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Transition analysis was performed on production-scale chromatography data in order to monitor column performance. Analysis of over 300 transitions from several different chromatography operations demonstrated the utility of the techniques presented. Several of the transitions analyzed occurred on columns with known integrity breaches. The techniques proved sensitive for detection of these breaches. Seven transition calculations are presented, which were combined to produce a single overall integrity value for each column. In addition, principal components analysis (PCA) was used to detect shifts in the transition pattern, including those attributed to integrity breaches. Besides detection of integrity breaches, transition analysis proved useful in monitoring column efficiency over multiple column uses.

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Introduction JWCAR029 is a CD19-directed 4-1BB chimeric antigen receptor (CAR) T cell product with a 4-1BB costimulatory domain, of which CD4 and CD8 CAR T cells are produced together and transfused in non-fixed ratio. We conducted a single arm, open-label, dose escalation Phase I trial of JWCAR029 in relapsed and refractory B-cell non-Hodgkin lymphoma (NCT03344367 and NCT03355859). Methods Eligible pts had confirmed B-cell NHL with R/R disease after ≥2 prior lines of therapy. All subjects received lymphodepleting chemotherapy prior to receiving JWCAR029. After lymphodepleting chemotherapy, JWCAR029 was administrated as a single infusion in escalating dose levels, from 25×106 CAR T cells (dose level 1, DL1), 50×106 CAR T cells (dose level 2, DL2), 100×106 CAR T cells (dose level 3, DL3) to 150×106 CAR T cells (dose level 4, DL4) according to mTPI-2 algorithm. Circulating blood counts, serum biochemistry, coagulation status, and cytokines were followed up after infusion. Cytokines were assessed on a Luminex platform. Tumor evaluation was evaluated per the Lugano criteria by PET-CT (Cheson, 2014) and safety and disease status was followed at approximately 1, 3, 6, 9, 12, 18 and 24 months after receiving JWCAR029. PK was measured by flow cytometry and real-time quantitative polymerase chain reaction system. All the adverse events were recorded for 24 months after infusion. The study was approved by Beijing Cancer Hospital and Shanghai Rui Jin Hospital Review Board with informed consent obtained in accordance with the Declaration of Helsinki. Results As of July 5, 2019, 44 patients were screened and 32 patients were enrolled and received treatment in two study sites in China. Twenty nine patients are evaluable and have been followed for at least 6 months: 20 diffuse large B cell lymphoma (DLBCL) and 9 follicular lymphoma, mantle cell lymphoma and extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue lymphoma. Median age was 52 years (range 29 to 68 years). The demographic characteristics of the patients are shown in Table 1. All patients received immunochemotherapy as induction and a median of four lines of salvage treatment (range 2 to 7). Eleven (34%) patients received bridging chemotherapy after T cell collection due to rapid tumor progression, followed by re-evaluation before CAR T cell infusion. Lymphodepletion consisted of fludarabine 25mg/m2/d and cyclophosphamide 250mg/m2/d on Day -4 to Day -2, followed by CAR T cell infusion on Day 0. Median time to peak CAR+ T cell expansion was 11 (8-15) days. No DLTs were reported. There were no treatment-related deaths. Seventeen patients (53.1%) reported cytokine release syndrome (CRS) with 16 grade 1 or 2 (50%) and 1 (3.1%) grade 3. No grade 4 or 5 CRS was observed. Main symptoms were fever (>39.0 degrees), fatigue, and muscle soreness. The rate of CRS was similar across dose level groups. Grade 1 and 2 neurotoxicity was observed in 5 patients (15.6%). No grade ≥3 neurotoxicity was reported. Most common adverse events (frequency >20%) included leukopenia (Gr 3-4: 21.9%, Gr 1-2: 43.8%), lymphopenia (Gr 1-2: 21.9%, Gr 3-4: 21.9%), neutropenia (Gr 1-2: 37.5%, Gr 3-4: 28.2%), thrombocytopenia (Gr 1-2: 21.9%, Gr 3-4: 3.1%), pyrexia (Gr 1-2: 21.9%) and immunoglobulins decreased (Gr 1: 28.1%). Among all 29 efficacy-evaluable patients (6 of DL1, 6 of DL2, 8 of DL3 and 9 of DL4), the best ORR was 89.7%; 85% for DLBCL patients. ORR/CRR of all evaluable patients at 1, 3 and 6 months were 86.2%/65.5%, 69%/62.1% and 58.6%/55.2%, respectively, and for the 20 DLBCL patients the ORR/CRR was 80%/60%, 55%/55%, and 45%/45%, respectively (Table 2). Conclusion Although longer follow-up is needed, the data from 29 evaluable patients in this Phase I trial have demonstrated high response rates and a favorable safety profile of JWCAR029 in relapsed and refractory B-cell non-Hodgkin lymphoma. A Ph II trial that further assess safety and efficacy of JWCAR029 in DLBCL and FL patients has been initiated and is open for enrollment. Disclosures Wang: JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Hao:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Yang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Lam:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Li:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zheng:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership.

