Abstract 72: Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin
Authors: Mazor Ronit , King Emily , Kishimoto Takashi Kei , Pastan Ira 
Published: July 2017

Abstract
Recombinant Immunotoxins (RITs) are genetically engineered proteins designed for cancer therapy. LMB-100 is a second generation RIT that is composed of a humanized Fab targeting mesothelin and a de-immunized fragment of Pseudomonas Exotoxin A. Due to the bacterial origin of the toxin, LMB-100 is immunogenic, although less immunogenic than first generation immunotoxins. Almost all patients treated with LMB-100 made anti-drug antibodies (ADAs) after two or more cycles of treatment that neutralized the RIT and greatly lowered efficacy. Kishimoto et al. demonstrated that Synthetic Vaccine Particles containing Rapamycin (SVP-R) inhibited the formation of ADAs, when administered with a foreign protein such as KLH or Pegsiticase. The SVP-R are taken up by macrophages and dendritic cells and increase the number of regulatory T cells in treated mice. Here we evaluated the efficacy of combination therapy of LMB-100 and SVP-R to eradicate mesothelin expressing tumors while preventing ADA formation. To evaluate if SVP-R can prevent ADAs against LMB-100, we immunized immune-competent mice with a combination of SVP-R and RIT and measured ADAs titers by ELISA and by a functional neutralization assay. We found that the treatment reduced ADA titers by more than 99%. To determine if the mice were tolerized to LMB-100, we treated the mice with two doses of SVP-R and six doses of LMB-100 to induce tolerance; and followed by nine challenges of LMB-100 given over 6 weeks. We found that the ADAs were reduced by 98%, indicating development of immunological tolerance to LMB-100. To show that the tolerance is transferable, we tolerized the mice with six doses of SVP-R and LMB-100 and adoptively transferred their splenocytes to naïve recipient mice. The recipient mice were challenged with six doses of LMB-100 and ADA titers were measured. We found that splenocytes from tolerized mice induced a 66% decrease in ADA formation in the recipient mice indicating that the tolerance was mediated by cells of the immune system. To demonstrate that immune suppression is useful in treating tumors, we implanted mouse breast cancer cells expressing human mesothelin into Balb/c mice with a normal immune system and treated them with LMB-100. LMB-100 produced tumor regressions when given before ADAs developed, but was inactive in mice with pre-existing ADAs. However, when mice with preexisting ADAs were treated with LMB-100 and SVP-R, anti-tumor activity was restored and ADAs suppressed (P≤0.0001). SVP-R are being evaluated in humans to prevent ADA to Pegsiticase, an enzyme for refractory gout treatment. Our data indicates that combining SVP-R with LMB-100 should be useful in treating cancer by allowing more treatment cycles and better efficacy. This approach can be used to increase the efficacy of other immunogenic agents such as CAR-T cells, antibody drug conjugates and viral gene therapy vectors
Citation Format: Ronit Mazor, Emily King, Takashi Kei Kishimoto, Ira Pastan. Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 72. doi:10.1158/1538-7445.AM2017-72