![Figure 1. Mechanism of rituximab-mediated depletion of CART123-CD20 and alemtuzumab depletion of CART123. (A) Schema of CART123-CD20 construct. (B) Surface expression of CD123-CAR-PE and CD20-PE-Cy7 on CART123-CD20 T cells by FC. (C) In vitro incubation of CD123+ MOLM14 cells with CART123 or CART123-CD20 have similar elevated inflammatory cytokine production as measured by FC, suggesting similar potency of both CD123-redirected CAR T-cell products. Minimal cytokine production occurs with coincubation of CAR T cells and CD123-Jurkat T cells. (D) Similar cytotoxicity is observed with in vitro coculture of MOLM14 cells with CART123 or CART123-CD20 at all effector:target (E:T) ratios, as measured by BLI. (E) Similar cytotoxicity is observed with coculture of MOLM14 cells with RNA-CART123 or CART123-CD20, as measured by BLI. (F) Rituximab depletes CART123-CD20 cells by CDC and by direct cellular cytotoxicity. Coculture of CART123-CD20 with increasing concentrations of rituximab in the absence or presence of 15% complement resulted in CART123-CD20 depletion only in the presence of complement and had direct cytotoxicity at highest concentration. Residual live CAR T cells were quantified by FC after 12 hours of coculture, and percent T-cell depletion was calculated. (G) Rituximab depletes CART123-CD20 by CDC within 4 to 12 hours of coculture. CART123-CD20 cells were incubated with rituximab at indicated concentrations in the presence of complement. Residual live CAR T cells were measured by FC at indicated time points for calculation of T-cell killing. (H) No evidence for ADCC was observed. CART123-CD20 cells (labeled with carboxyfluorescein diacetate succinimidyl ester [CFSE]) were cocultured with Dil-labeled macrophages and rituximab at the indicated concentrations. Phagocytosis after 2 hours (defined as CFSE+Dil+ singlet cells) was quantified by FC. Percent phagocytosis did not differ in the absence or presence of rituximab. (I) Similar to data in panel E, coculture of CART123 cells with alemtuzumab in the absence or presence of complement demonstrates CDC-mediated T-cell killing.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/129/17/10.1182_blood-2016-08-736041/4/blood736041f1.jpeg?Expires=1722542468&Signature=MvyrYfqfxIRgOGR6i~SrX-ETO7Zk4xDvGoZaendWfY8eOojGP9dahNChKLz5qhfEK3iULs9u1dsl4tEKFt6oI2Yowksv~Xmp6BjIxXM94Fc6nn-P8baQAf4cjjwCDQUW3U8e2rGh2VJrb~wJ20cc1VC0zrzPkeMJJJo2e9v1l4GzHjqfAPhVY1S6yhpfWwn~-j7jyF6HqC8OM9JQzk3w3pun3Lx34q9zW1j6Z9DlNbaAaWZqT1YAscsKFKobJb~yuOFELnUI8VkaW4NjhwG86~UWjIwQHdpNYJUr0W6p4P7Hwr4tKn1CfNtrTQjCf3imM1NiB8a0Lex1fYAR0oy32Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mechanism of rituximab-mediated depletion of CART123-CD20 and alemtuzumab depletion of CART123. (A) Schema of CART123-CD20 construct. (B) Surface expression of CD123-CAR-PE and CD20-PE-Cy7 on CART123-CD20 T cells by FC. (C) In vitro incubation of CD123+ MOLM14 cells with CART123 or CART123-CD20 have similar elevated inflammatory cytokine production as measured by FC, suggesting similar potency of both CD123-redirected CAR T-cell products. Minimal cytokine production occurs with coincubation of CAR T cells and CD123-Jurkat T cells. (D) Similar cytotoxicity is observed with in vitro coculture of MOLM14 cells with CART123 or CART123-CD20 at all effector:target (E:T) ratios, as measured by BLI. (E) Similar cytotoxicity is observed with coculture of MOLM14 cells with RNA-CART123 or CART123-CD20, as measured by BLI. (F) Rituximab depletes CART123-CD20 cells by CDC and by direct cellular cytotoxicity. Coculture of CART123-CD20 with increasing concentrations of rituximab in the absence or presence of 15% complement resulted in CART123-CD20 depletion only in the presence of complement and had direct cytotoxicity at highest concentration. Residual live CAR T cells were quantified by FC after 12 hours of coculture, and percent T-cell depletion was calculated. (G) Rituximab depletes CART123-CD20 by CDC within 4 to 12 hours of coculture. CART123-CD20 cells were incubated with rituximab at indicated concentrations in the presence of complement. Residual live CAR T cells were measured by FC at indicated time points for calculation of T-cell killing. (H) No evidence for ADCC was observed. CART123-CD20 cells (labeled with carboxyfluorescein diacetate succinimidyl ester [CFSE]) were cocultured with Dil-labeled macrophages and rituximab at the indicated concentrations. Phagocytosis after 2 hours (defined as CFSE+Dil+ singlet cells) was quantified by FC. Percent phagocytosis did not differ in the absence or presence of rituximab. (I) Similar to data in panel E, coculture of CART123 cells with alemtuzumab in the absence or presence of complement demonstrates CDC-mediated T-cell killing.