Background: JWCAR029 is the first IND approved CD19-targeted CAR T cell product by China National Drug Administration (CNDA) containing 4-1BB as the co-stimulatory factor with highly reproducible process and quality control that allows flat dose of CAR T cell infusion. To date, a total of 22 lots have been manufactured and 18 subjects have been infused in the ongoing multicenter, Phase 1 trial (NCT03344367 and NCT03355859) evaluating the safety and efficacy of JWCAR029 in adult relapsed or refractory B-cell Non-Hodgkin lymphoma patients. The process and quality control strategy for JWCAR029 contributes to the low variability in drug product quality attributes. Methods: Manufacturing of JWCAR029 begins with patient derived autologous T cells obtain via apheresis. JWCAR029 drug products were analyzed for viability, potency, subtype of T cells, copy numbers of lentiviral vector, and cell health related attributes using cellometer related bioassays, flow cytometry, and real-time quantitative polymerase chain reaction system (qPCR), respectively. Results: Process and quality of JWCAR029 started with an automated wash and T cell purification that results in pure CD3+ populations (median 99.56%, Inter Quartile Range [IQR] 99.22-99.86%). CD3+ T cells were transduced with lentiviral vector expressing a CD19-directed CAR with a 4-1 BB/CD3ζ endodomain. CAR+ T cells were cultured to a target cell dose and then formulated / cryopreserved for infusion. To reduce between-lot variance, the cryopreserved drug product (CDP) was packaged at fixed volume with a tight range of viable cell concentrations (CD3+: median 40.25 × 10^6 cells/mL, IQR 31.10-69.13 × 10^6 cells/mL, N=22) and CD3+CAR+ cell concentrations (median 27.25 × 10^6 cells/mL, IQR 23.57-33.10 × 10^6 cells/mL, N=22). JWCAR029 does not use a fixed ratio of CD4+CAR+ cells/CD8+CAR+ cells in the final CDP (median 1.18, IQR 0.70-1.95, N=22). In the ongoing, multicenter, single arm, open-label and dose escalation Phase 1 trial, JWCAR029 was administered as a flat dose at dose level 1 (DL1) of 2.5 × 10^7 CAR+ T cells (6 subjects), at dose level 2 (DL2) of 5.0 × 10^7 CAR+ T cells (9 subjects), or dose level 3 (DL3) of 1.0 × 10^8 CAR+ T cells (3 subjects). After infusion, stable expansion of CD4+ and CD8+ CAR+ T cells were observed and peak value was appeared at day 11 to day 15 after administration. Low occurrence rate and manageable cytokine release syndrome (CRS) and neurotoxicity (NT) with high complete response (CR) rate were observed with emerging dose: response relationship. Detailed PK, clinical safety, and efficacy data of JWCAR029 will be presented separately. Conclusion: In order to employ standardized and high quality cell therapy methods in a Chinese multi-center trial, JWCAR029 was developed to provide a CD19-directed 4-1BB CAR T cell product with highly controlled manufacturing and quality processes enables administration in adult relapsed or refractory B-cell Non-Hodgkin lymphoma subjects. These control strategies in manufacturing and quality processes facilitated to the low rates of CRS and NT. Disclosures Hao: JW Therapeutics: Employment, Equity Ownership. Cheng:JW Therapeutics: Employment, Equity Ownership. Gao:JW Therapeutics: Employment, Equity Ownership. Liu:JW Therapeutics: Employment, Equity Ownership. Lam:JW Therapeutics: Consultancy. Yao:JW Therapeutics: Employment, Equity Ownership; WuXi AppTec: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background: JWCAR029 is a CD19-directed 4-1 BB CAR T cell product, of which CD4 and CD8 CAR T cells are produced together and transfused in non-fixed ratio. A phase I, single-arm, open label study was conducted to evaluate the safety and efficacy of JWCAR029 in patients (pts) with relapsed or refractory B-NHL. Previously, preliminary data in six pts (Yan et al, Blood 2018 132:4187) showed high response rates and favorable safety profiles of JWCAR029. Herein, we presented the data of the Phase I trial of JWCAR029 (NCT03344367 and NCT03355859) in 29 pts with pharmacokinetics (PK), pharmacodynamics (PD), and anti-therapeutic antibody (ATA) evaluations. Methods: Eligible pts received lymphodepletion, with 25mg/m2 flu and 250mg/m2 cy, followed by a single dose of JWCAR029 