Anti-CD138 (Indatuximab) h(4-1BB-CD3ζ) CAR, pSBCAR1 (CAR-SB-LX0521)

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.1 CD138.CAR expression evaluated by flow cytometry in control T cells (Ctr-Ts) and in T cells transduced with the CD138 CAR constructs. (Indatuximab/BT062-CAR)

CD138-CAR1, CAR2, CAR3, and CAR4.CAR constructs, with an identical scFv Indatuximab/BT062 specific for CD138, but different hinges (IgG1.CH2.CH3, IgG1, CD8α),
transmembrane domains (CD28, CD8α), and costimulatory endodomains (CD28, 4-1BB); these are referred to as CD138-CAR1, CAR2, CAR3, and CAR4.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.2 Characterization of CD138.CAR-Ts.

Figure shows the frequency of CD8 and and central memory subsets (CD45RA+CCR7+) gated on CD3+ cells for Ctr-Ts and CD138.CAR-Ts generated from healthy donors (n = 3-6).

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig 3: CD138.CAR-Ts specifically lyse CD138+ target cells.

Figure shows the results of standard 51Cr release assays for CD138+cells (OPM-2 cells left panel) or CD138- tumor cells (Raji, right panel), at the indicted T cell (effector) to tumor cell (E:T) ratio. Symbols represent the mean ± SEM of CD138.CAR-Ts generated from 5 healthy donors (p < 0.0001, one-way ANOVA).

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.4 CD138.CAR-Ts specifically lyse CD138+ target cells.

Figure shows results of standard 51Cr release assays against other three CD138+ MM cell lines (U266, RPMI, MM.1S cells), at the 20:1 E:T ratio for Ctr-Ts or CD138.CAR-Ts

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.5 CD138.CAR-Ts specifically lyse CD138+ target cells.

Figure shows the percentage of residual tumor cells for CD138+ cells (OPM-2, left panel) or CD138- cells (Raji, right panel) co-cultured at 1:1 ratio with Ctr-Ts, or CD138.CAR-Ts.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.6 CD138.CAR-Ts specifically lyse CD138+ target cells.

Figure shows the percentage of residual tumor cells using other CD138+ MM cell lines (U266, RPMI, MM.1S cells), in co-cultures with Ctr-Ts or CD138.CAR-Ts at 1:1 ratio.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.7 IFNγ release quantification of CD138.CAR-Ts co-cluture with CD138+ target cells.

Figure shows the quantification of IFNγ released in the supernatant by Ctr-Ts or CD138.CAR-Ts co-cultured for 24 hrs with CD138+ cells (OPM-2, left panel) or CD138- cells (Raji, right panel) at a 1:1 ratio, and quantified by CBA assays.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.8 IFNγ release quantification of CD138.CAR-Ts co-cluture with CD138+ target cells.

shows the quantification of IFNγ released in the supernatant for three additional CD138+cell lines (U266, RPMI, MM.1S cells) by control T cells or by CD138.CAR-Ts (1-3 donors for each CAR).

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.9 CD138.CAR-T targeting of primary MM tumors.

Figure shows the percentage of residual MM tumor cells in experiments in which primary MM cells were co-cultured with autologous (MM patients derived) or allogeneic (healthy donor derived) Ctr-Ts and CD138.CAR-Ts.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.10 IFNγ and IL-2 release quantification of CD138.CAR-Ts co-cluture with co-cultured with autologous malignant plasma cells.

Figure shows the quantification of IFNγ, and IL-2 released in the supernatant by Ctr-Ts or CD138.CAR-Ts generated from MM patients co-cultured for 24 hrs with autologous malignant plasma cells, and quantified by CBA assays.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.11 CD138.CAR-T targeting of primary MM tumors.

Figure shows the flow plots for two representative donors of Ctr-Ts or CD138.CAR-Ts co-cultured with CD138+ RPMI MM cell line. SP cells, non-SP cells and T cells at the end of the 3 days co-culture are shown.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.12 CD138.CAR-T targeting of primary MM tumors.

Figure shows the flow plots for two representative donors of Ctr-Ts or CD138.CAR-Ts co-cultured with SP cells sorted from the RPMI MM cell line.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig.13 IFNγ and IL-2 release quantification of CD138.CAR-Ts co-cluture with co-cultured with sorted SP cells.

Figure shows the quantification of IFNγ and IL-2 released in the supernatant of Ctr-Ts or CD138. CAR-Ts from 2 representative donors co-cultured with sorted SP cells, and quantified by CBA-assay.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig. 14 In vivo antitumor activity of CD138.CAR-Ts.

Figure shows the bioluminescence signals(BLI) of Ctr-Ts and CD138.CAR-Ts. Each line shows the BLI for each mouse and the red dashed line the average of the BLI intensity for each group.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.

CAR Construction : Indatuximab(bt062) scfv-CD28-CD3ζ; Indatuximab(bt062) scfv-41BB-CD3ζ;

Fig. 14 In vivo antitumor activity of CD138.CAR-Ts.

Figure(B) shows the fold expansion of BLI as compared to day 14 (day of T cell injection) summarized as average (± SEM). Figure(C) shows the Kaplan-Meier survival curve.

Sun, C., Mahendravada, A., Ballard, B., Kale, B., Ramos, C., West, J., ... & Savoldo, B. (2019). Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma. Oncotarget, 10(24), 2369.