Combined Vaccination with B Cell Peptides Targeting Her-2/neu and Immune Checkpoints as Emerging Treatment Option in Cancer.

The application of monoclonal antibodies (mAbs), targeting tumor-associated (TAAs) or tumor-specific antigens or immune checkpoints (ICs), has shown tremendous success in cancer therapy. However, the application of mAbs suffers from a series of limitations, including the necessity of frequent administration, the limited duration of clinical response and the emergence of frequently pronounced immune-related adverse events. However, the introduction of mAbs has also resulted in a multitude of novel developments for the treatment of cancers, including vaccinations against various tumor cell-associated epitopes. Here, we reviewed recent clinical trials involving combination therapies with mAbs targeting the PD-1/PD-L1 axis and Her-2/neu, which was chosen as a paradigm for a clinically highly relevant TAA. Our recent findings from murine immunizations against the PD-1 pathway and Her-2/neu with peptides representing the mimotopes/B cell peptides of therapeutic antibodies targeting these molecules are an important focus of the present review. Moreover, concerns regarding the safety of vaccination approaches targeting PD-1, in the context of the continuing immune response, as a result of induced immunological memory, are also addressed. Hence, we describe a new frontier of cancer treatment by active immunization using combined mimotopes/B cell peptides aimed at various targets relevant to cancer biology.

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model.

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4+ and CD8+ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.

Clinical and Immunologic Responses to a B-Cell Epitope Vaccine in Patients with HER2/neu-Overexpressing Advanced Gastric Cancer-Results from Phase Ib Trial IMU.ACS.001.

PURPOSE: HER2/neu is overexpressed in up to 30% of gastroesophageal adenocarcinomas (GEA) and linked to poor prognosis. Recombinant mAbs to treat HER2/neu-overexpressing cancers are effective with limitations, including resistance and toxicity. Therefore, we developed a therapeutic B-cell epitope vaccine (IMU-131/HER-Vaxx) consisting of three fused B-cell epitopes from the HER2/neu extracellular domain coupled to CRM197 and adjuvanted with Montanide. This phase Ib study aimed to evaluate the optimal/safe dose leading to immunogenicity and clinical responses (https//clinicaltrials.gov/ct2/show/NCT02795988). PATIENTS AND METHODS: A total of 14 patients with HER2/neu-overexpressing GEA were enrolled, and dose escalation (10, 30, 50 µg) was performed in three cohorts (C). Immunogenicity was evaluated by HER2-specific Abs and cellular responses, clinical responses by CT scans according to RECIST version 1.1. RESULTS: IMU-131 was safe without vaccine-related significant local/systemic reactions or serious adverse events. A total of 11 of 14 patients were evaluable for changes in tumor size and vaccine-specific immune responses. One patient showed complete, 5 partial responses, and 4 stable diseases as their best response. HER2-specific IgG levels were dose dependent. In contrast to patients in C1 and C2, all patients in C3 mounted substantial HER2-specific Ab levels. In addition, cellular vaccine responses, such as Th1-biased cytokine ratios and reduced regulatory T cell numbers, were generated. Progression-free survival was prolonged in C3, correlating with the vaccine-specific humoral and cellular responses. CONCLUSIONS: IMU-131 was well tolerated and safe. The induced HER2-specific Abs and cellular responses were dose dependent and correlated with clinical responses. The highest dose (50 µg) was recommended for further evaluation in a phase II trial, with chemotherapy + IMU-131 or chemotherapy alone, which is currently ongoing.

A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors.

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment.

Activity of methylgerambullin from Glycosmis species (Rutaceae) against Entamoeba histolytica and Giardia duodenalis in vitro.

Entamoeba histolytica and Giardia duodenalis are widespread intestinal protozoan parasites which both spread via cysts that have to be ingested to infect a new host. Their environment, the small intestine for G. duodenalis and the colon for E. histolytica, contains only very limited amounts of oxygen, so both parasites generate energy by fermentation and substrate level phosphorylation rather than by oxidative phosphorylation. They both contain reducing agents able to reduce and activate nitroimidazole drugs such as metronidazole which is the gold standard drug to treat Entamoeba or Giardia infections. Although metronidazole works well in the majority of cases, it has a number of drawbacks. In animal models, the drug has carcinogenic activity, and concerns about a possible teratogenic activity remain. In addition, the treatment of G. duodenalis infections is hampered by emerging metronidazole resistance. Plant-derived drugs play a dominant role in human medicine, therefore we tested the activity of 14 isolated plant compounds belonging to seven different classes in vitro against both parasites. The tests were performed in a new setting in microtiter plates under anaerobic conditions. The compound with the highest activity was methylgerambullin, a sulphur-containing amide found in Glycosmis species of the family Rutaceae with an EC50 of 14.5 µM (6.08 µg/ml) after 24 h treatment for E. histolytica and 14.6 µM (6.14 µg/ml) for G. duodenalis. The compound was successfully synthesised in the laboratory which opens the door for the generation of new derivatives with higher activity.

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

INTRODUCTION: Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract. METHODS: First, we tested OLA in vitro. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells. RESULTS: Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4+CD25highFoxp3+ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses. CONCLUSION: Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.