Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodeling. While MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application due to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP over-expression following a myocardial infarction (MI) significantly reduced MMP activity and attenuated adverse left ventricular remodeling in a porcine model of MI. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.

The production of complex, yet well defined materials offers many opportunities in regenerative medicine, in which the mechanical and biological properties of the matrix must meet stringent requirements. Here we report the recombinant production of modular polypeptidic materials, based on the highly resilient protein resilin, which are equipped with multiple biologically active domains. The recombinant materials exhibit useful mechanical and cell adhesion behavior.

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC 80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADCresistant cell models retain sensitivity to other ADCs or standardof-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance.

The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a "ring-opened" linker. ADCs containing this hydrolyzed succinimide linker show equivalent cytotoxicity, improved in vitro stability, improved PK exposure, and improved efficacy as compared to their nonhydrolyzed counterparts. This method offers a simple way to improve the stability, exposure, and efficacy of maleimide-based ADCs.

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Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Elastomeric proteins are characterized by their large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Motivated by their unique mechanical properties, there has been tremendous research in understanding and manipulating elastomeric polypeptides, with most work conducted on the elastins but more recent work on an expanded set of polypeptide elastomers. Facilitated by biosynthetic strategies, it has been possible to manipulate the physical properties, conformation, and mechanical properties of these materials. Detailed understanding of the roles and organization of the natural structural proteins has permitted the design of elastomeric materials with engineered properties, and has thus expanded the scope of applications from elucidation of the mechanisms of elasticity to the development of advanced drug delivery systems and tissue engineering substrates.

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