Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

Differential protein-protein interactions underlie signaling mediated by the TCR and a 4-1BB domain-containing CAR.

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)-mediated signaling, but the number and type of protein interaction-mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.

Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

Synthesis and characterization of a matrix-metalloproteinase responsive silk-elastinlike protein polymer.

Silk-elastinlike protein polymers (SELPs) are recombinant polymers consisting of tandem repeats of silk (GAGAGS) and elastin (GVGVP) units. By modification of the length and composition of these repeats, the properties of SELP hydrogels can be controlled for specific applications including nucleic acid and virus delivery and tissue engineering. Here, the structure of SELPs is further modified to include a sequence that is sensitive to matrix-metalloproteinases (MMPs). MMPs are a ubiquitous family of extracellular matrix-modifying enzymes that are commonly associated with numerous vital processes. Increased levels of MMPs are found at high levels locally in many types of solid tumors. By modifying the SELP backbone with MMP-sensitive peptide sequences, a hydrogel that is degradable by MMPs was produced. The MMP-sensitivity of the polymer was examined by incubation with MMP-2 and MMP-9, which yielded complete cleavage of all full-length polymers by 36 hours and 48 hours, respectively, with no observable effect on unmodified SELP. Hydrogel sensitivity was tested by exposure to MMP-2 or MMP-9 for 2 weeks, during which samples were taken to analyze protein loss from the hydrogel and release of 100 nm fluorescent beads. Following the incubation period, hydrogels were tested in mechanical compression to examine the loss of hydrogel stiffness due to degradation. It was found that MMP-2 and MMP-9 caused 63% and 44% increased protein loss and 65% and 95% increased release from MMP-sensitive hydrogels, while the compressive modulus decreased by 41% and 29%. These results suggest the potential of MMP-responsive SELPs for localized delivery of bioactive agents where MMPs are overexpressed.

Locoregional CAR T cells for children with CNS tumors: Clinical procedure and catheter safety.

Central nervous system (CNS) tumors are the most common solid malignancy in the pediatric population. Based on adoptive cellular therapy's clinical success against childhood leukemia and the preclinical efficacy against pediatric CNS tumors, chimeric antigen receptor (CAR) T cells offer hope of improving outcomes for recurrent tumors and universally fatal diseases such as diffuse intrinsic pontine glioma (DIPG). However, a major obstacle for tumors of the brain and spine is ineffective T cell chemotaxis to disease sites. Locoregional CAR T cell delivery via infusion through an intracranial catheter is currently under study in multiple early phase clinical trials. Here, we describe the Seattle Children's single-institution experience including the multidisciplinary process for the preparation of successful, repetitive intracranial T cell infusion for children and the catheter-related safety of our 307 intracranial CAR T cell doses.

Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety.

Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation. SIGNIFICANCE: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1.

Silk-elastin-like hydrogel improves the safety of adenovirus-mediated gene-directed enzyme-prodrug therapy.

Recombinant silk-elastin-like protein polymers (SELPs) are well-known for their highly tunable properties on both the molecular and macroscopic hydrogel levels. One specific structure of these polymers, SELP-815K, has been investigated as an injectable controlled delivery system for the treatment of head and neck cancer via a gene-directed enzyme prodrug therapy (GDEPT) approach. Due to its pore size and gelation properties in vivo, SELP restricts the distribution and controls the release of therapeutic viruses for up to one month. It has been shown that SELP-mediated delivery significantly improves therapeutic outcome of the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system in xenograft models of human head and neck cancer. However little is known about potential benefits of this approach with regard to toxicity in the presence of a fully intact immune system. The studies presented here were designed to assess the change in toxicity of the SELP-mediated viral delivery compared to free viral injection in a non-tumor-bearing immune competent mouse model. Toxicity was assessed at 1, 2, 4, and 12 weeks via body weight monitoring, complete blood count (CBC), and blood chemistry. It was found that in the acute and subacute phases (weeks 1-4) there is significant toxicity in groups combining the virus and the prodrug, and matrix-mediated gene delivery with SELP demonstrates a reduction in toxicity from the 2 week time point through the 4 week time point. At the end of the subchronic phase (12 weeks), signs of toxicity had subsided in both groups. Based on these results, recombinant SELPs offer a significant reduction in toxicity of virus-mediated GDEPT treatment compared to free virus injection in the acute and subacute phases.

