Tolerogenic nanoparticles restore the antitumor PNAS PLUS activity of recombinant immunotoxins by mitigating immunogenicity Ronit Mazora, Emily M. Kinga, Masanori Ondaa, Nicolas Cuburub, Selamawit Addissiea,1, Devorah Crownc, Xiu-Fen Liua, Takashi Kei Kishimotod, and Ira Pastana,2 aLaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bLaboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; cLeidos Biomedical Research, Inc., National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and dSelecta Biosciences, Watertown, MA 02472 Contributed by Ira Pastan, December 11, 2017 (sent for review September 28, 2017; reviewed by Jack Ragheb and Amy S. Rosenberg) Protein-based drugs are very active in treating cancer, but their mans compared with its parental counterpart, SS1P. The changes efficacy can be limited by the formation of neutralizing antidrug included humanization of the antibody moiety, deletion of domain antibodies (ADAs). Recombinant immunotoxins are proteins that are II of PE38 that contains dominant T cell epitopes, and in- very effective in patients with leukemia, where immunity is sup- troduction of seven point mutations in domain III to silence B cell pressed, but induce ADAs, which compromise their activity, in patients epitopes (15). LMB-100 has shown excellent antitumor activity in with intact immunity. Here we induced a specific, durable, and animal models (16) and is now being evaluated in clinical trials for transferable immune tolerance to recombinant immunotoxins by the treatment of mesothelioma and pancreatic cancer (https:// combining them with nanoparticles containing rapamycin (SVP-R). clinicaltrials.gov/ct2/show/NCT02810418). Despite the deimmu- SVP-R mitigated the formation of inhibitory ADAs in naïve and sensi- nized toxin fragment, almost all patients develop ADAs after two tized mice, resulting in restoration of antitumor activity. The immune treatment cycles. tolerance is mediated by colocalization of the SVP-R and immunotoxin Rapamycin is an mTOR inhibitor that has both immunomod- to dendritic cells and macrophages in the spleen and is abrogated by ulatory properties and antitumor activity (17). Previous attempts depletion of regulatory T cells. Tolerance induced by SVPs was not to combine rapamycin with RIT were unsuccessful, because the MEDICAL SCIENCES blocked by checkpoint inhibitors or costimulatory agonist monoclonal doses required to suppress the immune response against RIT were antibodies that by themselves enhance ADA formation. toxic to the mice (18). It was recently reported that encapsulated rapamycin in synthetic vaccine nanoparticles (SVP-R) prevented mesothelin | rapamycin | antidrug antibodies | cancer | nanoparticle immune responses in mice against a variety of biological drugs, including adalimumab, PEGylated uricase, and coagulation factor rotein- and cell-based therapies have shown great potential in VIII (19, 20). While the exact mechanism for the immunogenicity Ptreating various cancers. However, the efficacy of these bi- reduction is not clear, it has been shown that injection of SVP ological therapies is often mitigated by elicited immune re- results in its accumulation in the liver, spleen, and draining lymph sponses to the actual therapy. Repeated administration of nodes (21). Nanoparticles are preferentially phagocytosed by immunogenic anticancer drugs, such as chimeric antigen re- antigen-presenting cells due to their size, charge, and shape (22). ceptor T cells (1), enzyme therapy (2), monoclonal antibodies (mAbs) (3), antibody drug conjugates (ADCs), recombinant Significance immunotoxins (4) and viral-based gene therapy vectors (5), lead to the formation of antidrug antibodies (ADAs), resulting in drug neutralization, accelerated clearance, or infusion-related Protein-based drugs are very active in treating cancer, but their efficacy is limited by the formation of neutralizing antidrug reactions and other serious adverse events (6). antibodies (ADAs). Recombinant immunotoxins are proteins Recombinant immunotoxins (RITs) are chimeric proteins active that are very effective in patients with leukemia, in whom in the treatment of several types of cancer (7, 8). RITs consist of a immunity is suppressed, but induce ADAs, which compromise portion of an antibody linked to a protein toxin, such as Pseudo- their activity, in patients with intact immunity. Here we used monas exotoxin A (PE38). PE38 is a very potent toxin, but because an immunomodulator that is encapsulated in a nanoparticle of its foreignness to the human immune system, it induces the delivery system (SVP-R) to induce specific immune tolerance to formation of ADAs that inactivate the RIT (9). The use of RITs in immunotoxins in mice. SVP-R induces immune tolerance, pre- patients whose immune systems are suppressed by cancer or by vents ADA formation, and prevents the drug neutralization chemotherapy has produced complete regressions and prolonged and clearance that results in restoration of its antitumor ac- survival of patients with chemoresistant hairy cell leukemia (10, 11). tivity. Importantly, the combination is also efficacious in mice In contrast, when PE38 is targeted to solid tumors, immune com- with preexisting antibodies, indicating that this approach can petent patients developed ADAs against the immunotoxin (12, 13). benefit patients who often have such antibodies. TheADAsneutralizetheRIT,dramatically accelerate its clear- ance, and prevent further treatment. With the coadministration of Author contributions: R.M., E.M.K., M.O., N.C., T.K.K., and I.P. designed research; R.M., systemic immunosuppressive drugs, a mesothelin-targeted RIT E.M.K., M.O., N.C., S.A., D.C., and X.