In Situ Vaccination with IL-12Fc and TLR Agonist - a Crucial Role for B Cells in Generating Anti-Tumor T Cell Immunity

Interleukin 12 (IL-12) is a potent proinflammatory cytokine that plays a central role in regulating both innate and adaptive immune responses. This cytokine has been tested as a cancer immunotherapy agent based on its impressive anti-tumor effects in animal models. Clinically, systemic delivery of IL-12 incurred dose-limiting toxicities because of its pharmaco-kinetic extremes of peaks and troughs, a result of its short serum half-life. Here, we evaluated mDF6006, a mouse IL-12 Fc-fusion protein which was designed to have a prolonged half-life with equivalent potency to recombinant mouse IL-12. We hypothesized that local intra-tumoral (IT) delivery of low doses IL-12Fc would lead to immune-therapeutic potential. To screen candidates for in situ vaccination, we have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting immune response is detected by the regression of the distant, untreated tumor. Using this assay for abscopal therapeutic effects we have tested a variety of immune stimulatory ligands and antibodies against immune checkpoints and T cell co-stimulatory targets, alone and in multiple combinations. Among these, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 ligand, and IL-12Fc has provided the most impressive results. TLRs, expressed in antigen-presenting cells and are components of the innate immune system that recognize molecular patterns on bacterial, fungal, or viral pathogens. In a mouse model of lymphoma, IT injection of CpG and IL-12Fc, combined to cause tumor eradication at both the injected site and the untreated tumor. All these results were dependent on T cells in the animal. During this therapy we observed a significant increase in activated B cells within the treated tumor and especially in its draining lymph nodes (dLN). This B cell stimulation was contributed by both the CpG and the IL12-Fc. Surprisingly, depletion of the animals of B cells by an anti-CD20 antibody completely ablated the abscopal therapeutic effect. B cells function as antigen-presenting cells, secrete cytokines and are precursors of antibody producing plasma cells. To evaluate which of these roles B cells play in the therapeutic efficacy of CpG and IL-12Fc we isolated B cells from the LN of the treated mice and co-cultured them with T cells. These B cells activated T cells in an antigen-specific manner that was MHC dependent. No such T cell activation occurred when MHC molecules were blocked with anti-MHC antibodies or when the cells were physically separated by a semi-permeable membrane. Thus, local CpG and IL12-Fc directly activated B cells to present tumor antigens to specific T cells in the tumor microenvironment, resulting ultimately in an abscopal tumor response. IL-12Fc and CpG are both currently in phase-1/2 trials as single agents and in combinations with checkpoint antibodies. Our results provide strong rationale for using low doses of IL-12Fc with CpG as an in situ therapeutic vaccine for lymphoma. Disclosures Levy: Virsti: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

In Situ Vaccination with Combination of Class B and Class C Toll-like Receptor 9 Agonist CpG Immune Adjuvant Nanoparticles to Induce a Systemic Anti-Lymphoma Response

