demonstrated robust proliferation in response to both nonspecific and antigen-specific stimulation and over one week of repeated antigen stimulation with NALM6 target cells (Figure 1D). We also confirmed that all CART19 cell groups demonstrated strong degranulation and cytokine production in response to nonspecific and antigen-specific stimulation, regardless of electroporation or PD-1 knockout (Figure 1E). In summary, our data demonstrate that Cas12a can be used as a gene editing tool to efficiently knock out therapeutically relevant genes in CART19 cell therapy. Additionally, Cas12a demonstrated improved knockout efficiency over Cas9 in three different genomic targets. PD-1 knockout via Cas9 or Cas12a reduced PD-1 expression on the CART19 cell surface, and PD-1 expression was almost completely ablated with Cas12a gene editing. Electroporation and PD-1 knockout did not impact the effector functions of the CART19 cells, including cytotoxicity, degranulation, cytokine secretion, or proliferation. In vivo studies assessing the antitumor efficacy and CART19 cell persistence are ongoing. Overall, Cas12a is a promising, efficient method of gene knockout to enhance the safety and efficacy of CART cells. Disclosures Sakemura: Humanigen: Patents &amp; Royalties. Cox:Humanigen: Patents &amp; Royalties. Kenderian:MorphoSys: Research Funding; Sunesis: Research Funding; Tolero: Research Funding; BMS: Research Funding; Juno: Research Funding; Gilead: Research Funding; Kite: Research Funding; Novartis: Patents &amp; Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents &amp; Royalties, Research Funding; Mettaforge: Patents &amp; Royalties; Lentigen: Research Funding.

Secondary donor-derived humanized CD19-modified CAR-T cells induce remission in relapsed/refractory mixed phenotype acute leukemia after allogeneic hematopoietic stem cell transplantation: a case report

BackgroundMixed phenotype acute leukemia (MPAL) is a rare leukemia and is regarded as a high-risk entity with a poor prognosis. Induction therapy of an acute lymphoblastic leukemia type or hybrid regimen and hematopoietic stem cell transplantation has been recommended for MPAL. However, the optimal therapies for relapsed or refractory MPAL remain unclear, especially for relapse after stem cell transplantation. Donor-derived chimeric antigen receptor T (CAR-T) cell therapy may be a promising therapeutic option for patients with MPAL who express target antigens and have relapsed after stem cell transplantation. However, recurrence remains a challenge, and reinfusion of CAR-T cells is not always effective. An infusion of secondary donor-derived humanized CD19-modified CAR-T cells may be effective in inducing remission.Case presentationWe report a case of MPAL with CD19 expression. The patient was treated with acute lymphoblastic leukemia-like induction and consolidation therapies but remained positive for SET-NUP214 fusion gene transcript. He subsequently underwent a haploidentical stem cell transplantation but relapsed within 6 months. He then underwent donor-derived CD19-targeted CAR-T cell therapy and achieved a sustained, complete molecular remission. Unfortunately, he developed a CD19-positive relapse after 2 years. Donor-derived humanized CD19-directed CAR-T cells induced a second complete molecular remission without severe cytokine release syndrome or acute graft-versus-host disease.ConclusionThis case demonstrated the efficacy and safety of humanized donor-derived CD19-modified CAR-T cell infusion for treating the recurrence of MPAL previously exposed to murine-derived CD19-directed CAR-T cells.

Humanized CD19-Specific Chimeric Antigen Receptor T Cells for Acute Lymphoblastic Leukemia

Introduction: Adoptive infusion of CD19-targeted chimeric antigen receptor T (CAR-T) cells has showed promising treatment effects for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). While immune response induced by murine single-chain variable fragment (scFv) of the CAR leads to its premature elimination, and thus increases the risk of leukemia relapse. Humanized scFv is expected to reduce the immunogenicity of CAR, thereby promoting the survival time as well as improving the therapeutic efficacy of CAR-T treatment. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19 CAR-T cells (hCART19s). Methods: A pilot phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (hCART19s) was performed with recruitment of children and adults with R/R B-ALL with or without prior exposure to a murine CD19 CAR-T cell product. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of humanized anti-CD19 scFv, the human CD8 transmembrane, CD8 hinge, 4-1BB costimulatory domain, CD3ζ intracellular regions and T2A-EGFRt sequence. After lymphodepletion chemotherapy with cyclophosphamide (750 mg/m2, day -5) and fludarabine (30 mg/m2/day, days -5 to -2), patients received a single dose of autologous hCART19s infusion at a dose of 1×106 cells/kg (body weight) on day 0. The post-infusion responses, toxicities, expansion and persistence of hCART19s in patients were observed and monitored. Results: 51 R/R B-ALL patients aged 3-69 yr were treated with hCART19s. 5 patients was diagnosed as Ph+ B-ALL. 2 patients had received prior allogeneic stem cell transplant (SCT). A total of 8 patients had central nervous system leukemia (CNSL), and 1 had testicular leukemia (TL). Among 46 patients without previous CAR-T therapy, 38 (82.6%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, while 2 of 5 patients, who relapsed after murine CAR-T infusion, achieved CR after hCART19s infusion. The rates of event-free survival and overall survival were 63% (95% confidence interval [CI], 46 to 75) and 79% (95% CI, 64 to 88), respectively, at 6 months and 44% (95% CI, 27 to 59) and 67% (95% CI, 51 to 79) at 12 months. Among the 40 patients with CR or CRi, 17 had a relapse before receiving additional anticancer therapy. 12 patients underwent allogeneic hematopoietic stem-cell transplantation (HSCT) while in remission, 10 were alive without relapse and 2 had a relapse after HSCT. During treatment, 37 (72.5%) patients developed grade 1-2 cytokine release syndrome (CRS), and 11 (21.6%) patients developed grade 3-5 CRS. Neurologic events occurred in 4 (7.8%) patients within 8 weeks after infusion. 2 (3.9%) patients had grade 3 neurologic events; no grade 4 events or cerebral edema were reported. Conclusions: This study demonstrated that hCART19s have high therapeutic efficacy and safety in children and adults with R/R B-ALL. More importantly, hCART19s was confirmed to exert anti-leukemic activities in patients who relapsed after murine CAR-T infusion. HSCT is a potential approach to reduce leukemia relapse in patients who achieved CR after CAR-T therapy. Disclosures No relevant conflicts of interest to declare.

