Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics.

BACKGROUND: Despite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge in the field of immunotherapy; thus, more optimized translational models could strongly influence clinical trial development. To address this unmet need, we designed a preclinical model reflecting the heterogeneity in melanoma patients' clinical responses that can be used to evaluate novel immunotherapies and synergistic combinatorial treatment strategies. Using our all-autologous humanized melanoma mouse model, we examined the efficacy of a novel engineered interleukin 2 (IL-2)-based cytokine variant immunotherapy. METHODS: To study immune responses and antitumor efficacy for human melanoma tumors, we developed an all-autologous humanized melanoma mouse model using clinically annotated, matched patient tumor cells and peripheral blood mononuclear cells (PBMCs). After inoculating immunodeficient NSG mice with patient tumors and an adoptive cell transfer of autologous PBMCs, mice were treated with anti-PD-1, a novel investigational engineered IL-2-based cytokine (nemvaleukin), or recombinant human IL-2 (rhIL-2). The pharmacodynamic effects and antitumor efficacy of these treatments were then evaluated. We used tumor cells and autologous PBMCs from patients with varying immunotherapy responses to both model the diversity of immunotherapy efficacy observed in the clinical setting and to recapitulate the heterogeneous nature of melanoma. RESULTS: Our model exhibited long-term survival of engrafted human PBMCs without developing graft-versus-host disease. Administration of an anti-PD-1 or nemvaleukin elicited antitumor responses in our model that were patient-specific and were found to parallel clinical responsiveness to checkpoint inhibitors. An evaluation of nemvaleukin-treated mice demonstrated increased tumor-infiltrating CD4+ and CD8+ T cells, preferential expansion of non-regulatory T cell subsets in the spleen, and significant delays in tumor growth compared with vehicle-treated controls or mice treated with rhIL-2. CONCLUSIONS: Our model reproduces differential effects of immunotherapy in melanoma patients, capturing the inherent heterogeneity in clinical responses. Taken together, these data demonstrate our model's translatability for novel immunotherapies in melanoma patients. The data are also supportive for the continued clinical investigation of nemvaleukin as a novel immunotherapeutic for the treatment of melanoma.

A high OXPHOS CD8 T cell subset is predictive of immunotherapy resistance in melanoma patients.

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS "CD8+ TOXPHOS cells." We validated that higher levels of OXPHOS in tumor- and peripheral blood-derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.

Immune-Checkpoint Blockade Opposes CD8+ T-cell Suppression in Human and Murine Cancer.

Immune-checkpoint blockade enhances antitumor responses against cancers. One cancer type that is sensitive to checkpoint blockade is squamous cell carcinoma of the head and neck (SCCHN), which we use here to study limitations of this treatment modality. We observed that CD8+ tumor-infiltrating lymphocytes (TILs) in SCCHN and melanoma express excess immune checkpoints components PD-1 and Tim-3 and are also CD27-/CD28-, a phenotype we previously associated with immune dysfunction and suppression. In ex vivo experiments, patients' CD8+ TILs with this phenotype suppressed proliferation of autologous peripheral blood T cells. Similar phenotype and function of TILs was observed in the TC-1 mouse tumor model. Treatment of TC-1 tumors with anti-PD-1 or anti-Tim-3 slowed tumor growth in vivo and reversed the suppressive function of multi-checkpoint+ CD8+ TIL. Similarly, treatment of both human and mouse PD-1+ Tim-3+ CD8+ TILs with anticheckpoint antibodies ex vivo reversed their suppressive function. These suppressive CD8+ TILs from mice and humans expressed ligands for PD-1 and Tim-3 and exerted their suppressive function via IL10 and close contact. To model therapeutic strategies, we combined anti-PD-1 blockade with IL7 cytokine therapy or with transfer of antigen-specific T cells. Both strategies resulted in synergistic antitumor effects and reduced suppressor cell function. These findings enhance our understanding of checkpoint blockade in cancer treatment and identify strategies to promote synergistic activities in the context of other immunotherapies.

Self-Assembling Peptide Gels for 3D Prostate Cancer Spheroid Culture.