Cell-Templated Silica Microparticles with Supported Lipid Bilayers as Artificial Antigen-Presenting Cells for T Cell Activation.

Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, a new platform technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation is presented. Artificial antigen presenting cells (aAPCs) are successfully created using supported lipid bilayers on various cell-templated silica microparticles with defined membrane fluidity and stimulating antibody density. This panel of aAPCs is used to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations are able to stimulate T cell growth, and preferentially promote CD8+ T cell growth over CD4+ T cell growth when compared to commercially available pendant antibody-conjugated particles. T cells cultured with HeLa- and red blood cell-templated aAPCs have a less-differentiated and less-exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica-supported lipid bilayers as an aAPC platform.

Macrophage silica nanoparticle response is phenotypically dependent.

Phagocytes are important players in host exposure to nanomaterials. Macrophages in particular are believed to be among the "first responders" and primary cell types that uptake and process nanoparticles, mediating host biological responses by subsequent interactions with inflammatory signaling pathways and immune cells. However, variations in local microenvironmental cues can significantly change the functional and phenotype of these cells, impacting nanoparticle uptake and overall physiological response. Herein we focus on describing the response of specific RAW 264.7 macrophage phenotypes (M1, INF-gamma/LPS induced and M2, IL-4 induced) to Stöber silica nanoparticle exposure in vitro and how this response might correlate with macrophage response to nanoparticles in vivo. It was observed that variations in macrophage phenotype produce significant differences in macrophage morphology, silica nanoparticle uptake and toxicity. High uptake was observed in M1, versus low uptake in M2 cells. M2 cells also displayed more susceptibility to concentration dependent proliferative effects, suggesting potential M1 involvement in in vivo uptake. Nanoparticles accumulated within liver and spleen tissues, with high association with macrophages within these tissues and an overall Th1 response in vivo. Both in vitro and in vivo studies are consistent in demonstrating that silica nanoparticles exhibit high macrophage sequestration, particularly those with Th1/M1 phenotype and in clearance organs. This sequestration and phenotypic response should be a primary consideration when designing new Stöber silica nanoparticle systems, as it might affect the overall efficacy.

A thermo-responsive protein treatment for dry eyes.

Millions of Americans suffer from dry eye disease, and there are few effective therapies capable of treating these patients. A decade ago, an abundant protein component of human tears was discovered and named lacritin (Lacrt). Lacrt has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium. Similar to other proteins placed on the ocular surface, the durability of its effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-responsive coacervate containing Lacrt would have better retention upon administration, we have constructed and tested the activity of a thermo-responsive Lacrt fused to an elastin-like polypeptide (ELP). Inspired from the human tropoelastin protein, ELP protein polymers reversibly phase separate into viscous coacervates above a tunable transition temperature. This fusion construct exhibited the prosecretory function of native Lacrt as illustrated by its ability to stimulate ß-hexosaminidase secretion from primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered via intra-lacrimal injection. Lacrt ELP fusion proteins undergo temperature-mediated assembly to form a depot inside the lacrimal gland. We propose that these Lacrt ELP fusion proteins represent a potential therapy for dry eye disease and the strategy of ELP-mediated phase separation may have applicability to other diseases of the ocular surface.

Genetically engineered nanocarriers for drug delivery.

Cytotoxicity, low water solubility, rapid clearance from circulation, and off-target side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or non-polymeric. This review summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins.

A hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody.

B-cell lymphomas continue to occur with a high incidence. The chimeric antibody known as Rituximab (Rituxan) has become a vital therapy for these patients. Rituximab induces cell death via binding and clustering of the CD20 receptor by Fcγ expressing effector cells. Because of the limited mobility of effector cells, it may be advantageous to cluster CD20 directly using multivalent nanostructures. To explore this strategy, this manuscript introduces a nanoparticle that assembles from a fusion between a single chain antibody and a soluble protein polymer. These hybrid proteins express in Escherichia coli and do not require bioconjugation between the antibody and a substrate. Surprisingly a fusion between an anti-CD20 single chain antibody and a soluble protein polymer assemble worm-like nanostructures, which were characterized using light scattering and cryogenic transmission electron microscopy. These nanoworms competitively bind CD20 on two B-cell lymphoma cell lines, exhibit concentration-dependent induction of apoptosis, and induce apoptosis better than Rituximab alone. Similar activity was observed in vivo using a non-Hodgkin lymphoma xenograft model. In comparison to Rituximab, systemic nanoworms significantly slowed tumor growth. These findings suggest that hybrid nanoworms targeted at CD20 may be useful treatments for B-cell related malignancies. Because of the ubiquity of antibody therapeutics, related nanoworms may have uses against other molecular targets.

Gold nanorod mediated plasmonic photothermal therapy: a tool to enhance macromolecular delivery.

Plasmonic photothermal therapy (PPTT) with gold nanostructures has been used to generate significant heat within tumors to ablate vasculature. Here we report the use of gold nanorod (GNR) mediated PPTT to induce moderate hyperthermia as a tool to enhance the delivery of macromolecules. GNRs were injected intravenously in a mouse sarcoma (S-180) tumor model. After 24h Evans blue dye (EBD) was injected and the right tumor was radiated with a laser diode for 10 min. EBD content in the right and left tumors were extracted in formamide, measured spectrophotometrically and expressed as a thermal enhancement ratio (TER). Enhanced delivery of EBD was observed (up to 1.8-fold) when tumor temperatures reached 43°C or 46°C. No statistical difference was observed between tumors at these two temperatures, though significant hemorrhage was observed in tumors and surrounding areas receiving the higher thermal dose (46°C). These results indicate that tumor directed PPTT may be used to induce moderate hyperthermia and therefore selectively increase the delivery of macromolecules with therapeutic anticancer drugs.

Fabrication of highly uniform nanoparticles from recombinant silk-elastin-like protein polymers for therapeutic agent delivery.

Here we generate silk-elastin-like protein (SELP) polymeric nanoparticles and demonstrate precise control over their dimensions using an electrospray differential mobility analyzer (ES-DMA). Electrospray produces droplets encompassing several polymer strands. Evaporation ensues, leading polymer strands to accumulate at the droplet interface, forming a hollow nanoparticle. The resulting nanoparticle size distributions, which govern particle yield, depend on buffer concentration to the -1/3 power, polymer concentration to the 1/3 power, and ratio of silk-to-elastin blocks. Three recombinantly tuned ratios of 8:16, 4:8, and 4:16, respectively named SELP-815K, SELP-47K, and SELP-415K, are employed, with the latter ratio resulting in a thinner shell and larger diameter for the nanoparticles than the former. The DMA narrows the size distribution by electrostatically classifying the aerosolized nanoparticles. These highly uniform nanoparticles have variations of 1.2 and 1.4 nm for 24.0 and 36.0 nm particles, respectively. Transmission electron microscopy reveals the nanoparticles to be faceted, as a buckling instability releases compression energy arising from evaporation after the shell has formed by bending it. A thermodynamic equilibrium exists between compression and bending energies, where the facet length is half the particle diameter, in agreement with experiments. Rod-like particles also formed from polymer-stabilized filaments when the viscous length exceeds the jet radius at higher solution viscosities. The unusual uniformity in composition and dimension indicates the potential of these nanoparticles to deliver bioactive and imaging agents.

Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy.

Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews recent developments in the synthesis and characterization of SELP hydrogels and their use for matrix-mediated gene delivery.