Anti-PD-1 synergizes with cyclophosphamide to induce potent anti-tumor vaccine effects through novel mechanisms

Programmed death-1 receptor (PD-1) is expressed on T cells following TCR activation. Binding of this receptor to its cognate ligands, programmed death ligand (PDL)-1 and PDL-2, down-regulates signals by the TCR, promoting T-cell anergy and apoptosis, thus leading to immune suppression. Here, we find that using an anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic antigen-specific immune responses and reveals novel activities of each agent in this combination. This strategy led to complete regression of established tumors in a significant percentage of treated animals, with survival prolongation. We show for the first time that combining CT-011 and CPM significantly increases the number of vaccine-induced tumor-infiltrating CD8(+) T cells, with simultaneous decrease in infiltrating Treg cells. Interestingly, we find that CT-011 prolongs Treg-cell inhibition induced by CPM, leading to a sustainable significant synergistic decrease of splenic and tumor-infiltrated Treg cells. Surprisingly, we find that the anti-tumor effect elicited by the combination of CT-011 and CPM is dependent on both CD8(+) and CD4(+) T-cell responses, although the antigen we used is a class I MHC-restricted peptide. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.     

Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch

Beta-amyloid (Abeta) peptide has been proposed to be a causal factor in Alzheimer's disease (AD). Currently being investigated, active and passive Abeta-immunotherapy significantly reduce Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Abeta42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Abeta antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Abeta-specific autoimmune Th1 cells. Thus, successful Abeta vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Abeta(1-15) and foreign T cell epitope PADRE (PADRE-Abeta(1-15)-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Abeta antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Abeta antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Abeta antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Abeta antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.     

Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice

Background

New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Aβ₂₈ conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576).

Methods

Mannan was purified, activated and chemically conjugated to Aβ₂₈ peptide. Humoral immune responses induced by the immunization of mice with mannan-Aβ₂₈ conjugate were analyzed using a standard ELISA. Aβ₄₂ and Aβ₄₀ amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry.

Results

Immunizations with low doses of mannan-Aβ₂₈ induced potent and long-lasting anti-Aβ humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Aβ antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Aβ₂₈ prevented Aβ plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Aβ₂₈ showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups.

Conclusion

Mannan conjugated to Aβ₂₈ provided sufficient adjuvant activity to induce potent anti-Aβ antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Aβ self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Aβ₂₈ immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Aβ antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.     

DNA epitope vaccine containing complement component C3d enhances anti-amyloid-beta antibody production and polarizes the immune response towards a Th2 phenotype

We have engineered a DNA epitope vaccine that expresses 3 self-B cell epitopes of Abeta(42) (3Abeta(1-11)), a non-self T helper (Th) cell epitope (PADRE), and 3 copies of C3d (3C3d), a component of complement as a molecular adjuvant, designed to safely reduce CNS Abeta. Immunization of mice with 3Abeta(1-11)-PADRE epitope vaccine alone generated only moderate levels of anti-Abeta antibodies and a pro-inflammatory T helper (Th1 phenotype) cellular immune response. However, the addition of 3C3d to the vaccine construct significantly augmented the anti-Abeta humoral immune response and, importantly, shifted the cellular immune response towards the potentially safer anti-inflammatory Th2 phenotype.     

Detection of the active components of calf thymus nuclear proteins (TNP), histones that are binding with high affinity to HIV-1 envelope proteins and CD4 molecules

VGV-1, a clinical-grade formulation of bovine thymus nuclear protein (TNP) has been demonstrated to possess anti-viral activity in HIV-1 patients in five clinical trials, one of which was placebo controlled double-blinded. However, to date molecular mechanisms remain to be identified. Using surface plasmon resonance we observed TNP components bind with high affinity to HIV-1 proteins involved in viral entry, gp41 and pg120, as well as the T cell HIV-1 receptor CD4. To identify protein components of TNP, gel electrophoresis was performed followed by tandem mass spectrometry (MS/MS). Searching of bovine protein databases revealed the presence of numerous histones. Further analysis of TNP by immunoaffinity chromatography using gp120 and CD4 molecules as targets followed by gel electrophoresis and MS/MS analysis confirmed these data, demonstrating that H1.1, H2B, H4, and H2A histones are the active component of TNP that bind to HIV envelop glycoprotein and its receptor. To conclusively demonstrate binding of histones to target proteins, we repeated the surface plasmon resonance experiments using commercially available bovine histones and demonstrated high-affinity interaction of histones with gp120, and CD4. The binding of histone proteins to CD4, as well as viral molecules has profound implications for basic understanding of immune functions as well as a possible mechanism of VGV-1 activity in AIDS patients.