Progress in prostate cancer research is presently limited by a shortage of reliable in vitro model systems. The authors describe a novel self-assembling peptide, bQ13, which forms nanofibers and gels useful for the 3D culture of prostate cancer spheroids, with improved cytocompatibility compared to related fibrillizing peptides. The mechanical properties of bQ13 gels can be controlled by adjusting peptide concentration, with storage moduli ranging between 1 and 10 kPa. bQ13's ability to remain soluble at mildly basic pH considerably improved the viability of encapsulated cells compared to other self-assembling nanofiber-forming peptides. LNCaP cells formed spheroids in bQ13 gels with similar morphologies and sizes to those formed in Matrigel or RADA16-I. Moreover, prostate-specific antigen (PSA) is produced by LNCaP cells in all matrices, and PSA production is more responsive to enzalutamide treatment in bQ13 gels than in other fibrillized peptide gels. bQ13 represents an attractive platform for further tailoring within 3D cell culture systems.

Award Winner in the Young Investigator Category, 2017 Society for Biomaterials Annual Meeting and Exposition, Minneapolis, MN, April 05-08, 2017: Lymph node stiffness-mimicking hydrogels regulate human B-cell lymphoma growth and cell surface receptor expression in a molecular subtype-specific manner.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, with multiple molecular subtypes. The activated B-cell-like DLBCL subtype accounts for roughly one-third of all the cases and has an inferior prognosis. There is a need to develop better class of therapeutics that could target molecular pathways in resistant DLBCLs; however, this requires DLBCLs to be studied in representative tumor microenvironments. The pathogenesis and progression of lymphoma has been mostly studied from the point of view of genetic alterations and intracellular pathway dysregulation. By comparison, the importance of lymphoma microenvironment in which these malignant cells arise and reside has not been studied in as much detail. We have recently elucidated the role of integrin signaling in lymphomas and demonstrated that inhibition of integrin-ligand interactions abrogated the proliferation of malignant cells in vitro and in patient-derived xenograft. Here we demonstrate the role of lymph node tissue stiffness on DLBCL in a B-cell molecular subtype specific manner. We engineered tunable bioartificial hydrogels that mimicked the stiffness of healthy and neoplastic lymph nodes of a transgenic mouse model and primary human lymphoma tumors. Our results demonstrate that molecularly diverse DLBCLs grow differentially in soft and high stiffness microenvironments, which further modulates the integrin and B-cell receptor expression level as well as response to therapeutics. We anticipate that our findings will be broadly useful to study lymphoma biology and discover new class of therapeutics that target B-cell tumors in physical environments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1833-1844, 2017.

Osteoarthritis: Pathology, Mouse Models, and Nanoparticle Injectable Systems for Targeted Treatment.

Osteoarthritis (OA) is a progressive, degenerative disease of articulating joints that not only affects the elderly, but also involves younger, more active individuals with prolonged participation in high physical-demand activities. Thus, effective therapies that are easy to adopt clinically are critical in limiting the societal burden associated with OA. This review is focused on intra-articular injectable regimens and provides a comprehensive look at existing in vivo models of OA that might be suitable for developing, testing, and finding a cure for OA by intra-articular injections. We first discuss the pathology, molecular mechanisms responsible for the initiation and progression of OA, and challenges associated with disease-specific targeting of OA. We proceed to discuss available animal models of OA and provide a detailed perspective on the use of mouse models in studies of experimental OA. We finally provide a closer look at intra-articular injectable treatments for OA, focusing on biomaterials-based nanoparticles, and provide a comprehensive overview of the various nanometer-size ranges studied.

Self-Assembly Protein Nanogels for Safer Cancer Immunotherapy.

Soluble antigen-based cancer vaccines have poor retention in tissues along with suboptimal antigen processing by dendritic cells. Multiple booster doses are often needed, leading to dose-limiting systemic toxicity. A versatile, immunomodulatory, self-assembly protein nanogel vaccine is reported that induces robust immune cell response at lower antigen doses than soluble antigens, an important step towards biomaterials-based safer immunotherapy approaches.

Self-assembled glycopeptide nanofibers as modulators of galectin-1 bioactivity.