1185PD - Parallel comparison of 4-1BB or CD28 co-stimulated CD19-targeted chimeric antigen receptor-T cells for B-cell non-Hodgkin lymphoma

BackgroundCD19-targeted chimeric antigen receptor-T (CAR-T) cells with CD28 or 4-1BB (28z CAR-T and BBz CAR-T) have shown great promise to treat relapsed or refractory (r/r) B cell acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin lymphoma (B-NHL). However, studies comparing the clinical outcomes of 28z CAR-T versus BBz CAR-T are lacking. Analysis of our previous double-arm clinical study of 28z CAR-T and BBz CAR-T in treatment of r/r B-ALL (18 cases for each arm) revealed that BBz CAR-T showed shorter initial response time, higher tumor-killing capacity, higher overall survival rate, longer persistence, and less severe cytokine release syndrome and neurotoxicity than 28z CAR-T. This study investigated the efficacy and toxicity of both CAR-T types in B-NHL patients. MethodsBBz CAR-T and 28z CAR-T were prepared under identical manufacturing processes. Patients with r/r B-NHL were pre-treated with fludarabine and cyclophosphamide, and infused with 28z or BBz CAR-T. Tumor burden, CAR-T persistence and adverse events were monitored. The CAR-T that induced dose-limiting toxicity in less than one patient was further evaluated based on dose escalation. ResultsSix patients were treated at the dose of 0.75-5×105/kg. Both types of CAR-T cells showed similar antitumor efficacies, with a complete response (CR) rate of 67% within three months. Cytokines including interleukin (IL)-6 and IL-10 were increased after CAR-T infusion. The peak levels of IL-6 and IL-10 were much higher in the 28z CAR-T cohort. Severe cytokine release syndrome (CRS), CAR-T cell related encephalopathy syndrome (CRES) (≥ grade 3), and one death occurred in the 28z CAR-T cohort, resulting in the termination of recruitment of this cohort. Six more patients received BBz CAR-T cells at a dose of 1×106/kg with one-month CR rate of 83% and only grade 1/2 adverse events. ConclusionsBBz CAR-T showed similar antitumor activity, but a more favorable safety profile, compared with 28z CAR-T. This study proved the competence of BBz CAR-T in B-NHL therapy, at least under our manufacturing process. Legal entity responsible for the studyPeking University Cancer Hospital & Institute. FundingImmunochina Pharmaceuticals Co., Ltd. DisclosureAll authors have declared no conflicts of interest.

Parallel Comparison of 4-1BB or CD28 Co-stimulated CD19-Targeted CAR-T Cells for B Cell Non-Hodgkin’s Lymphoma

CD19-targeted chimeric antigen receptor-T (CAR-T) cells with CD28 or 4-1BB (28z CAR-T and BBz CAR-T) have shown great promise to treat relapsed or refractory (r/r) B cell non-Hodgkin’s lymphoma (B-NHL). However, comparison of their clinical outcomes has never been reported. This study investigated their efficacy and adverse events in B-NHL therapy. Six patients with r/r B-NHL were initially enrolled and infused with 28z or BBz CAR-T cells at a dose of 0.75–5 × 105/kg. These CAR-T cells showed similar antitumor efficacies, with a complete response (CR) rate of 67% within 3 months. BBz CAR-T was well tolerated. However, severe cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome occurred in the 28z CAR-T cohort, resulting in the termination of further evaluation of 28z CAR-T. Three more patients were enrolled to investigate BBz CAR-T cells in-depth at an escalated dose (1 × 106/kg). All cases achieved CR within 3 months, and only grade 1/2 adverse events occurred. This study suggests that 4-1BB is more beneficial for the clinical performance of CAR-T cells than CD28 in CD19-targeted B-NHL therapy, at least under our manufacturing process.