3D bioprinting cowpea mosaic virus as an immunotherapy depot for ovarian cancer prevention in a preclinical mouse model.

Implantable polymeric hydrogels loaded with immunostimulatory cowpea mosaic virus (CPMV) were fabricated using digital light processing (DLP) printing technology. The CPMV-laden hydrogels were surgically implanted into the peritoneal cavity to serve as depots for cancer slow-release immunotherapy. Sustained release of CPMV within the intraperitoneal space alleviates the need for repeated dosing and we demonstrated efficacy against ovarian cancer in a metastatic mouse model.

Development of a novel multi­epitope vaccine against the pathogenic human polyomavirus V6/7 using reverse vaccinology.

BACKGROUND: Human polyomaviruses contribute to human oncogenesis through persistent infections, but currently there is no effective preventive measure against the malignancies caused by this virus. Therefore, the development of a safe and effective vaccine against HPyV is of high priority. METHODS: First, the proteomes of 2 polyomavirus species (HPyV6 and HPyV7) were downloaded from the NCBI database for the selection of the target proteins. The epitope identification process focused on selecting proteins that were crucial, associated with virulence, present on the surface, antigenic, non-toxic, and non-homologous with the human proteome. Then, the immunoinformatic methods were used to identify cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes from the target antigens, which could be used to create epitope-based vaccine. The physicochemical features of the designed vaccine were predicted through various online servers. The binding pattern and stability between the vaccine candidate and Toll-like receptors were analyzed through molecular docking and molecular dynamics (MD) simulation, while the immunogenicity of the designed vaccines was assessed using immune simulation. RESULTS: Online tools were utilized to forecast the most optimal epitope from the immunogenic targets, including LTAg, VP1, and VP1 antigens of HPyV6 and HPyV7. A multi-epitope vaccine was developed by combining 10 CTL, 7 HTL, and 6 LBL epitopes with suitable linkers and adjuvant. The vaccine displayed 98.35% of the world's population coverage. The 3D model of the vaccine structure revealed that the majority of residues (87.7%) were located in favored regions of the Ramachandran plot. The evaluation of molecular docking and MD simulation revealed that the constructed vaccine exhibits a strong binding (-1414.0 kcal/mol) towards the host's TLR4. Moreover, the vaccine-TLR complexes remained stable throughout the dynamic conditions present in the natural environment. The immune simulation results demonstrated that the vaccine design had the capacity to elicit robust immune responses in the host. CONCLUSION: The multi-parametric analysis revealed that the designed vaccine is capable of inducing sustained immunity against the selected polyomaviruses, although further in-vivo investigations are needed to verify its effectiveness.

Transcriptomics of Canine Inflammatory Mammary Cancer Treated with Empty Cowpea Mosaic Virus Implicates Neutrophils in Anti-Tumor Immunity.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-ß and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.

Combining energy-based focal ablation and immune checkpoint inhibitors: preclinical research and clinical trials.

Energy-based focal therapy (FT) uses targeted, minimally invasive procedures to destroy tumors while preserving normal tissue and function. There is strong emerging interest in understanding how systemic immunity against the tumor can occur with cancer immunotherapy, most notably immune checkpoint inhibitors (ICI). The motivation for combining FT and ICI in cancer management relies on the synergy between the two different therapies: FT complements ICI by reducing tumor burden, increasing objective response rate, and reducing side effects of ICI; ICI supplements FT by reducing local recurrence, controlling distal metastases, and providing long-term protection. This combinatorial strategy has shown promising results in preclinical study (since 2004) and the clinical trials (since 2011). Understanding the synergy calls for understanding the physics and biology behind the two different therapies with distinctive mechanisms of action. In this review, we introduce different types of energy-based FT by covering the biophysics of tissue-energy interaction and present the immunomodulatory properties of FT. We discuss the basis of cancer immunotherapy with the emphasis on ICI. We examine the approaches researchers have been using and the results from both preclinical models and clinical trials from our exhaustive literature research. Finally, the challenges of the combinatory strategy and opportunities of future research is discussed extensively.

Combining in situ vaccination and immunogenic apoptosis to treat cancer.

Immunization approaches are designed to stimulate the immune system and eliminate the tumor. Studies indicate that cancer immunization combined with certain chemotherapeutics and immunostimulatory agents can improve outcomes. Chemotherapeutics-based immunogenic cell death makes the tumor more recognizable by the immune system. In situ vaccination (ISV) utilizes established tumors as antigen sources and directly applies an immune adjuvant to the tumor to reverse a cold tumor microenvironment to a hot one. Immunogenic cell death and ISV highlight for the immune system the tumor antigens that are recognizable by immune cells and support a T-cell attack of the tumor cells. This review presents the concept of immunogenic apoptosis and ISV as a powerful platform for cancer immunization.

