N-dihydrogalactochitosan serves as an effective mucosal adjuvant for intranasal vaccine in combination with recombinant viral proteins against respiratory infection.

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. STATEMENT OF SIGNIFICANCE: We demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.

Single-cell RNA sequencing reveals localized tumour ablation and intratumoural immunostimulant delivery potentiate T cell mediated tumour killing.

BACKGROUND: Metastatic breast cancer poses great challenge in cancer treatment. N-dihydrogalactochitosan (GC) is a novel immunoadjuvant that stimulates systemic immune responses when administered intratumourally following local tumour ablation. A combination of photothermal therapy (PTT) and GC, referred to as localized ablative immunotherapy (LAIT), extended animal survival and generates an activated B cell phenotype in MMTV-PyMT mouse mammary tumour microenvironment (TME). However, how T cell populations respond to LAIT remains to be elucidated. METHODS: Using depletion antibodies, we studied the contributions of CD8+ and CD4+ T cells to the therapeutic effect of LAIT. Using single-cell RNA-sequencing (scRNAseq), we analysed tumour-infiltrating T cell heterogeneity and dissected their transcriptomes upon treatments of PTT, GC, and LAIT (PTT+GC). RESULTS: Loss of CD8+ T cells after LAIT abrogated the therapeutic benefits of LAIT. Ten days after treatment, proportions of CD8+ and CD4+ T cells in untreated TME were 19.2% and 23.0%, respectively. Upon LAIT, both proportions were increased to 25.5% and 36.2%, respectively. In particular, LAIT increased the proportions of naïve and memory cells from a resting state to an activated state. LAIT consistently induced the expression of co-stimulatory molecules, type I IFN responsive genes, and a series of antitumor cytokines, Ifng, Tnf, Il1, and Il17 in CD8+ and CD4+ T cells. LAIT also induced immune checkpoints Pdcd1, Ctla4, and Lag3 expression, consistent with T cell activation. Relevant to clinical translation, LAIT also upregulated genes in CD8+ and CD4+ T cells that positively correlated with extended survival of breast cancer patients. CONCLUSIONS: Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumour immunity.

Novel Immune Stimulant Amplifies Direct Tumoricidal Effect of Cancer Ablation Therapies and Their Systemic Antitumor Immune Efficacy.

Ablation therapies have emerged as an effective tool for destroying cancerous tissue, but for advanced and disseminated tumors their application remains mainly a palliative measure. However, it is becoming increasingly clear that this limitation can be redressed by the use of intratumoral immune stimulating agents for amplifying potential antitumor immune responses that are induced by ablation therapies. A novel immune stimulating drug IP-001, a specific variant of the N-dihydrogalactochitosan (GC) family of molecules, has shown to be effective against metastatic tumors, when combined with different forms tumor ablation. It acts as a multi-function immune stimulant both by directly inhibiting cell membrane repair and recycling of ablation-damaged tumor cells, and indirectly by sequestering ablation-released tumor antigens, as well as recruiting and stimulating antigen presenting cells to induce a potent Th1 type T cell response against the cancer. In this review, we briefly discuss the current applications of local ablation for cancer treatment and the effects of GC in combination with other ablation therapies, a therapeutic approach that is pioneering the field of Interventional Immuno-Oncology (IIO).

N-dihydrogalactochitosan-supported tumor control by photothermal therapy and photothermal therapy-generated vaccine.

Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival.

N-dihydrogalactochitosan as immune and direct antitumor agent amplifying the effects of photodynamic therapy and photodynamic therapy-generated vaccines.

It is becoming apparent that to obtain robust and prolonged antitumor responses in cancer immunotherapy, appropriate adjunct agents promoting both tumor antigen delivery and immune rejection enhancement are critically required. The semisynthetic biopolymer N-dihydrogalactochitosan (GC) is emerging as a promising such candidate. In the present study, the effects of GC were investigated when combined with cancer vaccines generated by photodynamic therapy (PDT) using mouse tumor model SCCVII (squamous cell carcinoma). The adjunct GC treatment was found to enhance therapeutic benefit obtained with PDT vaccine, while reducing the numbers of myeloid-derived suppressor cells. Another important property of GC is promoting directly the death of SCCVII cells sustaining injury from PDT mediated by various photosensitizers. This effect is extended to cells treated by cryoablation therapy (CAT) performed by exposure to -80 °C. A capacity of GC for preferential binding to PDT treated cells was demonstrated using fluorescence microscopy. In vitro testing with specific caspase-1 inhibitor revealed a pro-survival role of this enzyme in membrane lipid repair mechanisms following combined PDT plus GC treatment. In conclusion, GC represents a uniquely promising adjunct for various PDT protocols, photothermal and similar rapid tumor-ablating therapies.

N-dihydrogalactochitosan as a potent immune activator for dendritic cells.

Immunotherapy has become one of the fastest growing areas of cancer research. A promising in situ autologous cancer vaccine (inCVAX) uses a novel immune activator, N-dihydrogalactochitosan (GC), that possesses the ability to stimulate dendritic cells (DC). inCVAX is a combination treatment procedure involving treatment of the tumor with a thermal near-infrared laser to liberate whole cell tumor antigens, followed by injection of GC (a glucosamine polymer with galactose attached to the amino groups) into the treated tumor thereby inducing a systemic antitumor immune response. Regression of both the treated tumor and distant untreated metastases has been observed in both nonclinical and clinical settings following inCVAX. We studied the stimulatory action of GC on relatively immature DCs (DC2.4 cell line) in vitro. GC at 1 mg/mL was a potent stimulator for DC with limited toxicity, giving increased expression of major histocompatibility complex class 2, CD80, and CD11c. Confocal imaging also revealed qualitatively increased uptake of antigen (Texas red-labeled ovalbumin) by DCs after the introduction of GC. To visualize cellular uptake, GC was conjugated with FITC-fluorophore revealing its cellular internalization after 8 hours. In some cases GC was more effective than the toxic TLR4 agonist, lipopolysaccharide. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 963-972, 2017.

Advances in strategies and methodologies in cancer immunotherapy.

Since the invention of Coley's toxin by William Coley in early 1900s, the path for cancer immunotherapy has been a convoluted one. Although still not considered standard of care, with the FDA approval of trastuzumab, Provenge and ipilimumab, the medical and scientific community has started to embrace the possibility that immunotherapy could be a new hope for cancer patients with otherwise untreatable metastatic diseases. This review aims to summarize the development of some major strategies in cancer immunotherapy, from the earliest peptide vaccine and transfer of tumor specific antibodies/T cells to the more recent dendritic cell (DC) vaccines, whole cell tumor vaccines, and checkpoint blockade therapy. Discussion of some major milestones and obstacles in the shaping of the field and the future perspectives is included. Photoimmunotherapy is also reviewed as an example of emerging new therapies combining phototherapy and immunotherapy.

InCVAX--a novel strategy for treatment of late-stage, metastatic cancers through photoimmunotherapy induced tumor-specific immunity.

A novel, promising potential cancer vaccine strategy was proposed to use a two-injection procedure for solid tumors to prompt the immune system to identify and systemically eliminate primary and metastatic cancers. The two-injection procedure consists of local photothermal application on a selected tumor intended to liberate whole cell tumor antigens, followed by a local injection of an immunoadjuvant that consists of a semi-synthetic functionalized glucosamine polymer, N-dihydro-galacto-chitosan (GC), which is intended to activate antigen presenting cells and facilitate an increased uptake of tumor antigens. This strategy is thus proposed as an in situ autologous cancer vaccine (inCVAX) that may activate antigen presenting cells and expose them to tumor antigens in situ, with the intention of inducing a systemic tumor specific T-cell response. Here, the development of inCVAX for the treatment of metastatic cancers in the past decades is systematically reviewed. The antitumor immune responses of local photothermal treatment and immunological stimulation with GC are also discussed. This treatment approach is also commonly referred to as laser immunotherapy (LIT).

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Compensatory dendritic cell development mediated by BATF-IRF interactions.

The AP1 transcription factor Batf3 is required for homeostatic development of CD8α(+) classical dendritic cells that prime CD8 T-cell responses against intracellular pathogens. Here we identify an alternative, Batf3-independent pathway in mice for CD8α(+) dendritic cell development operating during infection with intracellular pathogens and mediated by the cytokines interleukin (IL)-12 and interferon-γ. This alternative pathway results from molecular compensation for Batf3 provided by the related AP1 factors Batf, which also functions in T and B cells, and Batf2 induced by cytokines in response to infection. Reciprocally, physiological compensation between Batf and Batf3 also occurs in T cells for expression of IL-10 and CTLA4. Compensation among BATF factors is based on the shared capacity of their leucine zipper domains to interact with non-AP1 factors such as IRF4 and IRF8 to mediate cooperative gene activation. Conceivably, manipulating this alternative pathway of dendritic cell development could be of value in augmenting immune responses to vaccines.

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice.

Subclinical doses of Paclitaxel (PTX) given 1day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu(+) tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8(+) T cells with enhanced lytic activity against neu(+) tumors. PTX treatment also enhances maturation marker expression on CD11c(+) DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8(+) T cells with greater lytic capability than DC's from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-gamma-secreting CD8(+) T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.