Galectins are carbohydrate-binding proteins that act as extracellular signaling molecules in various normal and pathological processes. Galectin bioactivity is mediated by specific non-covalent interactions with cell-surface and extracellular matrix (ECM) glycoproteins, which can enhance or inhibit signaling events that influence various cellular behaviors, including adhesion, proliferation, differentiation, and apoptosis. Here, we developed a materials approach to modulate galectin bioactivity by mimicking natural galectin-glycoprotein interactions. Specifically, we created a variant of a peptide that self-assembles into ß-sheet nanofibers under aqueous conditions, QQKFQFQFEQQ (Q11), which has an asparagine residue modified with the monosaccharide N-acetylglucosamine (GlcNAc) at its N-terminus (GlcNAc-Q11). GlcNAc-Q11 self-assembled into ß-sheet nanofibers under similar conditions as Q11. Nanofibrillar GlcNAc moieties were efficiently converted to the galectin-binding disaccharide N-acetyllactosamine (LacNAc) via the enzyme ß-1,4-galactosyltransferase and the sugar donor UDP-galactose, while retaining ß-sheet structure and nanofiber morphology. LacNAc-Q11 nanofibers bound galectin-1 and -3 in a LacNAc concentration-dependent manner, although nanofibers bound galectin-1 with higher affinity than galectin-3. In contrast, galectin-1 bound weakly to GlcNAc-Q11 nanofibers, while no galectin-3 binding to these nanofibers was observed. Galectin-1 binding to LacNAc-Q11 nanofibers was specific because it could be inhibited by excess soluble ß-lactose, a galectin-binding carbohydrate. LacNAc-Q11 nanofibers inhibited galectin-1-mediated apoptosis of Jurkat T cells in a LacNAc concentration-dependent manner, but were unable to inhibit galectin-3 activity, consistent with galectin-binding affinity of the nanofibers. We envision that glycopeptide nanofibers capable of modulating galectin-1 bioactivity will be broadly useful as biomaterials for various medical applications, including cancer therapeutics, immunotherapy, tissue regeneration, and viral prophylaxis.

Integrin-specific hydrogels as adaptable tumor organoids for malignant B and T cells.

Non-Hodgkin lymphomas are a heterogeneous group of lymphoproliferative disorders of B and T cell origin that are treated with chemotherapy drugs with variable success rate that has virtually not changed over decades. Although new classes of chemotherapy-free epigenetic and metabolic drugs have emerged, durable responses to these conventional and new therapies are achieved in a fraction of cancer patients, with many individuals experiencing resistance to the drugs. The paucity in our understanding of what regulates the drug resistance phenotype and establishing a predictive indicator is, in great part, due to the lack of adequate ex vivo lymphoma models to accurately study the effect of microenvironmental cues in which malignant B and T cell lymphoma cells arise and reside. Unlike many other tumors, lymphomas have been neglected from biomaterials-based microenvironment engineering standpoint. In this study, we demonstrate that B and T cell lymphomas have different pro-survival integrin signaling requirements (αvß3 and α4ß1) and the presence of supporting follicular dendritic cells are critical for enhanced proliferation in three-dimensional (3D) microenvironments. We engineered adaptable 3D tumor organoids presenting adhesive peptides with distinct integrin specificities to B and T cell lymphoma cells that resulted in enhanced proliferation, clustering, and drug resistance to the chemotherapeutics and a new class of histone deacetylase inhibitor (HDACi), Panobinostat. In Diffuse Large B cell Lymphomas, the 3D microenvironment upregulated the expression level of B cell receptor (BCR), which supported the survival of B cell lymphomas through a tyrosine kinase Syk in the upstream BCR pathway. Our integrin specific ligand functionalized 3D organoids mimic a lymphoid neoplasm-like heterogeneous microenvironment that could, in the long term, change the understanding of the initiation and progression of hematological tumors, allow primary biospecimen analysis, provide prognostic values, and importantly, allow a faster and more rational screening and translation of therapeutic regimens.

Integrin αvß3 acting as membrane receptor for thyroid hormones mediates angiogenesis in malignant T cells.

The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvß3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvß3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvß3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvß3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvß3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Gradated assembly of multiple proteins into supramolecular nanomaterials.

