Synthetic cytokine circuits that drive T cells into immune-excluded tumors.

Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

A vaccine targeting resistant tumours by dual T cell plus NK cell attack.

Most cancer vaccines target peptide antigens, necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore, tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells, but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding, enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably, this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary, highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations.

Biomaterial vaccines capturing pathogen-associated molecular patterns protect against bacterial infections and septic shock.

Most bacterial vaccines work for a subset of bacterial strains or require the modification of the antigen or isolation of the pathogen before vaccine development. Here we report injectable biomaterial vaccines that trigger potent humoral and T-cell responses to bacterial antigens by recruiting, reprogramming and releasing dendritic cells. The vaccines are assembled from regulatorily approved products and consist of a scaffold with absorbed granulocyte-macrophage colony-stimulating factor and CpG-rich oligonucleotides incorporating superparamagnetic microbeads coated with the broad-spectrum opsonin Fc-mannose-binding lectin for the magnetic capture of pathogen-associated molecular patterns from inactivated bacterial-cell-wall lysates. The vaccines protect mice against skin infection with methicillin-resistant Staphylococcus aureus, mice and pigs against septic shock from a lethal Escherichia coli challenge and, when loaded with pathogen-associated molecular patterns isolated from infected animals, uninfected animals against a challenge with different E. coli serotypes. The strong immunogenicity and low incidence of adverse events, a modular manufacturing process, and the use of components compatible with current good manufacturing practice could make this vaccine technology suitable for responding to bacterial pandemics and biothreats.

Ultrasound-triggered release reveals optimal timing of CpG-ODN delivery from a cryogel cancer vaccine.

Recently, several injectable scaffold-based cancer vaccines have been developed that can recruit and activate host dendritic cells (DCs) and generate potent antitumor responses. However, the optimal timing of adjuvant delivery, particularly of the commonly used cytosine-phosphodiester-guanine-oligonucleotide (CpG-ODN), for scaffold-based cancer vaccines remains unknown. We hypothesized that optimally timed CpG-ODN delivery will lead to enhanced immune responses, and designed a cryogel vaccine system where CpG-ODN release can be triggered on-demand by ultrasound. CpG-ODN was first condensed with polyethylenimine and then adsorbed to cryogels. Little adsorbed CpG-ODN was released in vitro. Ultrasound stimulation triggered continuous CpG-ODN release, at an enhanced rate even after ultrasound was turned off, with minimal burst release. In vivo, ultrasound stimulation four days post-vaccination induced a significantly higher antigen-specific cytotoxic T-lymphocyte (CTL) response compared to control mice. Furthermore, ultrasound stimulation at this time point generated a significantly higher IgG2a/c antibody titer than all the groups except ultrasound stimulation eight days post-vaccination. This optimal timing of ultrasound-triggered release coincided with peak DC accumulation in the cryogels. By enabling temporal control of vaccine components through release on-demand, this system is a promising platform to study the optimal timing of delivery of immunomodulatory agents for cancer vaccination.

SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma.

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of ideal target antigens that are both absolutely tumor specific and homogeneously expressed. We show that multi-antigen prime-and-kill recognition circuits provide flexibility and precision to overcome these challenges in the context of glioblastoma. A synNotch receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG), can be used to locally induce expression of a CAR. This enables thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific. Moreover, synNotch-regulated CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state. In immunodeficient mice bearing intracerebral patient-derived xenografts (PDXs) with heterogeneous expression of EGFRvIII, a single intravenous infusion of EGFRvIII synNotch-CAR T cells demonstrated higher antitumor efficacy and T cell durability than conventional constitutively expressed CAR T cells, without off-tumor killing. T cells transduced with a synNotch-CAR circuit primed by the CNS-specific antigen MOG also exhibited precise and potent control of intracerebral PDX without evidence of priming outside of the brain. In summary, by using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity, completeness, and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

Biomaterial-based scaffold for in situ chemo-immunotherapy to treat poorly immunogenic tumors.