The immune system recognizes and attacks tumors, but often the tumor is able to protect itself by generating a local immune suppressive environment that reduces antitumor immune response. Therapies such as immune checkpoint blockade antibodies are meant to overcome immune suppression and enable an effective antitumor immune response; however, many patients do not respond to immune checkpoint blockade therapy. Further immune manipulations are needed to improve the response to immune-based cancer treatments. Approaches that reverse the tumor-mediating immune suppression can be effective. This review discusses combining two such approaches, generating immunogenic cell death of tumor cells and treating recognized tumors with immune stimulatory reagents, called in situ vaccination or intratumoral immunotherapy.

Inactivated Cowpea Mosaic Virus for In Situ Vaccination: Differential Efficacy of Formalin vs UV-Inactivated Formulations.

Cowpea mosaic virus (CPMV) has been developed as a promising nanoplatform technology for cancer immunotherapy; when applied as in situ vaccine, CPMV exhibits potent, systemic, and durable efficacy. While CPMV is not infectious to mammals, it is infectious to legumes; therefore, agronomic safety needs to be addressed to broaden the translational application of CPMV. RNA-containing formulations are preferred over RNA-free virus-like particles because the RNA and protein, each, contribute to CPMV's potent antitumor efficacy. We have previously optimized inactivation methods to develop CPMV that contains RNA but is not infectious to plants. We established that inactivated CPMV has reduced efficacy compared to untreated, native CPMV. However, a systematic comparison between native CPMV and different inactivated forms of CPMV was not done. Therefore, in this study, we directly compared the therapeutic efficacies and mechanisms of immune activation of CPMV, ultraviolet- (UV-), and formalin (Form)-inactivated CPMV to explain the differential efficacies. In a B16F10 melanoma mouse tumor model, Form-CPMV suppressed the tumor growth with prolonged survival (there were no statistical differences comparing CPMV and Form-CPMV). In comparison, UV-CPMV inhibited tumor growth significantly but not as well as Form-CPMV or CPMV. The reduced therapeutic efficacy of UV-CPMV is explained by the degree of cross-linking and aggregated state of the RNA, which renders it inaccessible for sensing by Toll-like receptor (TLR) 7/8 to activate immune responses. The mechanistic studies showed that the highly aggregated state of UV-CPMV inhibited TLR7 signaling more so than for the Form-CPMV formulation, reducing the secretion of interleukin-6 (IL-6) and interferon-α (IFN-α), cytokines associated with TLR7 signaling. These findings support the translational development of Form-CPMV as a noninfectious immunotherapeutic agent.

In situ vaccination with cowpea mosaic virus elicits systemic antitumor immunity and potentiates immune checkpoint blockade.

BACKGROUND: In situ vaccination (ISV) is a cancer immunotherapy strategy in which immunostimulatory reagents are introduced directly into a tumor to stimulate antitumor immunity both against the treated tumor and systemically against untreated tumors. Recently, we showed that cowpea mosaic virus (CPMV) is a potent multi-toll-like receptor (TLR) agonist with potent efficacy for treating tumors in mice and dogs by ISV. However, ISV with CPMV alone does not uniformly treat all mouse tumor models tested, however this can be overcome through strategic combinations. More insight is needed to delineate potency and mechanism of systemic antitumor immunity and abscopal effect. METHOD: We investigated the systemic efficacy (abscopal effect) of CPMV ISV with a two-tumor mouse model using murine tumor lines B16F10, 4T1, CT26 and MC38. Flow cytometry identified changes in cell populations responsible for systemic efficacy of CPMV. Transgenic knockout mice and depleting antibodies validated the role of relevant candidate cell populations and cytokines. We evaluated these findings and engineered a multicomponent combination therapy to specifically target the candidate cell population and investigated its systemic efficacy, acquired resistance and immunological memory in mouse models. RESULTS: ISV with CPMV induces systemic antitumor T-cell-mediated immunity that inhibits growth of untreated tumors and requires conventional type-1 dendritic cells (cDC1s). Furthermore, using multiple tumor mouse models resistant to anti-programmed death 1 (PD-1) therapy, we tested the hypothesis that CPMV along with local activation of antigen-presenting cells with agonistic anti-CD40 can synergize and strengthen antitumor efficacy. Indeed, this combination ISV strategy induces an influx of CD8+ T cells, triggers regression in both treated local and untreated distant tumors and potentiates tumor responses to anti-PD-1 therapy. Moreover, serial ISV overcomes resistance to anti-PD-1 therapy and establishes tumor-specific immunological memory. CONCLUSIONS: These findings provide new insights into in situ TLR activation and cDC1 recruitment as effective strategies to overcome resistance to immunotherapy in treated and untreated tumors.