Biomaterials exhibiting precise ratios of different bioactive protein components are critical for applications ranging from vaccines to regenerative medicine, but their design is often hindered by limited choices and cross-reactivity of protein conjugation chemistries. Here, we describe a strategy for inducing multiple different expressed proteins of choice to assemble into nanofibres and gels with exceptional compositional control. The strategy employs 'ßTail' tags, which allow for good protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional ß-sheet fibrillizing peptides. Multiple different ßTail fusion proteins could be inserted into peptide nanofibres alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. The technology is illustrated by achieving precisely targeted hues using mixtures of fluorescent proteins, by creating nanofibres bearing enzymatic activity, and by adjusting antigenic dominance in vaccines.

Controllably degradable ß-sheet nanofibers and gels from self-assembling depsipeptides.

Self-assembled peptide materials have received considerable interest for a range of applications, including 3D cell culture, tissue engineering, and the delivery of cells and drugs. One challenge in applying such materials within these areas has been the extreme stability of ß-sheet fibrillized peptides, which are resistant to proteolysis, degradation, and turnover in biological environments. In this study, we designed self-assembling depsipeptides containing ester bonds within the peptide backbone. Beta-sheet fibrillized nanofibers were formed in physiologic conditions, and two of these nanofiber-forming depsipeptides produced hydrogels that degraded controllably over the course of days-to-weeks via ester hydrolysis. With HPLC, TEM, and oscillating rheometry, we show that the rate of hydrolysis can be controlled in a straightforward manner by specifying the amino acid residues surrounding the ester bond. In 3D cell cultures, depsipeptide gels softened over the course of several days and permitted considerably more proliferation and spreading of C3H10T1/2 pluripotent stem cells than non-degradable analogs. This approach now provides a reliable and reproducible means to soften or clear ß-sheet fibrillized peptide materials from biological environments.

A self-adjuvanting supramolecular vaccine carrying a folded protein antigen.

This work illustrates a strategy for the design of molecularly defined immunotherapies, using a blend of supramolecular peptide self-assembly and active site-directed protein capture.

Regulation of endothelial cell activation and angiogenesis by injectable peptide nanofibers.

RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. The results were validated in vivo using a mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, and increased ß(3) integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of ß(3) and MAPK/ERK pathway inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. The addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of ß(3) integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize biocompatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffold materials.

Fibrillized peptide microgels for cell encapsulation and 3D cell culture.

One of the advantages of materials produced by self-assembly is that in principle they can be formed in any given container to produce materials of predetermined shapes and sizes. Here, we developed a method for triggering peptide self-assembly within the aqueous phase of water-in-oil emulsions to produce spherical microgels composed of fibrillized peptides. Size control over the microgels was achieved by specification of blade type, speed, and additional shear steps in the emulsion process. Microgels constructed in this way could then be embedded within other self-assembled peptide matrices by mixing pre-formed microgels with un-assembled peptides and inducing gelation of the entire composite, offering a route towards multi-peptide materials with micron-scale domains of different peptide formulations. The gels themselves were cytocompatible, as was the microgel fabrication procedure, enabling the encapsulation of NIH 3T3 fibroblasts and C3H10T-1/2 mouse pluripotent stem cells with good viability.

A self-assembling peptide acting as an immune adjuvant.

The development of vaccines and other immunotherapies has been complicated by heterogeneous antigen display and the use of incompletely defined immune adjuvants with complex mechanisms of action. We have observed strong antibody responses in mice without the coadministration of any additional adjuvant by noncovalently assembling a T and B cell epitope peptide into nanofibers using a short C-terminal peptide extension. Self-assembling peptides have been explored recently as scaffolds for tissue engineering and regenerative medicine, but our results indicate that these materials may also be useful as chemically defined adjuvants. In physiological conditions, these peptides self-assembled into long, unbranched fibrils that displayed the epitope on their surfaces. IgG1, IgG2a, and IgG3 were raised against epitope-bearing fibrils in levels similar to the epitope peptide delivered in complete Freund's adjuvant (CFA), and IgM production was even greater for the self-assembled epitope. This response was dependent on self-assembly, and the self-assembling sequence was not immunogenic by itself, even when delivered in CFA. Undetectable levels of interferon-gamma, IL-2, and IL-4 in cultures of peptide-challenged splenocytes from immunized mice suggested that the antibody responses did not involve significant T cell help.