Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics.

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs.

Therapeutic HDAC inhibition in hypermutant diffuse intrinsic pontine glioma.

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with the development of hypermutant pediatric high-grade glioma, and confers a poor prognosis. While therapeutic histone deacetylase (HDAC) inhibition of diffuse intrinsic pontine glioma (DIPG) has been reported; here, we use a clinically relevant biopsy-derived hypermutant DIPG model (PBT-24FH) and a CRISPR-Cas9 induced genetic model to evaluate the efficacy of HDAC inhibition against hypermutant DIPG. We screened PBT-24FH cells for sensitivity to a panel of HDAC inhibitors (HDACis) in vitro, identifying two HDACis associated with low nanomolar IC50s, quisinostat (27 nM) and romidepsin (2 nM). In vivo, quisinostat proved more efficacious, inducing near-complete tumor regression in a PBT-24FH flank model. RNA sequencing revealed significant quisinostat-driven changes in gene expression, including upregulation of neural and pro-inflammatory genes. To validate the observed potency of quisinostat in vivo against additional hypermutant DIPG models, we tested quisinostat in genetically-induced mismatch repair (MMR)-deficient DIPG flank tumors, demonstrating that loss of MMR function increases sensitivity to quisinostat in vivo. Here, we establish the preclinical efficacy of quisinostat against hypermutant DIPG, supporting further investigation of epigenetic targeting of hypermutant pediatric cancers with the potential for clinical translation. These findings support further investigation of HDAC inhibitors against pontine high-grade gliomas, beyond only those with histone mutations, as well as against other hypermutant central nervous system tumors.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

Ex silico engineering of cystine-dense peptides yielding a potent bispecific T cell engager.

Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity (KD = 202 pM) PD-L1-binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development.

Medulloblastoma recurrence and metastatic spread are independent of colony-stimulating factor 1 receptor signaling and macrophage survival.

PURPOSE: Tumor infiltration by immunosuppressive myeloid cells or tumor-associated macrophages (TAMs) contributes to tumor progression and metastasis. In contrast to their adult counterparts, higher TAM signatures do not correlate with aggressive tumor behavior in pediatric brain tumors. While prominent TAM infiltrates exist before and after radiation, the degree to which irradiated macrophages and microglia support progression or leptomeningeal metastasis remains unclear. Patients with medulloblastoma often present with distant metastases and tumor recurrence is largely incurable, making them prime candidates for the study of novel approaches to prevent neuroaxis dissemination and recurrence. METHODS: Macrophage depletion was achieved using CSF-1 receptor inhibitors (CSF-1Ri), BLZ945 and AFS98, with or without whole brain radiation in a variety of medulloblastoma models, including patient-derived xenografts bearing Group 3 medulloblastoma and a transgenic Sonic Hedgehog (Ptch1+/-, Trp53-/-) medulloblastoma model. RESULTS: Effective reduction of microglia, TAM, and spinal cord macrophage with CSF-1Ri resulted in negligible effects on the rate of local and spinal recurrences or survival following radiation. Results were comparable between medulloblastoma subgroups. While notably few tumor-infiltrating lymphocytes (TILs) were detected, average numbers of CD3+ TILs and FoxP3+ Tregs did not differ between groups following treatment and tumor aggressiveness by Ki67 proliferation index was unaltered. CONCLUSION: In the absence of other microenvironmental influences, medulloblastoma-educated macrophages do not operate as tumor-supportive cells or promote leptomeningeal recurrence in these models. Our data add to a growing body of literature describing a distinct immunophenotype amid the medulloblastoma microenvironment and highlight the importance of appropriate pediatric modeling prior to clinical translation.

Optimal therapeutic targeting by HDAC inhibition in biopsy-derived treatment-naïve diffuse midline glioma models.

BACKGROUND: Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), have a dismal prognosis, with less than 2% surviving 5 years postdiagnosis. The majority of DIPGs and all DMGs harbor mutations altering the epigenetic regulatory histone tail (H3 K27M). Investigations addressing DMG epigenetics have identified a few promising drugs, including the HDAC inhibitor (HDACi) panobinostat. Here, we use clinically relevant DMG models to identify and validate other effective HDACi and their biomarkers of response. METHODS: HDAC inhibitors were tested across biopsy-derived treatment-naïve in vitro and in vivo DMG models with biologically relevant radiation resistance. RNA sequencing was performed to define and compare drug efficacy and to map predictive biomarkers of response. RESULTS: Quisinostat and romidepsin showed efficacy with low nanomolar half-maximal inhibitory concentration (IC50) values (~50 and ~5 nM, respectively). Comparative transcriptome analyses across quisinostat, romidepsin, and panobinostat showed a greater degree of shared biological effects between quisinostat and panobinostat, and less overlap with romidepsin. However, some transcriptional changes were consistent across all 3 drugs at similar biologically effective doses, such as overexpression of troponin T1 slow skeletal type (TNNT1) and downregulation of collagen type 20 alpha 1 chain (COL20A1), identifying these as potential vulnerabilities or on-target biomarkers in DMG. Quisinostat and romidepsin significantly (P < 0.0001) inhibited in vivo tumor growth. CONCLUSIONS: Our data highlight the utility of treatment-naïve biopsy-derived models; establishes quisinostat and romidepsin as effective in vivo; illuminates potential mechanisms and/or biomarkers of DMG cell lethality due to HDAC inhibition; and emphasizes the need for brain tumor-penetrant versions of potentially efficacious agents.

A TfR-Binding Cystine-Dense Peptide Promotes Blood-Brain Barrier Penetration of Bioactive Molecules.

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.

A potent peptide-steroid conjugate accumulates in cartilage and reverses arthritis without evidence of systemic corticosteroid exposure.

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.

A Protocol for the Generation of Treatment-naïve Biopsy-derived Diffuse Intrinsic Pontine Glioma and Diffuse Midline Glioma Models.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal tumor of the brainstem, most commonly affecting young children. Due to its location, surgical resection is not achievable, but consideration of a biopsy has become standard practice at children's hospitals with the appropriate neurosurgical expertise. While the decision to obtain a biopsy should be directed by the presence of atypical radiographic features that call the diagnosis of DIPG into question or the requirement of biopsy tissue for clinical trial enrollment, once this precious tissue is available its use for research should be considered. The majority of DIPG and diffuse midline glioma, H3 K27M-mutant (DMG) models are autopsy-derived or genetically-engineered, each of which has limitations for translational studies, so the use of biopsy tissue for laboratory model development provides an opportunity to create unique model systems. Here, we present a detailed laboratory protocol for the generation of treatment-naïve biopsy-derived DIPG/DMG models.

Calibration of fluorescence imaging for tumor surgical margin delineation: multistep registration of fluorescence and histological images.

Although a greater extent of tumor resection is important for patients' survival, complete tumor removal, especially tumor margins, remains challenging due to the lack of sensitivity and specificity of current surgical guidance techniques at the margins. Intraoperative fluorescence imaging with targeted fluorophores is promising for tumor margin delineation. To verify the tumor margins detected by the fluorescence images, it is necessary to register fluorescence with histological images, which provide the ground truth for tumor regions. However, current registration methods compare fluorescence images to a single-layer histological slide, which is selected subjectively and represents a single plane of the three-dimensional tumor. A multistep pipeline is established to correlate fluorescence images to stacked histological images, including fluorescence calibration and multistep registration. Multiple histological slices are integrated as a two-dimensional (2-D) tumor map using optical attenuation model and average intensity projection. A BLZ-100-labeled medulloblastoma mouse model is used to test the whole framework. On average, the synthesized 2-D tumor map outperforms the selected best slide as ground truth [Dice similarity coefficient (DSC): 0.582 versus 0.398, with significant differences; mean area under the curve (AUC) of the receiver operating characteristic curve: 88% versus 85.5%] and the randomly selected slide as ground truth (DSC: 0.582 versus 0.396 with significant differences; mean AUC: 88% versus 84.1% with significant differences), which indicates our pipeline is reliable and can be applied to investigate targeted fluorescence probes in tumor margin detection. Following this proposed pipeline, BLZ-100 shows enhancement in both tumor cores and tumor margins (mean target-to-background ratio: 8.64 ± 5.76 and 4.82 ± 2.79 , respectively).

A biobank of patient-derived pediatric brain tumor models.

Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments, better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children's Oncology Group ACNS02B3 study, we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number, and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies.