L-Pampo™, a Novel TLR2/3 Agonist, Acts as a Potent Cancer Vaccine Adjuvant by Activating Draining Lymph Node Dendritic Cells.

TLR agonists have emerged as an efficient cancer vaccine adjuvant system that induces robust immune responses. L-pampo™, a proprietary vaccine adjuvant of TLR2 and TLR3 agonists, promotes strong humoral and cellular immune responses against infectious diseases. In this study, we demonstrate that vaccines formulated with L-pampo™ affect the recruitment and activation of dendritic cells (DCs) in draining lymph nodes (dLNs) and leading to antigen-specific T-cell responses and anti-tumor efficacy. We analyzed DC maturation and T-cell proliferation using flow cytometry and ELISA. We determined the effect of L-pampo™ on DCs in dLNs and antigen-specific T-cell responses using flow cytometric analysis and the ELISPOT assay. We employed murine tumor models and analyzed the anti-tumor effect of L-pampo™. We found that L-pampo™ directly enhanced the maturation and cytokine production of DCs and, consequently, T-cell proliferation. OVA or OVA peptide formulated with L-pampo™ promoted DC migration into dLNs and increased activation markers and specific DC subsets within dLNs. In addition, vaccines admixed with L-pampo™ promoted antigen-specific T-cell responses and anti-tumor efficacy. Moreover, the combination of L-pampo™ with an immune checkpoint inhibitor synergistically improved the anti-tumor effect. This study suggests that L-pampo™ can be a potent cancer vaccine adjuvant and a suitable candidate for combination immunotherapy.

Intratumoral immunotherapy using a TLR2/3 agonist, L-pampo, induces robust antitumor immune responses and enhances immune checkpoint blockade.

BACKGROUND: Toll-like receptors (TLRs) are critical innate immune sensors that elicit antitumor immune responses in cancer immunotherapy. Although a few TLR agonists have been approved for the treatment of patients with early-stage superficial cancers, their therapeutic efficacy is limited in patient with advanced invasive cancers. Here, we identified the therapeutic role of a TLR2/3 agonist, L-pampo (LP), which promotes antitumor immunity and enhances the immune checkpoint blockade. METHODS: We generated LP by combining a TLR2 agonist, Pam3CSK4, with a TLR3 agonist, Poly (I:C). Immune responses to stimulation with various TLR agonists were compared. Tumor-bearing mice were intratumorally treated with LP, and their tumor sizes were measured. The antitumor effects of LP treatment were determined using flow cytometry, multiplexed imaging, and NanoString nCounter immune profiling. The immunotherapeutic potential of LP in combination with α-programmed cell death protein-1 (PD-1) or α-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) was evaluated in syngeneic MC38 colon cancer and B16F10 melanoma. RESULTS: The LP treatment induced a potent activation of T helper 1 (Th1) and 2 (Th2)-mediated immunity, tumor cell apoptosis, and immunogenic tumor cell death. Intratumoral LP treatment effectively inhibited tumor progression by activating tumor-specific T cell immunity. LP-induced immune responses were mediated by CD8+ T cells and interferon-γ, but not by CD4+ T cells and CD25+ T cells. LP simultaneously activated TLR2 and TLR3 signaling, thereby extensively changing the immune-related gene signatures within the tumor microenvironment (TME). Moreover, intratumoral LP treatment led to systemic abscopal antitumor effects in non-injected distant tumors. Notably, LP treatment combined with ɑPD-1 and ɑCTLA-4 further enhanced the efficacy of monotherapy, resulting in complete tumor regression and prolonged overall survival. Furthermore, LP-based combination immunotherapy elicited durable antitumor immunity with tumor-specific immune memory in colon cancer and melanoma. CONCLUSIONS: Our study demonstrated that intratumoral LP treatment improves the innate and adaptive antitumor immunity within the TME and enhances the efficacy of αPD-1 and αCTLA-4 immune checkpoint blockade.

Non-genetic engineering of cytotoxic T cells to target IL-4 receptor enhances tumor homing and therapeutic efficacy against melanoma.

