Additional file 2 of Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study
Creators: name: Zhang, Yunlin
name: Patel, Ruchi P.
name: Kim, Ki Hyun
name: Cho, Hyungwoo
name: Jo, Jae-Cheol
name: Jeong, Seong Hyun
name: Oh, Sung Yong
name: Choi, Yoon Seok
name: Kim, Sung Hyun
name: Lee, Ji Hyun
name: Angelos, Mathew
name: Guruprasad, Puneeth
name: Cohen, Ivan
name: Ugwuanyi, Ositadimma
name: Lee, Yong Gu
name: Pajarillo, Raymone
name: Cho, Jong Hyun
name: Carturan, Alberto
name: Paruzzo, Luca
name: Ghilardi, Guido
name: Wang, Michael
name: Kim, Soohwan
name: Kim, Sung-Min
name: Lee, Hyun-Jong
name: Park, Ji-Ho
name: Cui, Leiguang
name: Lee, Tae Bum
name: Hwang, In-Sik
name: Lee, Young-Ha
name: Lee, Yong-Jun
name: Porazzi, Patrizia
name: Liu, Dongfang
name: Lee, Yoon
name: Kim, Jong-Hoon
name: Lee, Jong-Seo
name: Yoon, Dok Hyun
name: Chung, Junho
name: Ruella, Marco
Publisher: name: figshare
Year: 2024
Subjects: subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Science Policy
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 2: Figure S1. h1218 antibody specificity characterization. A. Humanization of the chicken 1218 scFv by grafting the complementarity-determining regions (CDR) to human germline genes. Key framework region (FR) residues that were kept in the h1218 scFv were marked with *. B. Human HEK293 cell-based h1218 antibody binding (Retrogenix assay). Tested array included 2 control receptors and 16 potential hits derived from a previous binding screening using 5484 full-length human plasma membrane proteins and secreted proteins. h1218 showed specific binding to CD19 and to SUSD5, TMEM108 and GUCA2A on fixed (left) or live (right) cells. C. Confirmation of limited h1218 antibody binding ability to HEK293T WT cells or those overexpressed with TMEM-GFP or SUSD5-GFP by flow cytometry. D. Quantification of IFN-γ release from UTD or CART19 cells upon co-culture with WT HEK293T or those overexpressed with CD19, TMEM108, or SUSD5. E. Quantification of absorbance when various amounts of anti GUCA2A antibody, h1218 antibody or FMC63 antibody was added to GUCA2A secreted protein. All curves are represented as mean ± SEM. One-way ANOVA was performed with Tukey correction for multiple comparisons; **** p &lt; 0.0001, *** p &lt; 0.001, ** p &lt; 0.01, * p &lt; 0.05. Figure S2. FMC63- and h1218-CART19 cells demonstrated similar cytotoxic effects against four lymphoid cell lines at short term. A. Cytotoxicity on Raji, Pfeiffer, Toledo and Nalm6 after co-culture with UTD, FMC63-CART19 or h1218 CART19. B. Real-time kinetics analysis of Raji cell survival alone and in the presence of CART19-mediated cytotoxic activity over 48 hours (n=2 donors). Curves with error bars are represented as mean ± SEM. Figure S3. h1218-CART19 exhibited dose-dependent anti-tumor efficacy in xenograft models. A. 7 days after luciferase+ Raji cells intravenous injection (5x105 cells), vehicle, UTD and 4 doses (0.33x106, 1x106 and 3x106) of h1218-CART19 cells were administered. The tumor burden in mice bearing Raji was monitored over time. B. Tumor progression in low- and high-tumor-burden xenograft models. C. Replicate in vivo stress test experiment of FMC63- and h1218-CART19 cells against B-ALL Nalm6. (Left) Tumor burden in mice treated with UTD or CART19. (Right) Overall survival of the corresponding groups. Figure S4. AT101 phase I clinical trial design and patients. A. Schema of AT101 phase I clinical trial design. B. CONSORT diagram with patient screening, enrollment and treatment information. Figure S5. AT101 patient cell blood counts and ferritin levels. A. Patient-specific B cell count before and post AT101 infusion. B cell aplasia is defined as below 50 cells/μl. B. Patient-specific hemoglobin level over time. C. Patient-specific platelet level over time. D. Patient-specific ferritin level. Figure S6. AT101 patient response, progression-free survival, and overall survival. A. AT101 patient response 1 month (left) and 3 months (right) after infusion. B. Progression-free survival of patients treated with AT101 grouped based on response. C. Overall survival of patients treated with AT101 grouped based on response. Figure S7. AT101 patient serum cytokine levels. A. Level of perforin or sFas ligand quantified over time in individual patients. B. Level of MCP-4 or IP-10 quantified in individual patients. C. Level of IL-8 quantified in individual patients. D. Level of serum amyloid A quantified in individual patients.