-F.L. performed research; R.M., E.M.K., M.O., N.C., (SS1P) enabled 2 of 10 patients being treated for mesothelioma T.K.K., and I.P. analyzed data; and R.M., T.K.K., and I.P. wrote the paper. to receive more cycles of therapy, resulting in profound antitu- Reviewers: J.R., Eli Lilly and Company; and A.S.R., Food and Drug Administration. mor responses and prolonged survival (14). Therefore, RIT has Conflict of interest statement: T.K.K. is an employee and shareholder of Selecta Biosci- the potential to be a transformative therapy for chemotherapy- ences. All other authors declare no competing interests. refractory mesothelioma and other solid tumors if ADAs can be Published under the PNAS license. mitigated more broadly. 1Present address: NantKwest Inc., Woburn, MA 01801. LMB-100 is a second-generation RIT that has a humanized Fab 2To whom correspondence should be addressed. Email: [email protected]. A targeting mesothelin fused to a modified PE38 toxin (Fig. 1 ) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (15). LMB-100 was engineered to reduce immunogenicity in hu- 1073/pnas.1717063115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1717063115 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 Fig. 1. The combination of LMB-100 + SVP-R prevents the ADA response against LMB-100. (A) A ribbon diagram of LMB-100 and an illustration of SVP-R. (B) Mice were injected seven times every other week with LMB-100 or a combination of LMB-100 and SVP-R one, three, or seven times (indicated by arrows). Anti–LMB-100 antibodies were evaluated by ELISA (n = 8). (C) Mice were injected with LMB-100 and SVP-R as indicated by arrows (n = 7). (D) Mice were injected with LMB-100 and SVP-R as indicated by arrows. Final mean titer on week 10 is shown (n = 7). (E) Neutralization assay using plasma from the mice treated as shown in C (n = 7). KLM-1 cells were seeded and treated with plasma-LMB-100 mixture. Cell viability was assessed after 72 h. Curves represent mean of seven viability curves (n = 7, six replicas per samples). (F) Mice were injected with LMB-100 and SVP-R as indicated by arrows (n = 8). ELISA plates were coated with LMB-100, Fab, or anti–TAC-PE24. Plasma samples from week 6 were evaluated. The dilution factor for 50% of binding is shown. Lines indicate mean; error bars, SEM. For statistical analysis in B and C, the AUC for each curve was calculated, and AUCs were compared using one-way ANOVA. It has been speculated that the selective delivery of rapamycin to 100 (Fig. 1B), with a mean titer of 10,975 ± 2,372 at week 14, these immune organs generates a tolerogenic milieu that can in- indicating that LMB-100 is immunogenic in BALB/c mice. All duce specific immune tolerance to coadministered antigens (18). mice injected with LMB-100 + SVP-R had an undetectable titer Here we report preclinical studies supporting the use of SVP-R during the entire course of the experiment, indicating effective in patients to be treated with RITs. We demonstrate that SVP-R prevention of ADA formation. Furthermore, mice injected seven induces a long-lasting, specific, transferable, and Treg-dependent times with LMB-100 and given SVP-R with only the first three immune suppression and tolerance that prevents ADA formation injections had a mean titer of only 371 ± 301 at week 14, in- against LMB-100 in naïve mice and in mice with preexisting an- dicating induction of immune tolerance. This titer was significantly tibodies. This immune tolerance promotes the antitumor activity lower than that of control mice treated with LMB-100 alone at in immunocompetent mice that otherwise would be neutralized by both week 8 (P = 0.03) after only four doses and at week 14 (P = the ADAs. Furthermore, the combination of LMB-100 and SVP- 0.0006) after seven doses. The area under the curve (AUC) for R induces favorable cytotoxic activity in human mesothelioma and each mouse throughout the experiment, calculated to compare the pancreatic cancer cell lines, more potent than that induced by ADA responses (Fig. S1A), demonstrated a significant decrease in either agent alone. Finally, the immune tolerance persists when the mice given three doses (P = 0.001) or seven doses of SVP-R combined with anti–CTLA-4 checkpoint inhibitor or anti–OX-40 (P = 0.002).