Adoptive transfer of cytotoxic T lymphocytes (CTLs) has been used as an immunotherapy in melanoma. However, the tumor homing and therapeutic efficacy of transferred CTLs against melanoma remain unsatisfactory. Interleukin-4 receptor (IL-4R) is commonly up-regulated in tumors including melanoma. Here, we studied whether IL-4R-targeted CTLs exhibit enhanced tumor homing and therapeutic efficacy against melanoma. CTLs isolated from mice bearing melanomas were non-genetically engineered with IL4RPep-1, an IL-4R-binding peptide, using a membrane anchor composed of dioleylphosphatidylethanolamine. Compared to control CTLs, IL-4R-targeted CTLs showed higher binding to melanoma cells and in vivo tumor homing. They also exerted a more rapid and robust effector response, including increased cytokine secretion and cytotoxicity against melanoma cells and enhanced reprogramming of M2-type macrophages to M1-type macrophages. Moreover, IL-4R-targeted CTLs efficiently inhibited melanoma growth and reversed the immunosuppressive tumor microenvironment. These results suggest that non-genetically engineered CTLs targeting IL-4R have potential as an adoptive T cell therapy against melanoma.

Use of pre-S protein-containing hepatitis B virus surface antigens and a powerful adjuvant to develop an immune therapy for chronic hepatitis B virus infection.

A hepatitis B virus (HBV) vaccine has been developed using a new adjuvant and HBV surface antigens produced from a CHO cell line. The purified HBV surface antigens are composed of L protein, M protein, and S protein in a mixture of 20- and 40-nm-diameter particles and filamentous forms. This HBV surface antigen, formulated with L-pampo, a proprietary adjuvant, induced 10 times more antibody than the same antigen with alum and was capable of inducing strong immune responses in three different HBV transgenic mice. In spite of the presence of a large amount of HBV antigen in the blood, no antibody against HBV surface antigen was normally detected in these transgenic mice. After immunization, the HBV antigen was also cleared from the blood.

Adjuvant effect of CIA07, a combination of Escherichia coli DNA fragments and modified lipopolysaccharides, on the immune response to hepatitis B virus surface antigen.

CIA07 is an immunostimulatory agent composed of bacterial DNA fragments and modified lipopolysaccharide, which has antitumor activity against bladder cancer in mice. In this study, the adjuvant activity of CIA07 was evaluated using hepatitis B virus surface antigen (HBsAg) as the immunogen. Mice were immunized intramuscularly three times at 1-week intervals with HBsAg alone or in combination with alum, bacterial DNA fragments, modified lipopolysaccharide, CIA07 or CpG1826, and immune responses were assessed. At 1 week after the final injection, the HBsAg-specific total serum IgG antibody titer in CIA07-treated mice was 14 times higher than that in animals administered antigen alone, six times higher than in mice given alum or bacterial DNA fragments and twice as high as those treated with modified lipopolysaccharide or CpG1826, and remained maximal until 8 weeks postimmunization. Animals receiving antigen alone or plus alum displayed barely detectable HBsAg-specific serum IgG2a antibody responses. However, coadministration of CIA07 with antigen led to markedly enhanced serum IgG2a antibody titer and IFN-gamma(+) production in splenocytes, indicating that CIA07 effectively induces Th1-type immune responses. In addition, the number of HBsAg-specific CD8(+) T cells in peripheral blood mononuclear cells was elevated in CIA07-treated mice. These data clearly demonstrate that CIA07 is able to induce both cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant in therapeutic vaccines for hepatitis B virus infections.

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM.

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, Kd, are 0.89 and 0.18 microM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.

Primary malignant fibrous histiocytoma of the kidney.

Primary renal malignant fibrous histiocytoma is a rare tumor of the kidney. It is clinically and radiologically indistinguishable from a renal cell carcinoma. Even following radical surgery, the tumor shows a strong predilection for local recurrence and the prognosis is generally poor. We report on a 32-year-old man with malignant fibrous histiocytoma of the kidney who remained free of recurrence for 1 year after radical nephrectomy.