Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.

3D printed hydroxyapatite-nacre-starch based bone grafts: Evaluation of biological and mechanical properties.

The possibilities of utilizing nacre as a reinforcing material to manufacture 3D printed bone grafts are yet to be explored. This work reports the feasibility of fabricating 3D printed nacre-hydroxyapatitestarch composite bone graft substitutes, emphasizing the effects of nacre addition on biological and mechanical properties. Pressure-less extrusion-based 3D printing of ceramic-polymer viscous slurry is challenging due to the composition and process-parameter variations. To overcome these challenges, a dual extrusion solid freeform fabricator (SFF) has been designed. An increase in nacre loading improves the compressive strength from 9.5 ± 0.1 MPa to 11.7 ± 0.2 MPa, without any post-processing or sintering. Nacre's in vitro osteogenic properties lead to a slight increase in hFOB cellular attachment on the graft surface by day 11. The fabricated structures show good mechanical integrity during the dissolution study in simulated body fluid (SBF). These bone graft substitutes may be utilized to repair low load bearing skeletal defects.

Effects of chitosan-loaded hydroxyapatite on osteoblasts and osteosarcoma for chemopreventative applications.

Osteosarcoma remains one of the most common malignant primary bone tumors. Post-surgical defect repair combined with tumor suppression remains a major clinical challenge. Investigations of alternative treatments for osteosarcoma, while promising, have led to multi-drug resistance. These constraints of common treatment strategies have triggered the need for new therapeutic candidates in bone cancer treatment. Chitosan, a common biopolymer utilized in bone and tissue engineering applications, has recently been studied as a pro-apoptotic agent in metastatic cell lines like breast cancer, but has not been utilized in bone cancer applications. In this study, chitosan was directly loaded onto HA disks to evaluate its in vitro release and effects on human fetal osteoblast (hFOB) and human osteosarcoma (MG-63) cell lines. It is hypothesized that the sustained release of chitosan will decrease osteosarcoma cell proliferation and enhance proliferation of osteoblast cells. Through morphological characterization and MTT assay analysis, chitosan showed no toxicity to human fetal osteoblast (hFOB) cells. Chitosan was also shown to decrease human osteosarcoma cell viability by up to 96% compared to control samples. This suggests a pro-apoptotic mechanism against osteosarcoma as well as the potential clinical application of chitosan as a drug candidate in ceramic scaffolds at tumor resected sites.

Cytotoxic and osteogenic effects of crocin and bicarbonate from calcium phosphates for potential chemopreventative and anti-inflammatory applications in vitro and in vivo.

Delayed healing and nonhealing of bone defects or resected bone sites remains an important clinical concern in the biomedical field. Osteosarcoma is one of the most common types of primary bone cancers. Among calcium phosphates, hydroxyapatite (HA) and tricalcium phosphate (TCP) are the most widely used in various biomedical applications for bone reconstruction and replacement. In this study, crocin, saffron's natural bioactive and anti-inflammatory molecule, and bicarbonate, a neutralizing agent, were directly loaded onto HA disks to evaluate their in vitro release and effect on human osteoblast and osteosarcoma cell lines. This was assessed through release, initial toxicity, drug optimization, final toxicity studies and in vivo anti-inflammatory assessment through H&E indexing. It is hypothesized that the release of crocin, bicarbonate, and the dual release of both agents will decrease osteosarcoma cellular viability with no effect on osteoblast cells. A plateaued release of crocin and bicarbonate was achieved over seven weeks in physiological and acidic environments, where bicarbonate was shown to modulate the release of crocin. Through morphological characterization and MTT assay analysis, bicarbonate showed no toxicity to human fetal osteoblast (hFOB) cells and crocin significantly enhanced osteoblast proliferation. Through drug concentration optimization, all drug loaded samples decreased human osteosarcoma (MG-63) viability by 50% compared to control samples by Day 11, with clear changes in cell spreading and morphology. Moreover, 3D printed TCP scaffolds loaded with crocin and bicarbonate were tested in vivo in order to assess their preliminary effects on inflammation in a rat distal femur model at 4 days. Lower inflammatory cellular recruitment was achieved in the presence of crocin and bicarbonate, compared to the control. These results suggest a pro-apoptotic mechanism against osteosarcoma as well as anti-inflammatory properties of crocin and bicarbonate, elucidating a potential application for osteosarcoma regulation and wound healing for bone tissue regeneration applications.

Starch-Hydroxyapatite Composite Bone Scaffold Fabrication Utilizing a Slurry Extrusion-Based Solid Freeform Fabricator.

Significant efforts have been made to treat bone disorders through the development of composite scaffolds utilizing calcium phosphate (CaP) through additive manufacturing techniques. However, the incorporation of natural polymers with CaP during 3D printing is difficult and remains a formidable challenge in bone and tissue engineering applications. The objective of this study is to understand the use of a natural polymer binder system in ceramic composite scaffolds using a ceramic slurry-based solid freeform fabricator (SFF). This was achieved through the utilization of naturally sourced gelatinized starch with hydroxyapatite (HA) ceramic in order to obtain high mechanical strength and enhanced biological properties of the green part without the need for cross-linking or post processing. The parametric effects of solids loading, polycaprolactone (PCL) polymer addition, and designed porosity on starch-HA composite scaffolds were assessed through mechanical strength, microstructure, and in vitro biocompatibility utilizing human osteoblast cells. It was hypothesized that starch incorporation would improve the mechanical strength of the scaffolds and increase proliferation of osteoblast cells in vitro. Starch loading was shown to improve mechanical strength from 4.07 ± 0.66 MPa to 10.35 ± 1.10 MPa, more closely resembling the mechanical strength of cancellous bone. Based on these results, a reinforcing mechanism of gelatinized starch based on interparticle and apatite crystal interlocking is proposed. Morphological characterization utilizing FESEM and MTT cell viability assay showed enhanced osteoblast cell proliferation in the presence of starch and PCL. Overall, the utilization of starch as a natural binder system in SFF scaffolds was found to improve both green strength and in vitro biocompatibility.