Poorly immunogenic tumors, including triple negative breast cancers (TNBCs), remain resistant to current immunotherapies, due in part to the difficulty of reprogramming the highly immunosuppressive tumor microenvironment (TME). Here we show that peritumorally injected, macroporous alginate gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) for concentrating dendritic cells (DCs), CpG oligonucleotides, and a doxorubicin-iRGD conjugate enhance the immunogenic death of tumor cells, increase systemic tumor-specific CD8 + T cells, repolarize tumor-associated macrophages towards an inflammatory M1-like phenotype, and significantly improve antitumor efficacy against poorly immunogenic TNBCs. This system also prevents tumor recurrence after surgical resection and results in 100% metastasis-free survival upon re-challenge. This chemo-immunotherapy that concentrates DCs to present endogenous tumor antigens generated in situ may broadly serve as a facile platform to modulate the suppressive TME, and enable in situ personalized cancer vaccination.

Methoxyacetic acid inhibits histone deacetylase and impairs axial elongation morphogenesis of mouse gastruloids in a retinoic acid signaling-dependent manner.

BACKGROUND: Teratogenic potential has been linked to various industrial compounds. Methoxyacetic acid (MAA) is a primary metabolite of the widely used organic solvent and plasticizer, methoxyethanol and dimethoxyethyl phthalate, respectively. Studies using model animals have shown that MAA acts as the proximate teratogen that causes various malformations in developing embryos. Nonetheless, the molecular mechanisms by which MAA exerts its teratogenic effects are not fully understood. METHODS: Gastruloids of mouse P19C5 pluripotent stem cells, which recapitulate axial elongation morphogenesis of gastrulation-stage embryos, were explored as an in vitro model to investigate the teratogenic action of MAA. Morphometric parameters of gastruloids were measured to evaluate the morphogenetic effect, and transcript levels of various developmental regulator genes were examined to assess the impact on gene expression patterns. The effects of MAA on the level of retinoic acid (RA) signaling and histone deacetylase activity were also measured. RESULTS: MAA reduced axial elongation of gastruloids at concentrations comparable to the teratogenic plasma level (5 mM) in vivo. MAA at 4 mM significantly altered the expression profiles of developmental regulator genes. In particular, it upregulated the RA signaling target genes. The concomitant suppression of RA signaling using a pharmacological agent alleviated the morphogenetic effect of MAA. MAA at 4 mM also significantly reduced the activity of purified histone deacetylase protein. CONCLUSIONS: MAA impaired axial elongation morphogenesis in a RA signaling-dependent manner in mouse gastruloids, possibly through the inhibition of histone deacetylase.

Metabolic labeling and targeted modulation of dendritic cells.

Targeted immunomodulation of dendritic cells (DCs) in vivo will enable manipulation of T-cell priming and amplification of anticancer immune responses, but a general strategy has been lacking. Here we show that DCs concentrated by a biomaterial can be metabolically labelled with azido groups in situ, which allows for their subsequent tracking and targeted modulation over time. Azido-labelled DCs were detected in lymph nodes for weeks, and could covalently capture dibenzocyclooctyne (DBCO)-bearing antigens and adjuvants via efficient Click chemistry for improved antigen-specific CD8+ T-cell responses and antitumour efficacy. We also show that azido labelling of DCs allowed for in vitro and in vivo conjugation of DBCO-modified cytokines, including DBCO-IL-15/IL-15Rα, to improve priming of antigen-specific CD8+ T cells. This DC labelling and targeted modulation technology provides an unprecedented strategy for manipulating DCs and regulating DC-T-cell interactions in vivo.

Treating ischemia via recruitment of antigen-specific T cells.

Ischemic diseases are a leading cause of mortality and can result in autoamputation of lower limbs. We explored the hypothesis that implantation of an antigen-releasing scaffold, in animals previously vaccinated with the same antigen, can concentrate TH2 T cells and enhance vascularization of ischemic tissue. This approach may be clinically relevant, as all persons receiving childhood vaccines recommended by the Centers for Disease Control and Prevention have vaccines that contain aluminum, a TH2 adjuvant. To test the hypothesis, mice with hindlimb ischemia, previously vaccinated with ovalbumin (OVA) and aluminum, received OVA-releasing scaffolds. Vaccinated mice receiving OVA-releasing scaffolds locally concentrated antigen-specific TH2 T cells in the surrounding ischemic tissue. This resulted in local angiogenesis, increased perfusion in ischemic limbs, and reduced necrosis and enhanced regenerating myofibers in the muscle. These findings support the premise that antigen depots may provide a treatment for ischemic diseases in patients previously vaccinated with aluminum-containing adjuvants.

Anti-tumor immunity induced by ectopic expression of viral antigens is transient and limited by immune escape.

Immunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus- or host- "driver" oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed in vivo tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in Rag1-/- mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an "inflamed" or immune-activated tumor microenvironment and greater infiltration of CD8+ T cells compared to MOC2. By real-time PCR, we detected downregulation of E6 and E7 genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8+ T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and 'driver' status of target antigens to be considered in cancer vaccine design.

A facile approach to enhance antigen response for personalized cancer vaccination.

Existing strategies to enhance peptide immunogenicity for cancer vaccination generally require direct peptide alteration, which, beyond practical issues, may impact peptide presentation and result in vaccine variability. Here, we report a simple adsorption approach using polyethyleneimine (PEI) in a mesoporous silica microrod (MSR) vaccine to enhance antigen immunogenicity. The MSR-PEI vaccine significantly enhanced host dendritic cell activation and T-cell response over the existing MSR vaccine and bolus vaccine formulations. Impressively, a single injection of the MSR-PEI vaccine using an E7 peptide completely eradicated large, established TC-1 tumours in about 80% of mice and generated immunological memory. When immunized with a pool of B16F10 or CT26 neoantigens, the MSR-PEI vaccine eradicated established lung metastases, controlled tumour growth and synergized with anti-CTLA4 therapy. Our findings from three independent tumour models suggest that the MSR-PEI vaccine approach may serve as a facile and powerful multi-antigen platform to enable robust personalized cancer vaccination.

Injectable, Tough Alginate Cryogels as Cancer Vaccines.

A covalently crosslinked methacrylated (MA)-alginate cryogel vaccine has been previously shown to generate a potent response against murine melanoma, but is not mechanically robust and requires a large 16G needle for delivery. Here, covalent and ionic crosslinking of cryogels are combined with the hypothesis that this will result in a tough MA-alginate cryogel with improved injectability. All tough cryogels can be injected through a smaller, 18G needle without sustaining any damage, while covalently crosslinked-only cryogels break after injection. Cytosine-phosphodiester-guanine (CpG)-delivering tough cryogels effectively activate dendritic cells (DCs). Granulocyte macrophage colony-stimulating factor releasing tough cryogels recruit four times more DCs than blank gels by day 7 in vivo. The tough cryogel vaccine induces strong antigen-specific cytotoxic T-lymphocyte and humoral responses. These vaccines prevent tumor formation in 80% of mice inoculated with HER2/neu-overexpressing DD breast cancer cells. The MA-alginate tough cryogels provide a promising minimally invasive delivery platform for cancer vaccinations.

Covalent Conjugation of Peptide Antigen to Mesoporous Silica Rods to Enhance Cellular Responses.

Short peptides are the minimal modality of antigen recognized by cellular immunity and are therefore considered a safe and highly specific source of antigen for vaccination. Nevertheless, successful peptide immunotherapy is limited by the short half-life of peptide antigens in vivo as well as their weak immunogenicity. We recently reported a vaccine strategy based on dendritic cell-recruiting Mesoporous Silica Rod (MSR) scaffolds to enhance T-cell responses against subunit antigen. In this study, we investigated the effect of covalently conjugating peptide antigens to MSRs to increase their retention in the scaffolds. Using both stable thioether and reducible disulfide linkages, peptide conjugation greatly increased peptide loading compared to passive adsorption. In vitro, Bone Marrow derived Dendritic Cells (BMDCs) could present Ovalbumin (OVA)-derived peptides conjugated to MSRs and induce antigen-specific T-cell proliferation. Stable conjugation decreased presentation in vitro while reducible conjugation maintained levels of presentation as high as soluble peptide. Compared to soluble peptide, in vitro, expansion of OT-II T-cells was not affected by adsorption or stable conjugation to MSRs but was enhanced with reversible conjugation to MSRs. Both conjugation schemes increased peptide residence time in MSR scaffolds in vivo compared to standard bolus injections or a simple adsorption method. When MSR scaffolds loaded with GM-CSF and CpG-ODN were injected subcutaneously, recruited dendritic cells could present antigen in situ with the stable conjugation increasing presentation capacity. Overall, this simple conjugation approach could serve as a versatile platform to efficiently incorporate peptide antigens in MSR vaccines and potentiate cellular responses.

Adverse effect of valproic acid on an in vitro gastrulation model entails activation of retinoic acid signaling.

Valproic acid (VPA), an antiepileptic drug, is a teratogen that causes neural tube and axial skeletal defects, although the mechanisms are not fully understood. We previously established a gastrulation model using mouse P19C5 stem cell embryoid bodies (EBs), which exhibits axial patterning and elongation morphogenesis in vitro. Here, we investigated the effects of VPA on the EB axial morphogenesis to gain insights into its teratogenic mechanisms. Axial elongation and patterning of EBs were inhibited by VPA at therapeutic concentrations. VPA elevated expression levels of various developmental regulators, including Cdx1 and Hoxa1, known transcriptional targets of retinoic acid (RA) signaling. Co-treatment of EBs with VPA and BMS493, an RA receptor antagonist, partially rescued axial elongation as well as gene expression profiles. These results suggest that VPA requires active RA signaling to interfere with EB morphogenesis.

An in vitro gastrulation model recapitulates the morphogenetic impact of pharmacological inhibitors of developmental signaling pathways.

Certain chemical agents act as teratogens, causing birth defects and fetal deaths when pregnant women are exposed to them. The establishment of in vitro models that recapitulate crucial embryonic events is therefore vital to facilitate screening of potential teratogens. Previously, we created a three-dimensional culture method for mouse P19C5 embryonal carcinoma stem cells that, when cultured as embryoid bodies, display elongation morphogenesis resembling gastrulation, which is the critical event resulting in the germ layers and major body axes. Determination of how well this in vitro morphogenesis represents in vivo gastrulation is essential to assess its applicability as well as to identify limitations of the model for detecting teratogenic agents. Here, we investigated the morphological and molecular characteristics of P19C5 morphogenesis using pharmacological agents that are known to cause abnormal patterning in the embryo in vivo by inhibiting major developmental signaling--e.g., involving Wnt, Nodal, Bone morphogenic protein (Bmp), Fibroblast growth factor (Fgf), Retinoic acid, Notch, and Hedgehog pathways. Inhibitors of Wnt, Nodal, Bmp, Fgf, and Retinoic acid signaling caused distinct changes in P19C5 morphogenesis that were quantifiable using morphometric parameters. These five inhibitors, plus the Notch inhibitor, also altered temporal expression profiles of developmental regulator genes in a manner consistent with the in vivo roles of the corresponding signaling pathways. In contrast, the Hedgehog inhibitor did not have any impact on the process, suggesting an absence of active Hedgehog signaling in these embryoid bodies. These results indicate that the P19C5 in vitro gastrulation model is a promising tool to screen for teratogenic agents that interfere with many of the key developmental signals.

Evaluation of the Chinese version of the Disability of the Arm, Shoulder and Hand (DASH-HKPWH): cross-cultural adaptation process, internal consistency and reliability study.

UNLABELLED: The Disability of the Arm, Shoulder and Hand (DASH) was translated into Chinese by a physiotherapy team of the Prince of Wales Hospital, Hong Kong (DASH-HKPWH). OBJECTIVES: This study evaluated the cross-cultural adaptation process, face validity, internal consistency and reliability of the DASH-HKPWH. METHOD: Language officers and medical professionals from different fields were invited to translate and evaluate the face validity of the DASH-HKPWH. 88 patients were recruited to complete two DASH questionnaires on two occasions 1-2 weeks apart. RESULTS: Some adjustments were made to the translations based on the cultural and linguistic practice in Hong Kong. The face validity was satisfactory with a mean endorsement score of 3.2. The difference between the mean of DASH scores was not significant (t = -0.35, p = 0.73). The ICC (1,1) and Cronbach's alpha for the 30-item Disability/Symptom of the DASH-HKPWH was 0.77 and 0.94, respectively. CONCLUSION: The translation was valid and reliable and acceptably equivalent to the original version. The questionnaire is suitable for measuring changes experienced by patients with any upper extremity disorders.