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 moiety, deletion of domain (ADAs). Recombinant immunotoxins are proteins that are II of PE38 that contains dominant epitopes, and in- very effective in patients with , 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 (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 , 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 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 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). The mice tolerated treatment well, with no significant costimulatory agonist mAbs. weight loss (Fig. S1B).

Results Timing of SVP-R Immunization Is Critical for Immune Tolerance. To Combination of LMB-100 with SVP-R Prevents ADA Response. To determine the efficacy of SVP-R with an LMB-100 regimen sim- evaluate the effect of SVP-R on the ADA response to LMB-100 ilar to that used in human patients, mice were treated with suc- (Fig. 1A), BALB/c mice were injected every other week with cessive cycles of LMB-100. Each cycle consisted of three doses per LMB-100 or a combination of LMB-100 + SVP-R. LMB-100 has week every other day, and mice were injected with SVP-R once, mutations that diminish human, but not mouse, responses. Mice twice, or three times during the first and second cycles (Fig. 1C). injected with LMB-100 had a strong and rapid response to LMB- We found that a single dose of SVP-R per cycle was as effective as

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1717063115 Mazor et al. Downloaded by guest on September 26, 2021 three doses in preventing ADA formation (P = 0.003). The me- LMB-100 + SVP-R induces a specific immune tolerance that does PNAS PLUS dian titer in mice receiving LMB-100 alone was 47,926, compared not suppress the ability of the mice to mount an immune response with only 881, 1,958, and 993 in mice immunized with LMB-100 + against another antigen administered later. SVP-R given two, four, or six times, respectively, over the two To test whether the immune tolerance could be transferred treatment cycles. The ADA suppression was also maintained when from tolerant mice to naïve mice, donor mice were treated with mice were challenged with three additional cycles of LMB-100 in LMB-100, SVP-R, or LMB-100 + SVP-R for two cycles. Mice the absence of further SVP-R treatment. Six doses of LMB-100 + immunized with LMB-100 alone showed a mean titer of 4,521 ± SVP-R were well tolerated by the mice, with no significant weight 1,994, compared with 51 ± 25 in mice treated with LMB-100 + loss (Fig. S1C). SVP-R (Fig. S4). Splenocytes were isolated, pooled, and trans- We evaluated the effect of timing of SVP-R treatment by stag- ferred to naïve recipient mice (Fig. 2B). At 1 wk after cell in- gering the day of SVP-R injection. LMB-100 was injected on days jection, all recipient mice were challenged with two cycles of 1, 3, and 5 of each of five cycles, with SPV-R coadministered on LMB-100. Adoptive transfer of cells from mice immunized with day 1, day 3, days 1 and 3, days 3 and 5, or days 1, 3, and 5 of each LMB-100 followed by an LMB-100 challenge of recipient mice cycle (Fig. 1D). Control mice treated with LMB-100 showed a induced a mean titer of 4,884 ± 1,548, not significantly different mean titer of 44,132 at the end of five treatment cycles. In contrast, from the titer in mice receiving cells from vehicle-treated mice or mice that received SVP-R on day 1 showed significant decreases in no cells (mean titers of 4,571 ± 1,494 and 6,541 ± 3,079, re- ADA formation regardless of whether they received one, two, or spectively). Here, adoptive transfer did not induce substantial three SVP-R doses during each cycle, with mean titers of 1,413 ± immune memory. Because these three mice groups had similar 495 (P = 0.0007), 2,952 ± 1,320 (P = 0.001), and 1,979 ± 807 (P = mean titers, these mice are referred to as controls. 0.0007), respectively. Mice that received SVP-R on day 3 or days In contrast, adoptive transfer of 10 × 106 splenocytes from 3and5hadfinaltitersof29,341± 11,705 and 41,934 ± 9,725, mice immunized with a combination of LMB-100 + SVP-R de- respectively, indicating that cotreatment with SVP-R on the first creased the titers by 78–85% compared with the controls (P = day of each cycle is critical to prevent ADA formation. 0.007, 0.003, and 0.02, respectively). Adoptive transfer of 2.5 × SVP-R was also evaluated with the more immunogenic pre- 106 splenocytes reduced titers by 44–61%, but the difference was cursor of LMB-100, SS1P. Mice were injected with three doses of not statistically significant (P = 0.5). The mean titer of mice that SS1P at weeks 1, 3, and 7 (Fig. S2), and SVP-R was given at week received splenocytes from SVP-R–treated mice was not different 1. Three cycles of SS1P induced a mean ADA titer of 37,734 ± from that of control mice, indicating that tolerance induction 21,748, and a single cycle of SVP-R completely blocked these requires both LMB-100 and SVP-R in the donor mice and is not ADAs (P = 0.0001). due to a general immune suppression. MEDICAL SCIENCES