Introduction: Treatment of relapsed and refractory aggressive B cell lymphomas is challenging, especially after autologous stem cell transplant or chimeric antigen receptor T cell therapy. CpG oligodeoxynucleotides (CpGs) mimic bacterial DNA and bind to toll-like receptor 9 (TLR9). TLR9 is expressed in the endosomes of innate immune cells and B lymphocytes as well as B cell lymphomas. CpG binding to TLR9 in these cells leads to a pro-inflammatory response and induces apoptosis of lymphoma cells by altering NF-kB activation. However, clinical utility of CpGs is limited due to difficulties delivering the DNA directly to the innate immune cells and lymphoma cells, and these DNA strands are easily degraded in biological fluids. Therefore, we designed a nano-carrier to deliver two classes of CpGs to improve clinical efficacy of CpGs in treating lymphoma. Class B CpGs (B-CpGs) mainly stimulate B cells while class C CpGs (C-CpGs) act on both B cells and plasmacytoid dendritic cells. Though C-CpGs are the focus of current clinical trials, B-CpGs may have a special a role in the treatment of lymphoma due to their more potent, direct cytotoxic effect on the malignant lymphoma cells. Here, we evaluated the anti-lymphoma effect of the combination of B-CpG nanoparticles (BNP) and C-CpG nanoparticles (CNP) by optimizing lymphoma cell death and immune stimulation. Methods: BNP and CNP were synthesized by surface-functionalizing gold nanoparticles with modified B-CpGs and C-CpGs, followed by purification via centrifugation. We used a dual flank tumor murine lymphoma model to evaluate the local and systemic anti-lymphoma efficacy of the CpG NPs and screen for the most effective formulation. A20 lymphoma tumors were implanted on both flanks of Balb/c mice. When the tumors were 5-7mm in diameter, various treatments were injected intratumorally on days 1, 4, and 8 on the side with the larger tumor. Treatment groups include PBS, B-CpG, C-CpG, B+C-CpG, BNP, CNP, BNP+CNP. Free CpGs were injected at 50ug/ml in 50ul (2.5ug CpG per injection) and nanoparticles at 100nM (equivalent to 50ug/ml CpGs) in 50ul. Tumor growth were measured every other day. Event is defined as tumor volume > 3cm 3. Second, we used an advanced disease lymphoma model to evaluate intravenous delivery of combination NPs. We implanted A20 lymphoma cells on a flank tumor (on day -14), then we injected A20 cells intravenously to mimic advanced disease (on day -7). We treated the mice with intravenous (tail vein) injections of PBS, B+C-CpG, or BNP+CNP on days 1, 4, and 8. Results: We found that the combination of BNP and CNPs had the most significant reduction in total tumor volume (TTV) (treated + untreated tumor size) (393mm 3 on day 22). The PBS group demonstrated rapid tumor growth (TTV 3516mm 3). The experimental treatment groups, B-CpG (TTV 949mm 3),, C-CpG (TTV 2230mm 3), B+C-CpGs (TTV 1680mm 3), BNP (TTV 1224mm 3), and CNP (1894mm 3) had similar TTV growth curves. Separating the treated tumor and untreated tumor growth curves, we found that B-CpG and BNP along with combination of BNP+CNP had the best tumor suppression on the treated side. This is not surprising as B-CpG sequences are more directly cytotoxic to lymphoma cells versus C-CpGs. Interestingly, combination nanoparticle treatment (BNP+CNP) was the only group that had significantly reduced tumor growth of the untreated tumor compared with all other conditions (p<0.03) where no statistical difference was measured when the PBS treatment group with the other groups. The BNP+CNP treatment group also had significantly prolonged event free survival compared with all other treatment groups. Furthermore, in the advanced disease model, when BNP and CNP were both injected intravenously, the flank tumor was notably smaller (149mm 3) compared with the other two groups (B+C-CpG 1310mm 3 or PBS 1555mm 3). Importantly, there were no visible lymphoma nodules on liver in the nanoparticle treatment group while there are obvious nodules for both the PBS and the free CpG groups. Conclusions: Overall, the combination of class B and class C CpG nanoparticles can generate a strong in situ anti-lymphoma vaccination response, presumably through the combination of by combining the superior cytotoxicity of BNPs with the enhanced immune stimulation effect of the CNPs. Further evaluation of the combined mechanism of action of this dual CpG nanoparticle approach and toxicity studies are in progress. Disclosures Thaxton: Zylem biosciences: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Gordon: Zylem Biosciences: Patents & Royalties: Patents, No royalties; Bristol Myers Squibb: Honoraria, Research Funding.

Correspondence between formulations of Avrami and Gompertz equations for untreated tumor growth kinetics

The classical and modified equations of Kolmogorov-Johnson-Mehl-Avrami are compared with the equations of conventional Gompertz andMontijano-Bergues-Bory-Gompertz, in the frame of growth kinetics of tumors. For this, different analytical and numerical criteria are usedto demonstrate the similarity between them, in particular the distance of Hausdorff. The results show that these equations are similar fromthe mathematical point of view and the parameters of the Gompertz equation are explicitly related to those of the Avrami equation. It isconcluded that Modified Kolmogorov-Johnson-Mehl-Avrami and Montijano-Bergues-Bory-Gompertz equations can be used to describe thegrowth kinetics of unperturbed tumors.

Hampering brain tumor proliferation and migration using peptide nanofiber:siPLK1/MMP2 complexes

Aim: To develop a nonviral tool for the delivery of siRNA to brain tumor cells using peptide nanofibers (PNFs). Materials & methods: Uptake of PNFs was evaluated by confocal microscopy and flow cytometry. Gene silencing was determined by RT-qPCR and cell invasion assay. Results: PNFs enter phagocytic (BV-2) and nonphagocytic (U-87 MG) cells via endocytosis and passive translocation. si PLK1 delivered using PNFs reduced the expression of polo-like kinase 1 mRNA and induced cell death in a panel of immortalized and glioblastoma-derived stem cells. Moreover, targeting MMP2 using PNF:si MMP2 reduced the invasion capacity of U-87 MG cells. We show that stereotactic intra-tumoral administration of PNF:si PLK1 significantly extends the survival of tumor bearing mice comparing with the untreated tumor bearing animals. Conclusion: Our results suggest that this nanomedicine-based RNA interference approach deserves further investigation as a potential brain tumor therapeutic tool.

In Situ Vaccination with Cpg and Anti-OX40 Antibody: Preclinical Optimization for Clinical Translation

In-situ vaccination is a local intervention in which immune enhancing agents are injected locally into one site of tumor, triggering a T cell immune response locally that then travels to attack cancer throughout the body. We have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting systemic immune response, if any, is detected by the regression of the distant, untreated tumor. In this test for abscopal therapeutic effects, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG) - a TLR9 ligand - and agonist anti-OX40 antibody has provided impressive results. This combination lead to durable disease control and long-term treatment-free survival in multiple mouse models of cancer. CpG induced myeloid cells to secrete cytokines, which subsequently induced OX40 expression on T cells. Thus, we hypothesized that administration sequence and timing may affect the anti-tumor responses of in-situ vaccination. In order to screen for the best sequence and timing we implanted A20 lymphoma tumors bilaterally in opposite sides of the abdomen of Balb/C mice. After tumors were established, one tumor was injected at the selected sequence and timing with the test agents and the resulting immune response was monitored by the measuring growth of the distant, untreated tumor. As opposed to our usual schedule of three injections, even a single injection of CpG (50µg) and anti-OX40 (8µg) resulted in a fully protective systemic immune response. In addition, the cured animals were protected from re-challenge with the same A20 tumor but not unrelated tumors. Decreasing the dose even further to 10µg CpG and 1µg anti-OX40 partially preserved the therapeutic response with a long-term survival of 60%. Concurrent administration of CpG and anti-OX40 resulted in eradication of both local and distant disease. Sequential administration of CpG followed by anti-OX40 preserved the therapeutic efficacy. However, the opposite order of anti-OX40 followed by CpG significantly attenuated the therapeutic effect. While CpG followed by a 24- or 48-hour-delayed anti-OX40 treatment preserved the therapeutic efficacy, a 72h delay in anti-OX40 administration resulted in reduced therapeutic effect. These data demonstrate the importance of the administration sequence for fully protective anti-tumor immune responses. Our data suggest that the anti-OX40 antibody should be administered at the same time as CpG or with only a slight delay but not in the reverse order. Low-dose radiotherapy (2×2 Gy) is an effective treatment for patients with indolent non-Hodgkin's lymphoma. This treatment results in high response rates at the treated site. Since immune infiltrating cells in the tumor microenvironment are essential for in situ vaccination of CpG and anti-OX40 we aimed to assess the effect of adding radiation in our pre-clinical models of lymphoma. We found that the addition of 2x2 Gy radiation did not interfere with the induction of a protective immune response by of CpG and anti-OX40. Given the effectiveness of low dose radiotherapy for local control and its lack of interference with the immune related abscopal response in the pre-clinical model, we are including radiation in our current clinical trials. In addition, we have incorporated our findings in the preclinical model regarding dosing and scheduling of CpG and anti-OX40 antibody to the design of our current in situ vaccination lymphoma clinical trial. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

The influence of biliary drainage in patients with advanced pancreatic cancer receiving FOLFIRINOX.

378 Background: FOLFIRINOX (FFX) is a standard of care for patients (pts) with advanced pancreatic cancer (APC). In the original FFX report by Conroy (NEJM, 2011), pts with a biliary drainage were limited and the occurrence of cholangitis was not reported. In practice, however, we experience that a certain fraction of APC pts needs a biliary drainage and some of them have an elevated risk of cholangitis or febrile neutropenia (FN) during the course of FFX. We evaluated the influence of biliary drainage on the efficacy and safety of FFX. Methods: We used individual data from nationwide survey of FFX (JASPAC06). The JASPAC06 was a prospective registry of pts with FFX treated in clinical practice and enrolled 399 pts between December 2013 and November 2014 from 27 centers in Japan. We evaluated the associations of OS and PFS, as well as the frequencies of cholangitis and FN, with the use of biliary drainage. We excluded resected cases because an operative method with choledochojejunostomy was unknown. Results: Of 399 pts in the JASPAC06, 319 were eligible for this analysis and 80 resected cases were excluded. The use of biliary drainage was seen in 28% of pts (mainly bile duct stent inserted); primary dose reduction (modified FFX), 67%; previously untreated tumor, 77%; distant metastases, 76%; pancreas head, 47%. The main results are shown in the table. In summary, cholangitis was more frequent in pts with biliary drainage. Grade 3 or higher FN as well as CRP elevation was observed more frequently in pts with biliary drainage. There was no difference in PFS (median PFS, 7.3 vs. 6.5 mo; logrank, p=0.24) and OS (median OS, 12.3 vs. 12.2 mo; p=0.86) between the two groups. Conclusions: Our observation that the frequency of FN and CRP elevation was significantly higher in pts with biliary drainage indicates a higher risk of infection during the FFX treatment by using biliary drainage.[Table: see text]

Combination Lymphoma Immunotherapy Using Checkpoint Blockade and Intratumoral Virus-like Particles Containing CpG TLR9 Agonist

Checkpoint blockade of immune inhibitory pathways is an exciting therapeutic approach for the treatment of a variety of cancers and has become a standard of care for melanoma and other malignancies. Preliminary results suggest anti-PD-1 has modest single agent activity in patients with non-Hodgkin lymphomas, however there remains considerable room for treatment optimization and improvement through the use of unique immunotherapeutic combinations. We explored one such combination of PD-1 checkpoint blockade and CMP-001, a virus-like particle (VLP) containing a novel immunostimulatory CpG oligodeoxynucleotide (ODN) TLR9 agonist. Clinical grade CMP-001 is available and has been evaluated in over 700 subjects in non-cancer trials. In both preclinical and clinical studies, CMP-001 stimulates a strong and prolonged local Th1 cytokine response designed to enhance antigen presentation. Thus, with intratumoral injection, CMP-001 could augment the development of a tumor-specific T cell response that can be maintained by anti-PD-1 therapy. Syngeneic, immunocompetent mice were inoculated on both the left and right flanks subcutaneously with lymphoma (either A20 B cell lymphoma or E.G7-OVA T cell lymphoma). Anti-PD-1, or isotype control, was delivered intraperitoneally (i.p.) starting one week after tumor challenge and continued twice weekly. CMP-001, or saline control, was delivered intratumorally (i.t.) into the tumor on one flank also starting one week after tumor challenge for a total of 3 doses. Tumor growth and survival was followed. This experimental design allowed us to assess the local (treated tumor) and abscopal (untreated tumor) effect of therapy in two different lymphoma models. Intratumoral CMP-001 enhanced survival, and reduced tumor growth of both the treated and untreated tumors in the A20 lymphoma model. Each of these effects was enhanced with the addition of anti-PD-1 therapy (Figure 1). The anti-tumor effect on both the treated and untreated tumor was lost with depletion of either CD4 or CD8+ T cells demonstrating both cell populations contributed to the therapeutic effect. In the E.G7 model, CMP-001 treatment reduced growth of the local tumor - an effect that was modestly enhanced by anti-PD-1 therapy and dependent on CD8+ T cells. There was no significant response to therapy observed in the untreated E.G7 tumor, perhaps due to the rapid growth of the tumor before an abscopal immune response could develop. We conclude the combination of systemic anti-PD-1 and intratumoral injection of CMP-001 can enhance development of a systemic anti-tumor T cell response and is a promising immunotherapy combination worthy of clinical evaluation. A Phase 1b clinical trial of the combination of anti-PD-1 and CMP-001 is underway in advanced melanoma, and a Phase 1 clinical trial of the same combination is planned in lymphoma, based in part on these results. Figure 1 Figure 1. Disclosures Krieg: Checkmate Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weiner:Checkmate Pharmaceuticals: Consultancy, Research Funding.

Enhanced antitumor activity of cucurbitacin B combined with cerulenin in osteosarcoma

<p class="Abstract">In the present study, the effect of cucurbitacin B and cerulenin combination on osteosarcoma was investigated to develop an effective treatment regimen. The IC₅₀values of cerulenin and cucurbitacin B combination in the proportion of 1:1, 1:2 and 2:1 were 3.5, 7.2 and 8.6 μg/mL, respectively. The combination index (CI) values of &lt;0.95 for inhibition of growth and &lt;0.93 for inhibition of SENP5 expression clearly indicated synergism between the two. The Q-value for combination of 1 μg/mL cerulenin and 1 μg/mL cucurbitacin B was 1.2. AI calculated for tumor tissues treated with a combination of cucurbitacin B and cerulenin (26.2 ± 8.4%) was higher than that of the tissues treated separately with cucurbitacin B (14.5 ± 6.8%) or cerulenin (12.6 ± 7.5%). The AI for untreated tumor tissues was 4.2 ± 1.5%. Thus, the combination of cucurbitacin B and cerulenin can be a promising regimen in the treatment of osteosarcoma.</p>