Proactive Immunotherapeutic Approaches against Inflammatory Breast Cancer May Improve Patient Outcomes.

Inflammatory breast cancer (IBC) is highly metastatic at the onset of the disease with no IBC-specific treatments, resulting in dismal patient survival. IBC treatment is a clear unmet clinical need. This commentary highlights findings from a recent seminal approach in which pembrolizumab, a checkpoint inhibitor against programmed cell death protein 1 (PD-1), was provided to a triple-negative IBC patient as a neoadjuvant immune therapy combined with anthracycline-taxane-based chemotherapy. We highlight the findings of the case report and offer a perspective on taking a proactive approach to deploy approved immune checkpoint inhibitors. On the basis of our recently published research study, we propose in situ vaccination with direct injection of immunostimulatory agents into the tumor as an option to improve outcomes safely, effectively, and economically for IBC patients.

Multifunctional Plant Virus Nanoparticles for Targeting Breast Cancer Tumors.

Breast cancer treatment using plant-virus-based nanoparticles (PVNPs) has achieved considerable success in preclinical studies. PVNP-based breast cancer therapies include non-targeted and targeted nanoplatforms for delivery of anticancer therapeutic chemo and immune agents and cancer vaccines for activation of local and systemic antitumor immunity. Interestingly, PVNP platforms combined with other tumor immunotherapeutic options and other modalities of oncotherapy can improve tumor efficacy treatment. These applications can be achieved by encapsulation of a wide range of active ingredients and conjugating ligands for targeting immune and tumor cells. This review presents the current breast cancer treatments based on PVNP platforms.

Emerging Potential of Plant Virus Nanoparticles (PVNPs) in Anticancer Immunotherapies.

Cancer immunotherapies using plant virus nanoparticles (PVNPs) have achieved considerable success in preclinical studies. PVNP based nanoplatforms can be endogenous immune adjuvants and act as nanocarriers that stabilize and deliver cancer antigens and exogenous immune adjuvants. Although they do not infect mammalian cells, PVNPs are viruses and they are variably recognized by pathogen pattern recognition receptors (PRR), activate innate immune cells including antigen-presenting cells (APCs), and increase the expression of costimulatory molecules. Novel immunotherapy strategies use them as in situ vaccines (ISV) that can effectively inhibit tumor growth after intratumoral administration and generate expanded systemic antitumor immunity. PVNPs combined with other tumor immunotherapeutic options and other modalities of oncotherapy can improve both local and systemic anti-tumor immune responses. While not yet in clinical trials in humans, there is accelerating interest and research of the potential of PVNPs for ISV immune therapy for cancer. Thus, antitumor efficacy of PVNPs by themselves, or loaded with soluble toll-like receptor (TLR) agonists and/or cancer antigens, will likely enter human trials over the next few years and potentially contribute to next-generation antitumor immune-based therapies.

Cowpea Mosaic Virus Outperforms Other Members of the Secoviridae as In Situ Vaccine for Cancer Immunotherapy.

In situ vaccination for cancer immunotherapy uses intratumoral administration of small molecules, proteins, nanoparticles, or viruses that activate pathogen recognition receptors (PRRs) to reprogram the tumor microenvironment and prime systemic antitumor immunity. Cowpea mosaic virus (CPMV) is a plant virus that─while noninfectious toward mammals─activates mammalian PRRs. Application of CPMV as in situ vaccine (ISV) results in a potent and durable efficacy in tumor mouse models and canine patients; data indicate that CPMV outperforms small molecule PRR agonists and other nonrelated plant viruses and virus-like particles (VLPs). In this work, we set out to compare the potency of CPMV versus other plant viruses from the Secoviridae. We developed protocols to produce and isolate cowpea severe mosaic virus (CPSMV) and tobacco ring spot virus (TRSV) from plants. CPSMV, like CPMV, is a comovirus with genome and protein homology, while TRSV lacks homology and is from the genus nepovirus. When applied as ISV in a mouse model of dermal melanoma (using B16F10 cells and C57Bl6J mice), CPMV outperformed CPSMV and TRSV─again highlighting the unique potency of CPMV. Mechanistically, the increased potency is related to increased signaling through toll-like receptors (TLRs)─in particular, CPMV signals through TLR2, 4, and 7. Using knockout (KO) mouse models, we demonstrate here that all three plant viruses signal through the adaptor molecule MyD88─with CPSMV and TRSV predominantly activating TLR2 and 4. CPMV induced significantly more interferon ß (IFNß) compared to TRSV and CPSMV; therefore, IFNß released upon signaling through TLR7 may be a differentiator for the observed potency of CPMV-ISV. Additionally, CPMV induced a different temporal pattern of intratumoral cytokine generation characterized by significantly increased inflammatory cytokines 4 days after the second of 2 weekly treatments, as if CPMV induced a "memory response". This higher, longer-lasting induction of cytokines may be another key differentiator that explains the unique potency of CPMV-ISV.

Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer.

BACKGROUND: Inflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC. METHODS: Ten IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2-0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry. RESULTS: Two neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg+/CD8+ ratio and changes in CD8+Granzyme B+ T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3+ lymphocytes, decrease in FoxP3+/CD3+ ratio (p<0.04 for all comparisons), and no changes in CC-3+ immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy. CONCLUSIONS: Neoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients.

Inactivated Cowpea Mosaic Virus in Combination with OX40 Agonist Primes Potent Antitumor Immunity in a Bilateral Melanoma Mouse Model.

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure-function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wild-type CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

Multifunctional plant virus nanoparticles in the next generation of cancer immunotherapies.

Plant virus nanoparticles (PVNPs) have inherent immune stimulatory ability, and have been investigated as immune adjuvants to stimulate an anti-tumor immune response. The combination of immune stimulation, nanoparticle structure and the ability to deliver other therapeutic molecules provides a flexible platform for cancer immunotherapy. Researching multifunctional PVNPs and their modification will generate novel reagents for cancer immunotherapy. Here we review the properties of PVNPs, and their potential for clinical utilization to activate anti-tumor innate and lymphoid immune responses. PVNPs have potential utility for cancer immunotherapy as vaccine adjuvant, and delivery systems for other reagents as mono immunotherapy or combined with other immunotherapies. This review outlines the potential and challenges in developing PVNPs as cancer immunotherapy reagents.

Tumor-Derived Extracellular Vesicles in Cancer Immunoediting and Their Potential as Oncoimmunotherapeutics.

The tumor microenvironment (TME) within and around a tumor is a complex interacting mixture of tumor cells with various stromal cells, including endothelial cells, fibroblasts, and immune cells. In the early steps of tumor formation, the local microenvironment tends to oppose carcinogenesis, while with cancer progression, the microenvironment skews into a protumoral TME and the tumor influences stromal cells to provide tumor-supporting functions. The creation and development of cancer are dependent on escape from immune recognition predominantly by influencing stromal cells, particularly immune cells, to suppress antitumor immunity. This overall process is generally called immunoediting and has been categorized into three phases; elimination, equilibrium, and escape. Interaction of tumor cells with stromal cells in the TME is mediated generally by cell-to-cell contact, cytokines, growth factors, and extracellular vesicles (EVs). The least well studied are EVs (especially exosomes), which are nanoparticle-sized bilayer membrane vesicles released by many cell types that participate in cell/cell communication. EVs carry various proteins, nucleic acids, lipids, and small molecules that influence cells that ingest the EVs. Tumor-derived extracellular vesicles (TEVs) play a significant role in every stage of immunoediting, and their cargoes change from immune-activating in the early stages of immunoediting into immunosuppressing in the escape phase. In addition, their cargos change with different treatments or stress conditions and can be influenced to be more immune stimulatory against cancer. This review focuses on the emerging understanding of how TEVs affect the differentiation and effector functions of stromal cells and their role in immunoediting, from the early stages of immunoediting to immune escape. Consideration of how TEVs can be therapeutically utilized includes different treatments that can modify TEV to support cancer immunotherapy.

Differential effects of CD20+ B cells and PD-L1+ immune cells on pathologic complete response and outcome: comparison between inflammatory breast cancer and locally advanced breast cancer patients.

PURPOSE: This study evaluated epidemiologic and immune factors associated with pathologic complete response (pCR), breast cancer-specific survival (BCSS) and disease-free survival (DFS) outcomes in inflammatory (IBC) and locally advanced breast cancer (LABC) patients. METHODS: Tumor-infiltrating lymphocytes (TILs) and CD20+ B-cell frequencies (CD20+), and PD-L1 expression on tumor (PD-L1+carcinoma cells) and immune (PD-L1+TILs) cells were analyzed by immunohistochemistry along with clinicopathologic factors as modifiers of pCR and outcomes in 221 IBC and 162 LABC patients. Analysis included Kaplan-Meier curves and Cox proportional hazard models. RESULTS: IBC and LABC display similar levels of TILs, CD20+, and combined CD20+ and PD-L1+TILs (CD20+PD-L1+TILs), while LABC contained more PD-L1+TILs and PD-L1+ carcinoma cells. Absence of lymphovascular involvement, high TILs, PD-L1+ carcinoma cells, and combined CD20+ and PD-L1+ carcinoma cells correlated with pCR in IBC and LABC patients. High PD-L1+TILs correlated with pCR only in LABC; less lymph node involvement at diagnosis, CD20+ and CD20+PD-L1+TILs correlated with pCR only in IBC (P < 0.04, all comparisons). Achievement of pCR in IBC and LABC patients correlated with BCSS and DFS (P < 0.02). In multivariate analyses, pCR remained an independent prognostic factor of improved DFS in IBC and LABC patients, but of BCSS in only LABC. CD20+PD-L1+TILs remained an independent prognostic factor of improved DFS and BCSS only in IBC. CONCLUSION: CD20+PD-L1+TILs are an independent prognostic biomarker of improved outcomes in IBC, but not LABC. Selecting IBC patients by CD20 and PD-L1 status could stratify patients and potentially identify those in whom activating CD20 agents and anti-PD-1/PD-L1 therapy could be explored.

Cowpea mosaic virus stimulates antitumor immunity through recognition by multiple MYD88-dependent toll-like receptors.

Cowpea mosaic virus (CPMV), a non-enveloped plant virus, and empty CPMV (eCPMV), a virus-like particle (VLP) composed of CPMV capsid without nucleic acids, are potent in situ cancer vaccines when administered intratumorally (I.T.). However, it is unclear how immune cells recognize these nanoparticles and why they are immunogenic, which was investigated in this study. CPMV generated stronger selective induction of cytokines and chemokines in naïve mouse splenocytes and exhibited more potent anti-tumor efficacy than eCPMV. MyD88 is required for both CPMV- and eCPMV-elicited immune responses. Screening with human embryonic kidney (HEK)-293 cell toll-like receptor (TLR) reporter assays along with experiments in corresponding TLR-/- mice indicated CPMV and eCPMV capsids are recognized by MyD88-dependent TLR2 and TLR4. CPMV, but not eCPMV, is additionally recognized by TLR7. Secretion of type I interferons (IFNs), which requires the interaction between TLR7 and encapsulated single-stranded RNAs (ssRNAs), is critical to CPMV's better efficacy. The same recognition mechanisms are also functional in human peripheral blood mononuclear cells (PBMCs). Overall, these findings link CPMV immunotherapy efficacy with molecular recognition, provide rationale for how to develop more potent viral particles, accentuate the value of multi-TLR agonists as in situ cancer vaccines, and highlight the functional importance of type I IFNs for in situ vaccination.

Activity of immunoproteasome inhibitor ONX-0914 in acute lymphoblastic leukemia expressing MLL-AF4 fusion protein.

Proteasome inhibitors bortezomib and carfilzomib are approved for the treatment of multiple myeloma and mantle cell lymphoma and have demonstrated clinical efficacy for the treatment of acute lymphoblastic leukemia (ALL). The t(4;11)(q21;q23) chromosomal translocation that leads to the expression of MLL-AF4 fusion protein and confers a poor prognosis, is the major cause of infant ALL. This translocation sensitizes tumor cells to proteasome inhibitors, but toxicities of bortezomib and carfilzomib may limit their use in pediatric patients. Many of these toxicities are caused by on-target inhibition of proteasomes in non-lymphoid tissues (e.g., heart muscle, gut, testicles). We found that MLL-AF4 cells express high levels of lymphoid tissue-specific immunoproteasomes and are sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914, even in the presence of stromal cells. Inhibition of multiple active sites of the immunoproteasomes was required to achieve cytotoxicity against ALL. ONX-0914, an inhibitor of LMP7 (ß5i) and LMP2 (ß1i) sites of the immunoproteasome, and LU-102, inhibitor of proteasome ß2 sites, exhibited synergistic cytotoxicity. Treatment with ONX-0914 significantly delayed the growth of orthotopic ALL xenograft tumors in mice. T-cell ALL lines were also sensitive to pharmacologically relevant concentrations of ONX-0914. This study provides a strong rationale for testing clinical stage immunoproteasome inhibitors KZ-616 and M3258 in ALL.