ADA Response Is Neutralizing and Targets both the Fab and Toxin. To Depletion of Treg Cells. To study the role of Treg cells in SVP-R– determine whether ADAs can neutralize the immunotoxin, we induced immune tolerance, we depleted Treg cells in vivo after performed a functional in vitro neutralization assay using plasma SVP-R tolerance induction. Mice were injected with LMB- samples from mice injected with LMB-100 (15 doses), LMB-100 100 or LMB-100 + SVP-R three times. On days 15 and 16, Treg (15 doses) + SVP-R (six doses), or vehicle. Plasma samples were cells were depleted using an anti-CD25 (PC61)-depleting anti- mixed with various concentrations of LMB-100 and added to body (24), and then challenged with two more cycles of LMB-100 KLM-1 human pancreatic cells. The cells were very sensitive to (Fig. 2C). The depletion of Tregs abrogated the tolerogenic ef- LMB-100 with an IC50 of 1.1 ng/mL (Fig. 1E). Plasma from mice fect of SVP-R, increasing the mean titer from 416 ± 157 to immunized with LMB-100 alone inhibited the activity of LMB- 1,094 ± 304 (P = 0.04). This mean titer of 1,094 was similar to 100 and shifted the IC50 to 93.2 ng/mL (P < 0.0001), indicating that in mice that did not receive SVP-R (1,348 ± 399). that the ADAs are neutralizing. In contrast, incubation of LMB- 100 with plasma LMB-100 + SVP-R showed an IC50 50-fold Ig Subclasses. To study the effect of SVP-R on class switching, – lower (P < 0.0001) and not significantly different from the IC50 plasma samples were characterized for LMB-100 specific IgG and of LMB-100 incubated with plasma from vehicle-treated mice IgM antibodies (Fig. 2D). Immunization with LMB-100 induced (Fig. S3A). We observed a strong correlation between the anti– ADAs distributed across all IgG subclasses, with IgG1 the most 2 LMB-100 titers and IC50 (R = 0.96) (Fig. S3B). dominant. This subclass distribution is similar to the IgG subclass To determine whether the ADAs against LMB-100 target the distribution previously described after immunization with the Fab, the toxin fragment, or both, we assayed the plasma from mice parent immunotoxin SS1P (25). Immunization with LMB-100 + injected with five doses per week of LMB-100 alone or in combi- SVP-R induced an undetectable signal of LMB-100–specific IgG1, nation with SVP-R (n = 8) on plates coated with LMB-100, a IgG2a, IgG2b, or IgG3 antibodies. Interestingly, the levels of anti– human Fab, or an immunotoxin containing the same domain III of LMB-100 IgM antibodies were similar to the levels in mice im- the Exotoxin A (PE24) as found in LMB-100, fused to a mouse Fv munized with LMB-100 alone. These results indicate that SVP-R (anti–TacFv-PE24). Anti–LMB-100 plasma reacted strongly with prevents isotype switching, but does not prevent IgM production. the human Fab and less strongly with the deimmunized toxin, which had some of its murine B cell epitopes removed (23) (Fig. 1F). As LMB-100 + SVP-R Colocalize Preferentially on Dendritic Cells and expected, SVP-R reduced the response to both components. Macrophages. We hypothesize that specific immune tolerance requires the presence of the antigen in the same cells as the Combination of LMB-100 with SVP-R Induces a Specific and Transferable target cells of SVP-R. To determine the fate of SVP-R and Immune Tolerance. To determine whether the suppression of ADA LMB-100 in the spleen, after injection in vivo, we injected Alexa formation is a result of specific immune tolerance rather than a Fluor 488-labeled LMB-100– and Cy5-labeled SVP-R consecu- chronic immune suppression, mice were immunized with eight tively and isolated the splenocytes at 2 h postinjection (Fig. 3A). weekly injections of LMB-100 and three doses of SVP-R (i.v.) at Cell phenotype was analyzed using cellular markers according to weeks 1, 2, and 3. At week 4, mice were challenged with four the gating strategy shown in Fig. S5. We compared the uptake of weekly injections of ovalbumin and LMB-100 (S.C.) (Fig. 2A). The LMB-100 and SVP-R in macrophages, dendritic cells (DCs), + + combination of LMB-100 + SVP-R selectively inhibited ADA CD4 T cells, CD8 T cells, B cells, neutrophils, and monocytes formation against LMB-100, but did not affect the antibody re- (Fig. 3 B–D). We found that macrophages and DCs had the sponse to ovalbumin, resulting in similar anti-ovalbumin titers of highest uptake of both LMB-100 and SVP-R; 38% of the mac- 4,362 and 4,024. These results indicate that the combination of rophages and 13% of the DCs were positive for LMB-100, and

Mazor et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 Fig. 2. Combination of LMB-100 with SVP-R induces a specific, transferable, and regulatory T-cell–mediated immune tolerance. (A) Mice were injected three times weekly with LMB-100 (2.5 mg/kg i.v.) or a combination of LMB-100 with SVP-R (2.5 mg/kg i.v.). During weeks 4–8, mice were challenged with weekly doses of LMB-100 (i.v.) and ovalbumin (s.c.). Plasma was collected and analyzed for anti–LMB-100 and anti-ovalbumin antibodies by ELISA. For statistical analysis, AUC for each curve was calculated and analyzed using the Mann–Whitney U test. Error bars SEM, n = 13. (B) Mice were injected six times with vehicle, LMB-100, SVP-R, or LMB-100 + SVP-R. During week 4, splenocytes from donor mice were isolated and adoptively transferred to recipient naïve mice. Recipient mice were injected with LMB-100 six times. Plasma was collected and analyzed for anti–LMB-100 antibodies by ELISA. Results from two separate experiments with identical schedules were combined. n = 5–10. Error bars represent SEM. (C) Mice were injected with LMB-100 on days 1, 3, 5, 29, 31, 33, 43, 45, and 47. SVP-R was given on days 1, 3, and 5. Anti-mouse CD-25–depleting antibody (PC61) or isotype control were injected i.p. on days 15 and 16. Titers on day 55 are shown. (D) Plasma from mice that were injected seven times every other week with LMB-100 or a combination of LMB-100 + SVP-R. Anti–LMB-100 isotypes were analyzed using sandwich ELISA with subclasses IgG1, IgG2a, IgG2b, IgG3, and IgM specific to LMB-100 (n = 8).

29% of the macrophages and 11% of the DCs were positive for 0.002). This response was observed in three additional experi- SVP-R. Interestingly, 22% of the macrophages and 9% of the ments with groups of eight, eight, and four mice. DCs stained positive for both. This colocalization occurred even To evaluate whether SVP-R can induce a lasting immune though the two agents were injected separately. Relative cell tolerance that can prevent a response to later challenges in mice numbers were not changed (Table S1). with preexisting antibodies, the mice were challenged with three + − Monocytic cells expressing CD11bhigh, Ly6C , and Ly6G additional doses of LMB-100 (no SVP-R) during week 13 (Fig. have been involved in immunosuppressive activity (26, 27). We 4A). Titer evaluation at week 14 showed that administration of found that 3% of these cells demonstrated uptake of both LMB- LMB-100 + SVP-R during week 10 maintained a low titer of 100 and SVP-R. Finally, lymphocytes and neutrophils displayed 634 ± 269, which was significantly lower than the titer of mice the lowest percentages of colocalization (Fig. 3D). Taken to- treated with LMB-100 alone (11,505 ± 4,172; P = 0.0001). This gether, these results suggest that preferential uptake of SVP-R indicates that the LMB-100 + SVP-R combination on week and LMB-100 by professional antigen-presenting cells might 10 induced an immune tolerance that prevented the response to mediate the immune tolerance. a later LMB-100 challenge. We next evaluated whether SVP-R could also be used to The Combination of LMB-100 + SVP-R Prevents ADA Response in Mice therapeutically reduce high titers of preexisting antibodies. with Preexisting Antibodies. To determine whether SVP-R could Control mice from Fig. 4A that had an anti–LMB-100 antibody therapeutically reduce immunogenicity and induce immune tol- titer >10,000 induced by 12 doses of LMB-100 over the course of erance in mice with preexisting ADAs, mice were immunized six 14 wk were injected with LMB-100 or LMB-100 + SVP-R (Fig. times with LMB-100 during weeks 1 and 3 to induce preexisting 4B). Mice treated with the combination had a significant de- ADAs. At week 9, mice had a mean titer of 741 ± 66 and were crease in titer, from 31,114 ± 13,730 to 7,797 ± 4,558 (P = 0.02). divided into three groups with similar mean titers. At week 10, the To determine whether treatment of mice with preexisting an- groups were immunized with vehicle (PBS), LMB-100, or LMB- tibodies with the combination affected the number of antibody 100 + SVP-R (Fig. 4A). Titers were then evaluated at week 12. secreting plasma cells in the bone marrow (BM), we treated mice Challenge with LMB-100 alone induced a strong memory immune with preexisting antibodies with PBS, LMB-100, SVP-R, or LMB- response, resulting in a mean ADA titer of 9,808 ± 3,608. In 100 + SVP-R. Cells were collected from BM and spleen at 24 h contrast, challenge with LMB-100 + SVP-R not only prevented after injection and then assayed for the number of cells making the antibody increase, but also decreased the titer (257 ± 121) anti–LMB-100 antibodies by ELISpot (Fig. 4 C and D). The mice compared with the preboost titer (738 ± 320; P = 0.003) and had similar numbers of antibody-secreting cells (mean, 9.6 ± 6.7 compared with mice injected with PBS at week 12 (502 ± 143; P = SFC/1E6 cells) in BM and no detectable spots in spleens. These

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Fig. 3. LMB-100 and SVP-R colocalize preferentially on DCs and macrophages. (A) Dye-conjugated SVP-Cy5 and LMB-100-Alexa Fluor 488 were injected i.v. alone or in combination (n = 3–4 mice per group). Spleen cells were analyzed by FACS at 2 h after injection for dye-conjugate uptake. (B and C) Representative + + + + FACS plots show gating for macrophages (F4/80 CD11b ) and DCs (CD11c MHC-II ), and in vivo uptake by the gated populations. Red quadrants indicate the + percent of positive cells analyzed for each experimental condition. (D) Summary of SVP-R and LMB-100 in vivo uptake by macrophages, DCs, monocytes, CD4

+ MEDICAL SCIENCES T cells, B cells, neutrophils and CD8 T cells. The gating strategy for all cells is shown in Fig. S5.

results indicate that SVP-R does not affect antibody-secreting vival rate (i.e., time to reach 600 mm3; P = 0.0001) (Fig. 5D). plasma cells residing in the BM. These experiments were repeated two more times using seven mice per group with similar results. However, mice treated with The Combination of LMB-100 + SVP-R Restores Antitumor Activity of LMB-100 + SVP-R had decreased weight, perhaps due to in- LMB-100 in Mice with Preexisting Anti–LMB-100 Antibodies. To study creased exposure to LMB-100 as a result of preventing neu- the activity of LMB-100 and SVP-R in immunocompetent tumor- tralizing ADAs (Fig. S8). bearing mice, the AB-1 mouse mesothelioma cell line (28) was stably transfected with human mesothelin (AB1-L9; Fig. S6). AB1- SVP-R Does Not Accelerate Tumor Growth Rate. To test whether L9 cells inoculated into BALB/c mice grew rapidly, reaching treating mice with SVP-R interferes with tumor immunity and/or 600 mm3 in 15 d (Fig. 5A). To evaluate antitumor activity, tumor- enhances tumor growth, we inoculated the CT26 (murine colon bearing mice were therapeutically treated six times with LMB-100, carcinoma) and 66C14 (murine breast cancer) cell lines in the SVP-R, or LMB-100 + SVP-R, when the tumors reached a mean flanks of immune competent BALB/c mice and compared the size of 199 mm3. Mice treated with LMB-100 showed significant growth rates in SVP-R–treated mice and PBS-treated mice (Fig. tumor growth inhibition (P = 0.003 for AUC of tumor growth 5 E and F). SVP-R delayed the growth of CT26 tumors and curves compared with PBS-treated mice), with 1 of 7 mice achieving showed no change in tumor growth in 66C14 tumors. complete remission. Mice treated with SVP-R showed only a minor tumor growth delay (P = 0.05). LMB-100 + SVP-R induced the SVP-R Enhances the Cytotoxic Activity of LMB-100 in Human Cell Lines. most significant tumor growth inhibition (P = 0.0003), resulting in a Because rapamycin has also been reported to have antitumor ac- 13-fold decrease in tumor size on day 20. Due to the relatively short tivity, we measured the cytotoxic activity of SVP-R on human immunization schedule, all mice had either very low or undetect- mesothelioma cells (HAY) and human pancreatic cells (KLM-1) in able titers when evaluated on day 18 of the experiment (Fig. S7), so vitro. We found that SVP-R had modest cytotoxic activity by itself no significant in vivo neutralization of LMB-100 was observed. (Fig. 6A) in both cell lines. However, when combined with LMB- To study the activity of LMB-100 and SVP-R in mice with 100, 5 μg/mL of SVP-R improved the cytotoxic activity of LMB- preexisting antibodies, mice were first immunized with LMB- 100, shifting the IC50 on KLM-1 cells from 1.1 ng/mL to 0.1 ng/mL ± 100 four times to induce an average baseline titer of 2,597 2,080 (Fig. 6B),andonHAYcells.1μg/mL of SVP-R improved the IC50 before inoculation with AB1-L9. At 5 d after tumor inoculation, from 2.9 ng/mL to 0.9 ng/mL (Fig. 6C). HAY cell viability was also when the tumors reached a mean of 135 mm3, mice were treated evaluated by staining with crystal violet after a 72-h incubation with with two cycles of three injections with LMB-100 or vehicle (Fig. SVP-R (2 μg/mL) and LMB-100 (0.4 ng/mL), followed by in- 5B) with or without SVP-R administered on the first day of each cubation for 72 h with no drug (Fig. 6D). The combination was cycle (every other week). We found that the tumors treated with more effective than either drug alone in killing cells. LMB-100 alone did not respond to treatment and had a similar growth rate as PBS-treated tumors. We attribute the lack of re- SVP-R Activity Is Not Diminished by Checkpoint Inhibitors or sponse to LMB-100 to the high ADA titer (Fig. 5C), which neu- Costimulatory Agonists. We next investigated whether anti– tralized the activity of LMB-100. CTLA-4 antagonist antibody and anti–OX-40 agonist antibody In contrast, mice treated with LMB-100 + SVP-R had an ex- can enhance the formation of ADAs against LMB-100, and cellent response to LMB-100 and did not develop high ADA whether such ADAs could be blocked by SVP-R. Mice (n = 8) titers. Mice treated with LMB-100 + SVP-R had a higher sur- were injected with five weekly doses of LMB-100, along with an

Mazor et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 Fig. 4. Combination of LMB-100 with SVP-R induces immune tolerance in mice with preexisting antibodies. (A) Female BALB/c mice were injected six times with LMB-100 (2.5 mg/kg i.v.) on weeks 1 and 3 to induce a titer of ADA against LMB-100. On week 10, mice were challenged with three doses of LMB-100, vehicle (PBS), or LMB-100 + SVP-R. The mice treated with LMB-100 + SVP-R were challenged with three additional doses of LMB-100 on week 12. Plasma was collected and analyzed for LMB-100 ADAs by ELISA. n = 7 or 12. Error bars represent SEM. (B) Female BALB/c mice were injected 12 times with LMB-100 over the course of 14 wk to induce a high titer of ADA against LMB-100. In week 15, mice were immunized with LMB-100 or LMB-100 + SVP-R. ADA titers prechallenge and postchallenge are shown. (C and D) BM and spleen were isolated from mice with preexisting ADA and were challenged with PBS, LMB-100, SVP-R, or LMB-100 + SVP-R. BM cells and splenocytes (100,000 cells/well) were seeded in ELISpot plates precoated with LMB-100 (n = 8).

anti-mouse CTLA-4 antibody or an anti–OX-40 antibody given on anti-immunotoxin immune response in patients (29), and B and the fifth day of every week (Fig. 7). We found that both antibodies T cell suppression using a combination of cyclophosphamide and substantially enhanced the formation of anti–LMB-100 ADA ti- pentostatin (14). The success of this approach was limited by the ters compared with treatment with LMB-100 alone (P = 0.001 for toxicity of the immunosuppressive agents, and while some pa- anti-CTLA-4 and P = 0.02 for anti-OX-40). Injection of SVP-R on tients exhibited a dramatic delay in ADA formation, most pa- the same days as LMB-100 resulted in either elimination (i.e., tients developed strong ADA responses that halted treatment. mean titer below the limit of detection) in the mice treated with anti–CTLA-4 or a dramatic 12-fold decrease in titer in the mice Immune Tolerance Mechanism. Unlike general immune suppressive treated with anti–OX-40. SVP-R activity was not compromised by therapies, SVP-R has a strong affinity to accumulate in the lymph the activity of the immune checkpoint inhibitors or costimulatory nodes and spleen (21), where it is selectively endocytosed by agonists. These experiments were repeated two more times with antigen-presenting cells (Fig. 3). This localization translates to a n = 5andn = 3, with similar results. dramatic reduction in toxicity and enhancement of a tolerogenic effect. Rapamycin-conditioned DCs can become tolerogenic and Discussion promote the differentiation of Tregs (30–32). In this study, we have The immune response to RITs is a major factor limiting their shown that SVP-R specifically targets professional phagocytes, such efficacy against solid tumors in cancer patients with intact immune as macrophages, DCs, and, to a lesser extent, monocytes. Impor- systems. In the present study, we have established the preclinical tantly, LMB-100 specifically targets professional phagocytes and basis of specific immune tolerance for RITs using rapamycin en- colocalizes with SVP-R. These results support the finding that SVP- capsulated in SVP-R. These nanoparticles are composed of a R can induce tolerogenic DCs in vivo (19), but does not preclude biodegradable poly (lactic acid) core with a corona of surface additional involvement of macrophages. The tolerance was abro- PEGylation. We demonstrate that SVP-R produces a long-lasting, gated after depletion of Tregs by 14 d after SVP-R treatment (Fig. specific, and transferable immune tolerance that prevents ADA 2C), supporting the mechanism of myeloid cell tolerance mediated formation against LMB-100 in naïve mice and reduces ADAs in by Treg cells. Of note, the antibody used for Treg depletion (24) + + mice with preexisting antibodies. Induction of immune tolerance was recently shown to efficiently deplete CD25 CD4 Treg cells, − + to LMB-100 resulted in restoration of its antitumor activity in a but not CD25 Foxp3 Treg cells (33). The finding that a CD25- syngeneic mesothelioma tumor model in immunocompetent mice targeting antibody negated SVP-R activity but a CTLA4-targeting that otherwise would be neutralized by ADAs. antibody did not may be explained by the dosing and schedule regimen or by differences in mechanism of the two antibodies. Immune Suppression vs. Tolerance. Previous studies have evaluated While SVP-R effectively inhibited IgG antibody responses, we several immune suppression approaches to reducing the immu- observed that specific IgM antibodies were not affected by SVP-R nogenicity of RITs in patients. These approaches include B cell (Fig. 2D). Similarly, Kishimoto et al. (19) injected C57BL6 mice depletion using , which was ineffective in preventing with KLH and SVP-R and found that while SVP-R induced

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1717063115 Mazor et al. Downloaded by guest on September 26, 2021 PNAS PLUS

Fig. 5. Combinations of SVP-R and LMB-100 restores neutralized antitumor activity. (A) AB1-L9 cells were inoculated into mice and treated with PBS, LMB-100, or MEDICAL SCIENCES SVP-R as indicated by arrows (n = 7). (B) Mice were immunized with LMB-100 four times to induce a baseline titer and then inoculated with AB1-L9. Mice were treated with vehicle, LMB-100, or SVP-R as indicated by arrows. Tumor size was measured using a caliper (n = 7). (C) Plasma from days 5 and 19 was analyzed for anti–LMB-100 antibodies by ELISA. Titer was interpolated at 10% of the signal. (D) Mice were treated as described in C. The experiment was terminated on day 31. The Kaplan–Meier plot shows the time to the experimental endpoint (once tumor volume was >400 mm3 or if a mouse lost >30% of its body weight). n = 7. (E) Mice were inoculated with CT26 cells on day 1 and treated with SVP-R or vehicle on days 10 and 16. Values indicate average tumor size (n = 7). Error bars represent SEM. (F) Mice were inoculated with 66C14 cells on day 1 and treated with SVP-R or vehicle on days 10, 12, and 14. Values indicate average tumor size (n = 5). For statistical analysis, AUC for each curve was calculated, and AUCs were compared using one-way ANOVA. Error bars represent SEM.

a >99% decrease in anti-KLH IgG, it only reduced 50% of the antigens. We found that mice that were tolerized by injections of + IgM. This indicates that SVP-R prevents CD4 -mediated class LMB-100 and SVP-R mounted an immune response to a second switching, but not the initial (T cell-independent) activation of B antigen injected s.c. (Fig. 2A). That the mice had an immune cell and IgM production. These results support either a Treg- response to the second immunogen but not to LMB-100, even mediated mechanism or a lack of T cell help. though both were administered at the same time, dose, and A major differentiator between immune suppression and tol- frequency during the challenge phase, indicates the induction of erance is the ability to mount an immune response against other specific tolerance to LMB-100 rather than global suppression of

Fig. 6. SVP-R enhances the cytotoxic activity of LMB-100 in human cell lines. KLM-1 and HAY cells were seeded in 96-well plates and treated with various concentrations of SVP-R, LMB-100, or LMB-100 + SVP-R. After 72 h, cell viability was assessed using WST-8 or crystal violet. Viability curves were fitted to each

sample and IC50 was calculated. (A) Cytotoxic activity of SVP-R in both cell lines. (B) Activity of LMB-100 in KLM-1 cells with or without 5 μg/mL of rapamycin encapsulated in SVP. (C) Activity of LMB-100 in HAY cells with or without 1 μg/mL of rapamycin encapsulated in SVP. Curves show a mean of six replicates. Error bars represent SEM. (D) Representative well images taken after HAY cells were fixed and stained with crystal violet.

Mazor et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 anticancer effect as single agents and in combinations for multiple types of tumors (reviewed in ref. 37). Our finding that SVP-R improved the cytotoxic and antitumor activity of the immuno- toxin (Figs. 5A and 6) is supported by a recent synergy screen that identified the mTOR inhibitor everolimus as the best enhancer of immunotoxin activity among 459 small molecules (38). Encapsu- lation of rapamycin in 150 nm SVP adds an additional benefit, as nanoparticles enhance permeability and retention in tumors (39, 40). The slow release of rapamycin at the tumor site could syn- ergize with the targeted immunotoxin. Together, the improved antitumor activity and reduced immunogenicity provide compel- Fig. 7. SVP-R activity is not diminished by checkpoint inhibitor antibodies. ling support for this combination in human clinical trials. BALB/c mice were immunized weekly with LMB-100 or LMB-100 + SVP-R five times (2.5 mg/kg i.v.), and at 5 d after each injection were immunized with SVP-R Did Not Affect Tumor Immunogenicity. SVP-R must be anti-mouse CTLA-4 antagonist (A) or anti–OX-40 antagonist (B) or vehicle (i.p.). Plasma samples were collected on day 6 of each week, and LMB-100 coadministered with antigen to confer immune tolerance (18). ADA titer was evaluated using direct ELISA. n = 8. Error bars represent SEM. Injection of SVP-R even 2 d after LMB-100 was ineffective in The experiments were repeated with n = 3 and 5, with similar results. inducing immune tolerance (Fig. 1D). Importantly, SVP-R alone did not cause the tumors in immune competent mice to grow faster (Fig. 5 A, E, and F). These observations alleviate a po- the immune system. Immune suppression is commonly mediated tential safety concern of the SVP-R inducing tolerance against by drugs that have no lasting effect on the immune system after the tumor or making the tumor grow faster. the cessation of therapy. In contrast, immune tolerance involves the induction of regulatory cells that actively maintain tolerance Triple Combination with Checkpoint Inhibitors. The groundbreaking in the absence of drugs. We observed that transfer of splenocytes advancements in with checkpoint inhibi- isolated from mice treated with the combination of LMB-100 + tors and costimulatory agonists, coupled with clinical observations SVP-R (Fig. 2B) prevented ADA formation in naïve recipient that tumor killing by immunotoxins may induce immune re- mice. Taken together, our data suggest that the combination of cruitment and activation in the tumor microenvironment (14), LMB-100 + SVP-R induces immune tolerance. have led us to hypothesize that the combination of LMB-100 with checkpoint inhibitors may provide synergistic antitumor activity. Therapeutic Activity in a Preexisting Antibody Model. Preexisting To that end, Leshem et al. (41) evaluated the antitumor effect of antibodies that target and neutralize therapeutic proteins are a combining immunotoxins and checkpoint inhibitor antibodies in hindrance in many therapeutic regimens. Our findings that SVP-R mice. They found that intratumoral LMB-100 injection combined not only were effective in controlling the boost in anti–LMB- with anti-mouse CTLA-4 antibody induced dramatic tumor 100 titers, but also actually demonstrated a striking prolonged eradication and resistance to secondary tumor challenges. tolerance (Fig. 4), indicate that a combination of RITs with SVP-R We evaluated the effect of anti–CTLA-4 and anti–OX-40 an- may be useful in patients with preexisting antibodies or even in tibodies on the onset and intensity of ADA formation against patients who participated in previous clinical trials with SS1P, LMB-100, and the ability of SVP-R to prevent these responses. LMB-100, or . Many patients in these We found that both anti–CTLA-4 checkpoint inhibition and trials initially responded to immunotoxin therapy, but the response anti–OX-40 costimulatory agonist expedited and intensified the was halted due to ADA formation (7, 34). formation of LMB-100 ADAs (Fig. 7). Importantly, SVP-R given The decrease in titers was not due to the depletion of long-lived on the day of injection of LMB-100 completely eradicated these BM plasma cells that secrete ADAs (Fig. 4C). It is possible that exacerbated immunogenicity responses. Future work needs to the reduction in titers is associated with inhibition of short-lived address the effect of SVP-R on the synergistic antitumor activity plasma cells from memory B cell activation, or perhaps with of immunomodulatory mAbs with LMB-100. clearance of the circulating ADAs through immune complex for- The anti–CTLA-4 mAb used in our study has been studied for mation. These results agree with work by Zhang et al. (20) anticancer activity in CT26 and MC38 (mouse colon carcinoma) showing that mixing SVP-R with recombinant factor VIII and tumor models (42), and it is thought to act by reducing Treg cells coinjecting the mixture in mice with factor VIII inhibitors resulted and concomitantly activating effector T cells at the tumor site in a very low immune response. A similar decrease in ADA was (42). The anti–OX-40 mAb used in the present study has been observed in mice with preexisting antibodies against adeno- studied for anticancer activity in CT26 and MOSEC (mouse associated virus after treatment with nonencapsulated rapamycin ovarian) tumor models (43, 44). It is speculated that the mech- and prednisolone (35). anism of action involves activation of the NFKB pathway and, + In our mouse studies, we observed a wide range of ADA titers consequently, CD4 T cell activation. The fact that these im- (741–47,926) associated with multiple treatment schedules. For mune stimulatory mAbs did not compromise the tolerogenic example, in Fig. 2C a mean titer of 741 was detected at 9 wk after activity of SVP-R suggests that the tolerogenic signal is not six doses of LMB-100. The maximal titer of 47,926 was measured overridden by these immunotherapeutic antibodies. at 1 wk after the final injection in an experiment that used 15 doses. The control titer in the Treg experiment (1,348 ± 399) Concluding Remarks was measured after nine doses of LMB-100. The titer cannot be LMB-100 is currently being evaluated in clinical trials for treat- compared with any other experiment in this study, because we ment of mesothelioma and pancreatic cancer (https://clinicaltrials. did not use this schedule in any other experiment. Nevertheless, gov/ct2/show/NCT02810418). Immunogenicity likely will be a the titer after nine doses is lower than expected and may be major obstacle to the maintenance of therapeutic blood levels explained by variations in age, food, and microbiota. after multiple treatment cycles, as has been previously observed with several immunotoxins (12). Moxetumomab has completed Rapamycin and Cancer. The mTOR signaling network contains phase 3 of clinical evaluation, demonstrating 50% immunogenicity various tumor-suppressor genes and proto-oncogenes, including in patients with hairy cell leukemia during Phases 1 and 2 (https:// PTEN, PIK3, and AKT (reviewed in ref. 36). Rapamycin and clinicaltrials.gov/ct2/show/NCT01829711). Thus, there is an unmet other mTOR inhibitors are being evaluated in clinical trials for need to mitigate immunogenicity to unlock the full potential of

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1717063115 Mazor et al. Downloaded by guest on September 26, 2021 RITs. SVP-R is currently being evaluated in combination with a ADA against the Fab or the toxin fragments. ELISA plates were coated with 2 μg/mL PNAS PLUS PEGylated uricase in a Phase 2 clinical trial in patients with of the Fab portion of LMB-100 (generously provided by Roche), or a 2 μg/mL symptomatic gout (https://clinicaltrials.gov/ct2/show/NCT02648269). RIT containing a murine scFv that targets an irrelevant epitope (anti-Tac) The strategy that we describe here represents a novel and safe linked to the deimmunized toxin fragment of LMB-100. ADA determination was performed as described above. approach to unlocking the full potential of RIT therapy for solid The OD of the wells was read immediately after the addition of H2SO4 stop tumors. These results may have implications for other highly for- solution at a wavelength of 450 nm with subtraction at 650 nm. Titers were eign, life-saving therapeutic proteins as well. calculated based on a four-parameter logistic curve-fit graph and in- terpolated on the half maximal value of the anti–LMB-100 (IP12) (15) or anti- Materials and Methods ovalbumin (clone TOSG1C6; BioLegend) standard curves. LMB-100 and SVP-R. LMB-100 was manufactured by Roche as described pre- viously (45) and provided for these studies through a Collaborative Research Transfection of Cell Line with Human Mesothelin and Tumor Inoculation. The and Development Agreement (2791). SVP-R was manufactured by Selecta AB-1 mouse mesothelioma cell line (Sigma-Aldrich) was stably transfected μ Bioscience as described previously, with a rapamycin content of 500 g/mL (19). with human mesothelin cDNA (41) using Lipofectamine LTX/PLUS reagents (Invitrogen) in accordance with the manufacturer’s protocol. The transfected Animal Experiments. Female BALB/cAnNCr mice (age 8–14 wk) were used for cells were sorted three times for the top 5% expressing cells by FACS. LMB- all experiments. All mouse experiments followed National Institutes of 100/SS1P–sensitive single clones were then isolated from the population of Health guidelines approved by the Animal Care and Use Committee of the sorted cells. Clone AB1-L9 (5 × 106) was inoculated in BALB/c mice in 100 μL National Cancer Institute (Animal Protocol LMB-071). Mice were injected i.v. of PBS. When tumor volume reached 200 mm3, tumors were excised. with antigens and SVP-R unless described otherwise. Mice were injected Digested tumors were prepared as described previously (47). according to the schedules indicated in each experiment (with RIT injected To make single clones of AB1-L9 cells, digested tumors were diluted 5 min after SVP-R), and plasma samples were collected by mandibular (0.5 cells/100 μL) and aliquoted 100 μL in a 96-well culture dish with selection bleeding. Mouse weight was measured weekly. All mouse studies were reagent. Fifteen single clones were obtained, and the clones with the performed with age-matched control groups. highest GeoMean values were selected. The final clone was injected s.c. in × 6 For tumor experiments, female BALB/c mice were inoculated with 1 10 BALB/c mice; >95% of the tumors were grown in BALB/c mice. AB1-L9 cells (Fig. S6)or1× 106 CT26 cells (American Type Culture Collection) × 6 in RPMI medium in the flank, or 0.5 10 66C14 cells in IMDM medium in the B-Cell ELISpot. BM extracted from the femurs of eight immunized mice was mammary pad. Tumor sizes were measured using a caliper every 2 or 3 d. washed, filtered through a 70-mm mesh, and lazed to eliminate RBCs. Cells Mice with a tumor burden >10% of body weight were euthanized. No an- were resuspended in warm RPMI supplemented with heat-inactivated FCS, imals were excluded from the statistical analysis (41). 1% L-glutamine, and 1% penicillin-streptomycin. PVDF plates (0.45 μm; μ Depletion of Treg cells was performed by i.p. injection of 200 g of anti- μ

Mabtech) were coated with 2 g/mL LMB-100 for 18 h, washed, and blocked MEDICAL SCIENCES mouse CD25 depleting antibody (clone PC61) or isotype control (clone with assay media at 37 °C for 2 h. Six replicas of each BM sample were TNP6A7) (both purchased from BioXcell), as described previously (24). seeded at a concentration of 100,000 cells/well and then incubated for 4 h. Anti–CTLA-4 (Roche IgG2A; clone 9D9) was generously provided by Roche Spots indicating anti–LMB-100 antibody-secreting B cells were detected us- Pharmaceuticals, and anti-OX40 (clone OX-86; InVivoPlus) was purchased ing a capture anti-mouse Ig biotinylated antibody (Mabtech) followed by from BioXcell. Antibodies were diluted in PBS, and 5 mg/kg was injected i.p. ALP and BCIP/NTP substrate (KPL). as in the indicated schedules. Spots were counted by computer-assisted image analysis (ImmunoSpot 5.0; Cellular Technology). Results are shown in SFC/1E6 cells. Cytotoxicity and Neutralization Assay. The KLM1 pancreatic cell line was provided by Dr. U. Rudloff (National Cancer Institute). HAY mesothelioma Flow Cytometry. Spleens were dissected from mice immunized with Alexa cells were provided by the Stehlin Foundation for Cancer Research. Cells Fluor 488-labeled LMB-100, Cy5-labeled SVP-R, or both and also from un- were cultured in RPMI medium supplemented with 10% FCS, 1% L-glutamine, treated mice. Splenocytes were extracted by injecting 3 mL of medium and 1% penicillin-streptomycin. Cells were seeded in 96-well flat-bottom supplemented with Liberase, DNAs, and collagenase (all from Roche) to the plates (5,000 cells/well) for 24 h, then treated with various concentra- spleen, followed by a 10-min incubation at 37 °C. Spleens were minced, tions of LMB-100, SVP-R, and both in four replicates. Cell viability was passed through a 70-mm mesh, and washed, and then RBCs were lysed. All assessed 72 h later using a WST cell viability assay (Dojindo Molecular cells were >90% viable as assessed by trypan blue staining. Cells were fixed, Technologies) in accordance with the manufacturer’s instructions. Color washed, and stained as described previously (48) using the following anti- change was evaluated at optical density (OD) 450 nm. OD reads were bodies, obtained from BioLegend: CD3 (clone 17A2), CD4 (clone GK1.5), CD8 normalized at 0–100% viability, with 100% viability representing no treat- (clone 53–5.8), CD19 (clone 6D5), B220 (clone RA3, 6B2), CD11c (clone N418), ment and 0% representing treatment with staurosporine (Sigma-Aldrich)- IAIE (clone M5/114.15.2), CD11b (clone M1/70,), Ly6G (clone 1A85), and Ly6C positive control. (clone HK1.4). Data were collected on a FACSCanto II flow cytometer (BD Neutralization assays were performed using KLM1 cells as described Bioscience) and analyzed with FlowJo version X (Tree Star). previously (46). Serum samples from 21 mice were diluted 1:50.

ELISA. Statistical Analysis. Statistical analysis and graphing were done using GraphPad Total Ig anti LMB-100 and anti-ovalbumin antibodies. Plasma samples were col- Prism software. Multiple comparisons of parametric variables were performed – lected into heparinized tubes, spun, and frozen until titer evaluation. Total Ig by one-way ANOVA. The Mann Whitney U test was used for comparisons of ’ ’ anti–LMB-100 and anti-ovalbumin antibodies were measured by direct ELISA two nonparametric variables, and Friedman stestwithDunns multiple com- as described previously (46). parisons was used for comparisons of multiple nonparametric variables. Isotype determination of anti–LMB-100 and total Ig. ELISA plates (Thermo Fisher Scientific) were coated with 2 μg/mL LMB-100 or polyclonal donkey anti- ACKNOWLEDGMENTS. We thank Dr. Chin-Hsien Tai for the structural model mouse IgG (Jackson ImmunoResearch Laboratories). Plates were blocked, of LMB-100; Drs. Thomas Waldmann, Jay Berzofsky, Jonathan Ashwell, and Dan Fowler for their helpful advice; Ms. Deborah Glass for assay support; and serial dilutions of plasma were incubated for 1 h. Captured antibodies in and the Selecta formulation and bioanalytical team for preparing the SVP-R. the plasma were bound by goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, and This work was funded in part by the Intramural Research Program of the IgM isotyping kits at dilutions of 1:3,000, 1:4,000, 1:4,000, 1:3,000, and Center for Cancer Research, National Cancer Institute; by the Mesothelioma 1:16,000, respectively (Sigma-Aldrich). Anti-goat IgG (H + L) HRP (1:15,000; Applied Research Foundation; and by a Collaborative Research and Devel- Jackson ImmunoResearch Laboratories) was used for detection. opment Agreement (02991) with Selecta Biosciences.

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