at one of four dose levels (DL1, 25×106 cells; DL2, 50×106 cells; DL3, 100×106 cells; DL4, 150×106 cells). Blood samples were collected and analyzed for PK, PD, and ATA at a central lab per protocol defined time points. The existence and duration of CAR T cells (PK) were measured by validated flow cytometry and qPCR assays. CD4 and CD8 subpopulation of CAR+ T cells were detected by cetuximab targeting EGFRt as a marker co-expressed with CAR transgene in fresh peripheral blood. In parallel, batched frozen blood samples collected from each pt were detected for integrated CAR transgene by qPCR at the same protocol defined time points. Plasma ATA against murine CD19 scFv (FMC63) was measured with a validated electrochemiluminescent (ECL) assay. Results: As of July 5, 2019, blood samples from 29 pts who received JWCAR029 treatment with a minimum follow-up of 6 M (median, 6 M) were evaluable in the analysis. From DL1 to DL4, median Cmax, Tmax and AUC0-28 for JWCAR029 transgene detected by qPCR did not differ among dose levels (Table 1). CD4/CD8 ratio (range, 0.23-5.50) at cryopreserved drug product of JWCAR029 was not associated with best response of CR/PR at 6 M. Greater in vivo expansion was detected by both qPCR and flow cytometry in pts with best response of CR/PR than those with SD/PD at 6 M (Table 1). Higher concentration of CD8+CAR+T cells than CD4+CAR+T cells were detected in PB by flow cytometry for all treated pts (Cmax median= 30.6 vs 5.64). At 3 M, 81.5% (22/27) and 48.2% (13/27) pts had detectable CD8+ and CD4+ CAR+ T cells, respectively. Of those pts with detectable CAR+ T cells at 3 M, 70% (14/20) and 35% (7/20) had detectable CD8+ and CD4+ CAR+ cells at 6 M, respectively. Significantly higher Cmax and AUC0-28 were observed in patients with ≥ Grade 1 CRS (Cmax median= 85004 vs 16328, P<0.01; AUC median=536543 vs 141731, P<0.01). And relatively higher Cmax and AUC0-28 were found in patients with NT (Cmax median= 116112 vs 40391; AUC median=711306 vs 301035). 27.5% of pts (8/29) had detectable ATA in plasma, of which 25% (2/8) pts had pre-existing antibodies before CAR T cell infusion. 6 pts developed antibodies without pre-existing antibodies and were considered treatment-induced. The median time for treatment-induced antibody development was 6 M (range, 3-12). Increasing level of antibodies were detected at median time of 6 M (range, 6-6) for pts who had pre-existing antibodies and were considered treatment-boosted. No significant differences in PK profiles of JWCAR029 transgene levels were found between ATA negative group and treatment-induced ATA positive group (Cmax median= 44497 vs 50032; AUC median= 420635 vs 313654; Fig.1). Although the sample size of the treatment-boosted subgroup was small, there was a trend for lower expansion of CAR T cells in pts who had pre-existing ATA than pts who did not develop ATA (Cmax median= 3051 vs 44497; AUC median = 16437 vs 420635;Fig.1). In ATA positive subgroup, 100% (8/8) pts responded with CR rate of 75% (6/8). 6 M-response rate was 65.5% (5/8) for ATA positive subgroup and 57.1% (12/21) for ATA negative subgroup. Incidence of ≥ Grade 1 CRS or NT was indistinguishable between ATA positive and negative subgroups, 50% (4/8) in ATA positive vs 57.1% (12/21) negative. Conclusion: Preliminary data from JWCAR029 Phase I study has demonstrated that pts with best response of CR/PR at 6 months had a relatively higher CAR T cell expansion. Current data suggested that the prevalence of pre-existing ATA may compromise CAR+T PK profile. No association of the presence or boost of ATA with efficacy or safety of JWCAR029 was observed. Further exploration of ATA and clinical outcomes will be studied in the ongoing pivotal Phase 2 study in 70 pts with B-NHL. Disclosures Hao: JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Wang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zhou:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Yang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Wang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Lam:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Li:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zheng:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership.