Stimulation Of Antigen-presenting Cells Research Articles

Abstract 3327: L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors

Abstract Introduction: Immunotherapy is a promising therapeutic approach for a wide range of cancers. However, some tumors called "cold tumors" lack infiltrating T-cells and are not responsive to immune checkpoint inhibitors (ICIs), leading to worse clinical outcomes. This study demonstrates that L-pampo, a novel TLR2/3 agonist, promotes an anti-tumor immune response in cold tumors. Also, combining L-pampo with ICIs can improve the anti-tumor efficacy more safely than standard-of-care (SoC) chemotherapies. Method: We utilized syngeneic tumor models using CT26 colon cancer and 4T1 triple-negative breast cancer (TNBC). L-pampo was administered alone or in combination with ICIs such as anti-PD-1 (aPD-1) and anti-CTLA-4 (aCTLA-4) antibodies to tumor-bearing mice (n=8/group). Tumor size was measured every three days, and the survival rate was monitored until day 60. On day 21 or 27 after transplantation, we analyzed the tumor weight and tumor-infiltrating immune cells. Furthermore, we assessed the respective anti-tumor efficacies and safety of L-pampo and SoC chemotherapies (FOLFOX and paclitaxel). Result: In the CT26 colon cancer model, L-pampo effectively reduced the tumor size compared to the vehicle group (83.7%). The combination of L-pampo and ICI reduced tumor size by 86.1% (with aCTLA4) and 90.5% (with aPD-1), while the ICI alone did not effectively inhibit tumor growth (aPD-1; 17.3%, aCTLA-4; 23.6%). Notably, the triple concurrent treatment with L-pampo, aPD-1 and aCTLA-4 reduced tumor size by 96.6% and induced completed tumor regression in 1 out of 8. In the 4T1 TNBC model, L-pampo reduced tumor size by 64.2% compared to the vehicle treatment, and combined treatment with L-pampo and a-PD-1 suppressed tumor growth compared to aPD-1 alone (9.6% vs. 61.5%). Additionally, L-pampo improved median overall survival in each tumor model (CT26; 25.5 vs. 35.5, 4T1; 29.5 vs. 53.5 days). To compare the anti-tumor efficacy of L-pampo and conventional cytotoxic chemotherapy, we treated tumor-bearing mice with FOLFOX or paclitaxel as SoC regimen, respectively. Compared with each chemotherapy, L-pampo revealed comparable antitumor efficacies (Tumor growth inhibition CT26; 75.6% vs. 70.6%, 4T1; 65.5% vs. 58.8%). Interestingly, L-pampo inhibited tumor growth without significant adverse effects. In contrast, conventional chemotherapies induced acute hematological side effects such as leukopenia/neutropenia/thrombocytopenia and rapid weight loss. Furthermore, L-pampo increased the active cytotoxic T-cells infiltration through antigen-presenting cells stimulation within the tumors. Conclusion: This study suggests that L-pampo, a novel TLR2/3 agonist, can be a potent anti-tumor agent, improving the anti-tumor efficacy of ICIs in cold tumors with enhanced anti-tumor immunity and minimal toxicities than conventional chemotherapies. Citation Format: Guentae Kim, Yoonki Heo, Hye Jung Kim, Sejung Park, Byung Cheol Ahn, Won Suk Lee, Chan Kim, Hong Jae Chon, Eunyoung Chun, Jung Sun Yum. L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3327.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

Mn2+/CpG Oligodeoxynucleotides Codecorated Black Phosphorus Nanosheet Platform for Enhanced Antitumor Potency in Multimodal Therapy.

Manganese ions (Mn2+)-coordinated nanoparticles have emerged as a promising class of antitumor nanotherapeutics, capable of simultaneously disrupting the immunosuppressive tumor microenvironment (TME) and triggering the stimulator of interferon genes (STING) pathway-dependent antitumor immunity. However, the activation of STING signaling by Mn2+-based monotherapies is suboptimal for comprehensive stimulation of antigen presenting cells and reversal of immunosuppression in the TME. Here, we report the design of a Mn2+/CpG oligodeoxynucleotides (ODNs) codecorated black phosphorus nanosheet (BPNS@Mn2+/CpG) platform based on the Mn2+ modification of BPNS and subsequent adsorption of synthetic CpG ODNs. The coordination of Mn2+ significantly improved the stability of BPNS and the adsorption of CpG ODNs. The acidic TME and endosomal compartments can disrupt the Mn2+ coordination, triggering pH-responsive release of CpG ODNs and Mn2+ to effectively activate the Toll-like receptor 9 and STING pathways. As a result, M2-type macrophages and immature dendritic cells were strongly stimulated in the TME, thereby increasing T lymphocyte infiltration and reversing the immunosuppression within the TME. Phototherapy and chemodynamic therapy, utilizing the BPNS@Mn2+/CpG platform, have demonstrated efficacy in inducing immunogenic cell death upon 808 nm laser irradiation. Importantly, the treatment of BPNS@Mn2+/CpG with laser irradiation exhibited significant therapeutic efficacy against the irradiated primary tumor and effectively suppressed the growth of nonirradiated distant tumor. Moreover, it induced a robust immune memory, providing long-lasting protection against tumor recurrence. This study demonstrated the enhanced antitumor potency of BPNS@Mn2+/CpG in multimodal therapy, and its proof-of-concept application as a metal ion-modified BPNS material for effective DNA/drug delivery and immunotherapy.

Antigenicity and adjuvanticity co-reinforced personalized cell vaccines based on self-adjuvanted hydrogel for post-surgical cancer vaccination

Cancer vaccine-based postsurgical immunotherapy is emerging as a promising approach in patients following surgical resection for inhibition of tumor recurrence. However, low immunogenicity and insufficient cancer antigens limit the widespread application of postoperative cancer vaccines. Here, we propose a “trash to treasure” cancer vaccine strategy to enhance postsurgical personalized immunotherapy, in which antigenicity and adjuvanticity of purified surgically exfoliated autologous tumors (with whole antigen repertoire) were co-reinforced. In the antigenicity and adjuvanticity co-reinforced personalized vaccine (Angel-Vax), polyriboinosinic: polyribocytidylic acid (pIC) and tumor cells that have undergone immunogenic death are encapsulated in a self-adjuvanted hydrogel formed by cross-linking of mannan and polyethyleneimine. Angel-Vax exhibits an enhanced capacity on antigen-presenting cells stimulation and maturation compared to its individual components in vitro. Immunization with Angel-Vax provokes an efficient systemic cytotoxic T-cell immune response, contributing to the satisfied prophylactic and therapeutic efficacy in mice. Furthermore, when combined with immune checkpoint inhibitors (ICI), Angel-Vax effectively prevented postsurgical tumor recurrence, as evidenced by an increase in median survival of approximately 35% compared with ICI alone. Unlike the cumbersome preparation process of postoperative cancer vaccines, the simple and feasible approach herein may represent a general strategy for various kinds of tumor cell-based antigens in the inhibition of postsurgical tumor relapse by reinforced immunogenicity.

Development of fast dissolving polymer-based microneedles for delivery of an antigenic melanoma cell membrane

Delivery of cancer cell membranes (CM) is a new approach for the activation of the immune system and the induction of immunotherapy of cancer. Local delivery of melanoma CM into skin can induce efficient immune stimulation of antigen presenting cells (APCs), such as dendritic cells. In the current study, fast dissolving microneedles (MNs) were developed for the delivery of melanoma B16F10 CM. Two polymers were tested for the fabrication of MNs: poly(methyl vinyl ether-co-maleic acid) (PMVE-MA) and hyaluronic acid (HA). The incorporation of CM in MNs was achieved through coating of the MNs using a multi-step layering procedure or the micromolding technique. The CM loading and its stabilization were improved by adding sugars (sucrose and trehalose) and a surfactant (Poloxamer 188), respectively. In an ex vivo experiment, both PMVE-MA and HA showed fast dissolutions (<30 s) after insertion into porcine skin. However, HA-MN showed better mechanical properties, namely improved resistance to fracture when submitted to a compression force. Overall, a B16F10 melanoma CM-dissolving MN system was efficiently developed as a promising device suggesting further studies in immunotherapy and melanoma applications.

Abstract 6392: Combination of pulsed electric field, immunotherapy and cisplatin significantly prolongs survival in an orthotopic breast cancer mouse model

Abstract Introduction: Pulsed Electric Field (PEF) therapy is a novel approach to treat cancer by providing focal tumor ablation while sparing sensitive structures within the tumor micro-environment. Previous studies have demonstrated that PEF induces immunogenic cell death by activating inflammatory signaling pathways and stimulation of antigen presenting cells withing the tumor micro-environment. PEF ablation provides a tumor focused in situ vaccination strategy that combines with checkpoint blockade. We hypothesized that PEF ablation should synergize with chemotherapy and anti-PD1 in a model where checkpoint inhibitors are inactive. Experimental Design: To test this hypothesis, we utilized a highly aggressive syngeneic orthotopic triple negative breast cancer 4T1 mouse model that is non-responsive to anti-PD1 and only marginally responsive to cisplatin to determine if this combination treatment could prolong survival. Thirty-two Balb/c mice were inoculated in the mammary fat pad with 200,000 4T1 cells. Once tumors were established (5mm in diameter), animals were randomized to the following groups: (A) Sham/IgG control (n=8; needle inserted but no PEF), (B) PEF only (n=8), (C) cisplatin + anti-PD-1 (n=8) and (D) PEF + cisplatin + anti PD-1 (n=8). Tumors in groups B and D were treated once with PEF. Anti-PD1 and isotype matched control IgG was administered once per week (200μg, i.p injections) starting on the day of PEF (Group D) for four weeks. Cisplatin was administered (2mg/kg, i.v) once per week for four weeks starting on the day of PEF (d=0) for four weeks. All the mammary fat pad tumors were resected when they achieved a tumor volume of ~200 mm3 to recapitulate post-resection metastatic relapse (See Schematic 1). Results and Conclusions: All mice in the Sham-control IgG group needed to be euthanized by day 43 due to metastases in multiple organs including lungs, bones, liver and brain. All mice in the cisplatin + anti PD-1 group had to be euthanized by day 74. Interestingly, all groups that were treated with PEF survived much longer. On day 90, 28% of mice in the PEF alone group were alive and 43% were alive in the PEF + anti-PD1 + Cisplatin (see Figure 1). The rank-log p-value for the Kaplan-Meier survival curve was statistically significant. In conclusion, our results demonstrate that PEF treatment in combination with anti-PD1 and Cisplatin significantly prolongs survival in a highly aggressive triple negative breast tumor mouse model that is non-responsive to immunotherapy. These findings support evaluation of PEF in cancer patients that are receiving combination of checkpoint blockade and chemotherapy. Citation Format: Chiara Pastori, Mukta Wagh, Ebtesam Nafie, Fatima Murad, Mohit Trikha, Robert Neal. Combination of pulsed electric field, immunotherapy and cisplatin significantly prolongs survival in an orthotopic breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6392.

Construction of a prognostic prediction model for renal clear cell carcinoma combining clinical traits

Kidney renal clear cell carcinoma (KIRC) is one of the common malignant tumors of the urinary system. Patients with different risk levels are other in terms of disease progression patterns and disease regression. The poorer prognosis for high-risk patients compared to low-risk patients. Therefore, it is essential to accurately high-risk screen patients and gives accurate and timely treatment. Differential gene analysis, weighted correlation network analysis, Protein–protein interaction network, and univariate Cox analysis were performed sequentially on the train set. Next, the KIRC prognostic model was constructed using the least absolute shrinkage and selection operator (LASSO), and the Cancer Genome Atlas (TCGA) test set and the Gene Expression Omnibus dataset verified the model’s validity. Finally, the constructed models were analyzed; including gene set enrichment analysis (GSEA) and immune analysis. The differences in pathways and immune functions between the high-risk and low-risk groups were observed to provide a reference for clinical treatment and diagnosis. A four-step key gene screen resulted in 17 key factors associated with disease prognosis, including 14 genes and 3 clinical features. The LASSO regression algorithm selected the seven most critical key factors to construct the model: age, grade, stage, GDF3, CASR, CLDN10, and COL9A2. In the training set, the accuracy of the model in predicting 1-, 2- and 3-year survival rates was 0.883, 0.819, and 0.830, respectively. The accuracy of the TCGA dataset was 0.831, 0.801, and 0.791, and the accuracy of the GSE29609 dataset was 0.812, 0.809, and 0.851 in the test set. Model scoring divided the sample into a high-risk group and a low-risk group. There were significant differences in disease progression and risk scores between the two groups. GSEA analysis revealed that the enriched pathways in the high-risk group mainly included proteasome and primary immunodeficiency. Immunological analysis showed that CD8 (+) T cells, M1 macrophages, PDCD1, and CTLA4 were upregulated in the high-risk group. In contrast, antigen-presenting cell stimulation and T-cell co-suppression were more active in the high-risk group. This study added clinical characteristics to constructing the KIRC prognostic model to improve prediction accuracy. It provides help to assess the risk of patients more accurately. The differences in pathways and immunity between high and low-risk groups were also analyzed to provide ideas for treating KIRC patients.

Enhanced Immunogenicity of an Influenza Ectodomain Matrix-2 Protein Virus-like Particle (M2e VLP) Using Polymeric Microparticles for Vaccine Delivery.

In this study, we demonstrate how encapsulating a conserved influenza ectodomain matrix-2 protein virus-like particle (M2e5x VLP) into a pre-crosslinked bovine serum albumin (BSA) polymeric matrix enhances in vitro antigen immunogenicity and in vivo efficacy. The spray-dried M2e5x VLP-loaded BSA microparticles (MPs) showed enhanced stimulation of antigen presenting cells (APCs), as confirmed through nitrite production and increased antigen–cell interactions seen in real time using live-cell imaging. Next, to further boost the immunogenicity of M2e5x VLP microparticles, M2e5x MPs were combined with Alhydrogel® and monophosphoryl lipid-A (MPL-A®) adjuvant microparticles. M2e5x VLP MPs and the combination VLP M2e5x VLP + Alhydrogel® + MPL-A® MPs elicited a significant increase in the expression of antigen-presenting molecules in dendritic cells compared to M2e5x VLP alone. Lastly, for preliminary evaluation of in vivo efficacy, the vaccine was administered in mice through the skin using an ablative laser. The M2e5x VLP + Alhydrogel® + MPL-A® MPs were shown to induce high levels of M2e-specific IgG antibodies. Further, a challenge with live influenza revealed heightened T-cell stimulation in immune organs of mice immunized with M2e5x VLP + Alhydrogel® + MPL-A® MPs. Hence, we utilized the advantages of both VLP and polymeric delivery platforms to enhance antigen immunogenicity and adaptive immunity in vivo.

Myeloid antigen-presenting cell niches sustain antitumor Tcells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

A 4-chemokine signature to predict intermediate immunogenicity in homologous recombination repair deficient tumors.

2546 Background: Treatment responses to immune checkpoint blockade (ICB) associate with T cell tumor inflammation. Tumors with mismatch repair deficiency display an inflammatory tumor phenotype and respond to ICB. Similarly, tumors with homologous recombination repair deficiency (HR-d) may be primed for ICB treatment. We have previously shown that a panel of 4 chemokines identifies a subclass of pancreatic cancer with markers of T cell-inflammation. Here, we evaluated this 4-chemokine signature in cancer types with HR-d molecular subclasses. Methods: We combined paired transcriptomes and genomic data of breast (n = 699), ovarian (n = 174) and prostate (n = 457) cancers from the Cancer Genome Atlas and tumor-enriched pancreas cancers (n = 121) to evaluate the 4-chemokine signature in HR-d vs. HR-proficient tumors across these 4 cancers. Metrics of antitumor immunity were also compared. Results: Across tumor types, elevated expression of the 4-chemokine signature (chemokine-hi) associated with transcriptional hallmarks of a T cell-mediated antitumor response, including antigen presenting cell stimulation, antigen presentation, and T cell activity. In tumors with a predominant COSMIC signature 3, which associates with HR-d, the 4-chemokine signature predicted intermediate levels of T cell-inflammation. Conclusions: These data suggest that 1) the 4-chemokine signature may be a clinically relevant biomarker in identifying subclasses of tumours responsive to immunotherapies, and that 2) HR-d tumors harbor intermediate immunogenicity. Correlation of treatment responses to immunotherapies with the 4-chemokine signature is needed validate its predictive value as a biomarker for treatment stratification with immunotherapies.[Table: see text]

Co-delivery of PSMA antigen epitope and mGM-CSF with a cholera toxin-like chimeric protein suppressed prostate tumor growth via activating dendritic cells and promoting CTL responses

Subunit vaccines derived from tumor antigens play a role in tumor therapy because of their unique advantages. However, because of the weak immunogenicity of peptides in subunit vaccines, it is difficult to trigger an effective cytotoxic T lymphocyte (CTL) response, which is critical for cancer therapy. A requirement for the activation of CTL cells by exogenous antigens is the stimulation of antigen presenting cells (APC) with the help of adjuvants and cross-presentation to T lymphocytes. Standard nonconjugated adjuvant-peptide mixtures do not ensure co-targeting of the antigen and the adjuvant to the same APC, which limits the effects of adjuvants. In this study, a fusion protein consisting of murine granulocyte-macrophage colony stimulating factor (mGM-CSF) fused with CTA2 (A2 subunit of cholera toxin) was generated and assembled with CTB-PSMA624-632 (prostate specific membrane antigen (PSMA) peptide 624–632 fused to CTB) to obtain a cholera toxin-like protein. The chimeric protein retained the biological activity of mGM-CSF and had stronger GM1 binding activity than (CTB-PSMA624-632)5. C57BL/6J mice immunized with the CT-like chimeric protein exhibited delayed tumor growth following challenge with human PSMA-EGFP-expressing RM-1 cells. Experiment results showed that the CT-like chimeric protein could induce the maturation of DC cells and improve CTL responses. Overall, these results indicate that the nasal administration of a CT-like chimeric protein vaccine results in the development of effective immunity against prostate tumor cells and might be useful for future clinical anti-tumoral applications.

Abstract PS4-14: Immunological analysis of the combination therapy of nivolumab, paclitaxel and bevacizumab in patients with HER2-negative MBC in NEWBEAT trial (WJOG9917BTR)

Abstract Background: Synergistic antitumor effect of combined anti-PD-1 antibody and anti-VEGF agent has been expected, based on previous preclinical data. We have conducted NEWBEAT trial to evaluate efficacy of triple combination regimen of nivolumab + paclitaxel + bevacizumab in patients (pts)with HR+ HER2- MBC or metastatic TNBC, and clinical results were presented in SABCS 2019. A biomarker study (WJOG9917BTR) was conducted to evaluate the VEGF and immune status of these patients. Methods: HER2-negative breast cancer patients in the NEWBEAT trial were enrolled. To explore the biomarkers for the triple combination treatment, immune status and its dynamics were evaluated with multicolor flowcytometry, multiplex ELISA in peripheral blood before and after treatment. Results: Among the 57 patients who were enrolled to the NEWBEAT trial, 50 patients were registered to the biomarker study. The expression of Ki67 and inducible T-cell co-stimulator (ICOS) on T cells increased after treatment, indicating induction of the T cell proliferation and activation. In responder (defined as patients with progression-free survival longer than 1 year, n = 30), the number of naïve CD4+ T cells at pretreatment were higher and effector memory CD4+ T cells were lower than non-responder. On the other hand, CD86+ myeloid DC at pretreatment were lower in non-responder pts. The median concentration of VEGF-A in serum before treatment was 116.065 pg/ml (range: 0-740.23) and decreased below 37 pg/ml at day 8 after treatment. Although serum VEGF-A level is inversely correlated with clinical outcome of pts with anti-PD-1 antibody in previous reports, in this trial VEGF-A high subgroup had better objective response than VEGF-A low subgroup, suggesting that blockade of VEGF by bevacizumab may overcome immunosuppression via VEGF signaling. Interestingly, in recurrent pts , the number of VEGFR-2+ CD4+ T cells / Monocyte were higher, and PD-L1+ CD4+ T cells / Monocyte / myeloid Dendritic Cell tended to be higher than in de novo stage IV pts. These results suggested that immune status of recurrent pts were more immunosuppressive than de novo stage IV pts, and that it might be more effective by the combination therapy to block the VEGF and PD-1 pathways. Moreover, the changes of immune status and dynamics on CD8+ T cells were not observed, suggesting that the therapeutic strategy of breast cancer might require the re-activation of CD8+ effector T cells through the stimulation of antigen-presenting cells followed by modification of CD4+ helper T cells.Conclusions: Our analysis showed the different immune status depending stage, subtype and response in advanced breast cancer pts. The dynamic decrease of serum VEGF-A concentration and high expression of VEGFR-1 or VEGFR-2 in the immune suppressive cells in advanced breast cancer pts suggested that combination treatment with bevacizumab might clinically overcome the immune suppression via inhibition of VEGF-A. (UMIN000029590) Citation Format: Yukinori Ozaki, Shigehisa Kitano, Junji Tsurutani, Tsutomu Iwasa, Masato Takahashi, Toru Mukohara, Norikazu Masuda, Manabu Futamura, Hironobu Minami, Koji Matsumoto, Hidetaka Kawabata, Makiko Yamashita, Kenichi Yoshimura, Toshimi Takano. Immunological analysis of the combination therapy of nivolumab, paclitaxel and bevacizumab in patients with HER2-negative MBC in NEWBEAT trial (WJOG9917BTR) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-14.

Stability Studies of the Vaccine Adjuvant U-Omp19.

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens.To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates.This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

Abstract 4437: Anti-tumor immunotherapeutic strategy for uveal melanoma by combining photodynamic therapy with ROCK inhibitor

Abstract Uveal melanoma (UM) is the most frequent intraocular malignant tumor among adults. Although UM is a rare disease, the prognosis of patients with UM is poor since nearly 50% of patients develop metastatic disease, and the overall survival outcome is dismal. Here, we suggest a novel and optimal treatment regime for UM not only offering lower burden to patients with high applicability but also preventing lethal metastatic disease by empowering the immune system through the combination of local Photodynamic therapy (PDT) and ROCK inhibitor. PDT is a topical treatment modality with the minimally invasive therapeutic method and now broadly used in a treatment option for the eyes. PDT with chlorin e6 based formulated FIC elicits immunogenic cell death (ICD) of cancer cells, and hallmarks of ICD stimulate antigen-presenting cells (APCs). Furthermore, ripasudil, one of the ROCK inhibitors, is clinically approved for the treatment of glaucoma and ocular hypertension in the form of an eye drop solution. Ripasudil is repurposed as an anticancer compound based on the immuno-stimulatory effect of ROCK inhibition. A combination of FIC-PDT with ripasudil mediated stimulation of APCs leads primed cytotoxic T cell infiltration into the tumor microenvironment (TME) and eventually augment anti-tumor immunity. Moreover, the combinational strategy sensitizes PD-1/PD-L1 immune checkpoint blockade (ICB) therapy to improve survival rates over time and inhibit tumor growth of the distant tumor by generating a systemic anti-tumor immune response. Collectively, our therapeutic strategy may lead to a new treatment option for UM by suppressing the primary tumor effectively and preventing the metastatic disease. Citation Format: Seohyun Kim, In-San Kim. Anti-tumor immunotherapeutic strategy for uveal melanoma by combining photodynamic therapy with ROCK inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4437.

Cytopeutics® umbilical cord mesenchymal stromal cells (Cyto-MSC) for patients with grade II–IV acute graft-versus-host disease: a phase I/II clinical study – protocol overview

Background & Aim Background Graft-versus-host disease (GVHD) is a devastating complication following allogeneic hematopoietic stem cell transplantation (HSCT) mediated by stimulation of antigen presenting cells (APCs) which leads to donor T-lymphocytes activation and target tissue destruction, particularly affecting the skin, gastrointestinal tract, and liver in acute setting. In recent years, researchers have discovered that the application of mesenchymal stromal cells (MSCs) as salvage treatment among steroid refractory GVHD patients improves outcomes without long-term risk association. On the other hand, the use of MSCs concurrently with steroids as front-line treatment for acute GVHD has yet to be researched on. We hypothesize that this approach, as the MSCs will be administered at earlier stage of the disease, will increase survival rate and reduce mortality among aGVHD patients. Objective In this study, we aim to determine the efficacy and safety of allogeneic infusion of Cytopeutics® umbilical cord-derived mesenchymal stromal cells (Cyto-MSC) in combination of standard corticosteroid therapy as front-line approach for treatment of grade II-IV acute GVHD patients. Methods, Results & Conclusion Study Design This is a phase I/II clinical study involving patients who underwent an allogeneic HSCT for malignant or non malignant haematological disorders and developed grade II-IV acute GVHD. A total of 40 eligible patients will be recruited in this study. For Phase I open labelled study, 5 eligible patients will be recruited to receive Cyto-MSC (5 × 106 MSC per kg bodyweight) and standard treatment. Meanwhile, for Phase II double blinded placebo controlled study, another 35 eligible patients will be recruited and randomized into 2 study groups where 15 patients will be assigned into Group A to receive Cyto-MSC (5 × 106 MSC per kg bodyweight) and standard treatment, meanwhile another 20 patients will be assigned into Group B to receive Placebo and standard treatment. Cyto-MSC or Placebo will be administered at Day 1 and Day 4. Another infusion of Cyto-MSC or Placebo will be given at Day 7 if the patient shows no or partial response based on GVHD grading criteria. All patients will be assessed up until 6 months follow-up which include medical history, clinical and physical evaluations, pathology investigations, biomarkers and immune cell subsets analysis, as well as quality of life questionnaires.

Synergistic effect of dual targeting vaccine adjuvant with aminated β-glucan and CpG-oligodeoxynucleotides for both humoral and cellular immune responses

Presently, clinically approved adjuvants (such as aluminum salts) fail to induce cellular immune responses, which is crucial to defend against intracellular pathogens (including HIV, malaria, tuberculosis and Ebola) and cancer. However, Freund’s complete adjuvant potently stimulates both humoral and cellular immune responses, accompanying by high toxicity and severe side reactions. Here in this work, a CpG-oligodeoxynucleotides (CpG-OND) crosslinked aminated β-glucan-Ovalbumin dual targeting nanoparticle (CpG-OND-AG-OVA) is prepared through a simple and mild ionic complexation method. The aminated β-glucan plays dual roles as antigen presenting cells (APCs) targeted carrier and immunopotentiator (targeting and activating dectin-1 on APCs). Meanwhile, CpG-OND also plays dual roles as ionic crosslinker and immunopotentiator (targeting and activating Toll-like receptor 9 in APCs). The adjuvant activity of the particles is evaluated through in vitro and in vivo experiments. The particles significantly enhance uptake and sustained proteolytic processing of antigens, and result in APCs maturation, inducing robust Th1 and Th2-type immune responses comparable to Freund’s adjuvant without obvious toxicity. The potent adjuvant activity of the nanoparticles may originate from dual targeting synergistic effects between aminated β-glucan and CpG-OND. Accordingly, the dual targeting nanoparticles may be a promising vaccine adjuvant for inducing robust humoral and cellular immune responses against infectious diseases and cancers. Statement of SignificanceAn ideal adjuvant for subunit vaccine should act as both a carrier to enhance the uptake, sustained processing and cytosolic delivery of antigens, and an immunopotentiator to stimulate antigen presenting cells (APCs) for activation of naive T cells. Additionally, it should be easy to obtain and safe with negligible toxicity. Unfortunately, both synthetic and natural polymers that have been developed into antigen delivery system cannot completely fulfill the requirements. In the present study, the authors design nanoparticles with aminated β-glucan and CpG-oligodeoxynucleotides (CpG-OND) through a simple and mild method. β-Glucan (a dectin-1 and TLR2 targeted PAMP) and CpG-OND (a TLR9 targeted PAMP) are readily accessible. Aminated β-glucan plays dual roles in the nanoparticle as APCs targeted carrier and immunopotentiator. Meanwhile, CpG-OND also plays dual roles as crosslinker and APCs targeted immunopotentiator. By making use of synergistic effect of the dual targeting vaccine adjuvant with aminated β-glucan and CpG-OND, the nanoparticles induce robust antigen specific immune responses comparable to Freund’s adjuvant without obvious toxicity.

B lymphocytes can be activated to act as antigen presenting cells to promote anti-tumor responses.

Immune evasion by tumors includes several different mechanisms, including the inefficiency of antigen presenting cells (APCs) to trigger anti-tumor T cell responses. B lymphocytes may display a pro-tumoral role but can also be modulated to function as antigen presenting cells to T lymphocytes, capable of triggering anti-cancer immune responses. While dendritic cells, DCs, are the best APC population to activate naive T cells, DCs or their precursors, monocytes, are frequently modulated by tumors, displaying a tolerogenic phenotype in cancer patients. In patients with cervical cancer, we observed that monocyte derived DCs are tolerogenic, inhibiting allogeneic T cell activation compared to the same population obtained from patients with precursor lesions or cervicitis. In this work, we show that B lymphocytes from cervical cancer patients respond to treatment with sCD40L and IL-4 by increasing the CD80+CD86+ population, therefore potentially increasing their ability to activate T cells. To test if B lymphocytes could actually trigger anti-tumor T cell responses, we designed an experimental model where we harvested T and B lymphocytes, or dendritic cells, from tumor bearing donors, and after APC stimulation, transplanted them, together with T cells into RAG1-/- recipients, previously injected with tumor cells. We were able to show that anti-CD40 activated B lymphocytes could trigger secondary T cell responses, dependent on MHC-II expression. Moreover, we showed that dendritic cells were resistant to the anti-CD40 treatment and unable to stimulate anti-tumor responses. In summary, our results suggest that B lymphocytes may be used as a tool for immunotherapy against cancer.

Computer-aided prediction of antigen presenting cell modulators for designing peptide-based vaccine adjuvants

BackgroundEvidences in literature strongly advocate the potential of immunomodulatory peptides for use as vaccine adjuvants. All the mechanisms of vaccine adjuvants ensuing immunostimulatory effects directly or indirectly stimulate antigen presenting cells (APCs). While numerous methods have been developed in the past for predicting B cell and T-cell epitopes; no method is available for predicting the peptides that can modulate the APCs.MethodsWe named the peptides that can activate APCs as A-cell epitopes and developed methods for their prediction in this study. A dataset of experimentally validated A-cell epitopes was collected and compiled from various resources. To predict A-cell epitopes, we developed support vector machine-based machine learning models using different sequence-based features.ResultsA hybrid model developed on a combination of sequence-based features (dipeptide composition and motif occurrence), achieved the highest accuracy of 95.71% with Matthews correlation coefficient (MCC) value of 0.91 on the training dataset. We also evaluated the hybrid models on an independent dataset and achieved a comparable accuracy of 95.00% with MCC 0.90.ConclusionThe models developed in this study were implemented in a web-based platform VaxinPAD to predict and design immunomodulatory peptides or A-cell epitopes. This web server available at http://webs.iiitd.edu.in/raghava/vaxinpad/ will facilitate researchers in designing peptide-based vaccine adjuvants.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Guérin (BCG) is safe and effective for protection against children's severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Abstract 3647: Therapeutic activity of effector function-enhanced, non-fucosylated anti-CD40 antibodies in preclinical immune-competent rodent tumor models

Abstract Recent success of immune checkpoint inhibitors (CPIs) in the clinic underscores the role of cancer immune surveillance and the therapeutic potential of targeting immune receptors. Unlike CPIs, many members of the TNF receptor superfamily (TNFRSF), e.g., CD40, 4-1BB, OX-40, and GITR deliver agonistic signals to immune cells that may directly activate antitumor immune responses and, in combination with CPIs, may improve outcomes of active immunotherapy. Sugar-Engineered Antibody (SEA)-CD40 is a non-fucosylated, humanized IgG1 anti-human CD40 monoclonal antibody currently being evaluated in a phase 1 clinical trial in solid tumors and lymphoma. It binds FcγR3A with higher affinity and demonstrates enhanced immune stimulatory activity compared to its parent dacetuzumab. An SEA version of an anti-mouse CD40 antibody (mouse IgG2a), SEA-1C10, has been generated. As expected, SEA-1C10 bound to mouse FcγRIV, the functional homolog of human FcγR3A in mice, at a higher affinity than parent 1C10. Enhanced FcγRIV engagement by SEA-1C10 translated into stronger antitumor activity in the CD40 (-) B16F10 melanoma and CD40 (+) A20 lymphoma models. In the A20 model, tumor free mice that survived the initial A20 tumor implantation and SEA-1C10 treatment were re-challenged with live A20 cells. Twenty nine out of thirty one such tumor free mice rejected A20 tumor re-engraftment, suggesting that a strong memory immune response was elicited by SEA-1C10 treatment. The SEA-1C10/anti PD-1 combination also showed greater antitumor activity compared to SEA-1C10 or anti-PD-1 alone, supporting the hypothesis that stimulation of antigen-presenting cells by agonistic CD40 signaling can complement immune checkpoint inhibition. In addition, a transgenic (TG) mouse strain has been generated in which the endogenous Cd40 locus is inactivated by the targeted insertion of a human-mouse chimeric CD40 gene. This allows for incorporation of the extracellular domain of human CD40 while keeping the transmembrane and cytoplasmic domains of mouse CD40. Lineage-restricted expression of the CD40 TG on B cells and monocytes was confirmed. CD40 TG cells responded to SEACD40-mediated agonistic signaling in vitro by secreting CD40 signature cytokines and up-regulating the costimulatory molecules CD86 and MHC class II. Treatment of CD40 TG animals with SEA-CD40 resulted in cytokine induction and B-cell depletion. Similar to studies in human PBMCs and non-human primates, SEA-CD40 induced more robust cytokine production and immune cell activation than its fucosylated parent dacetuzumab. These experimental systems will be further applied to address clinically important questions including mechanism(s) of anti-tumor effects, biomarkers indicating immune activation, dosing strategies, and timing of combinatorial regimens with CPIs. Citation Format: Shyra J. Gardai, Weiping Zeng, Che-Leung Law. Therapeutic activity of effector function-enhanced, non-fucosylated anti-CD40 antibodies in preclinical immune-competent rodent tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2017-3647