Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic.

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer.

Papain-like cysteine proteases are composed of 11 human cysteine cathepsins, originally located in the lysosomes. They exhibit broad specificity and act as endopeptidases and/or exopeptidases. Among them, only cathepsins B, H, C, and X/Z exhibit exopeptidase activity. Recently, cysteine cathepsins have been found to be present outside the lysosomes and often participate in various pathological processes. Hence, they have been considered key signalling molecules. Their potentially hazardous proteolytic activities are tightly regulated. This review aims to discuss recent advances in understanding the structural aspects of these four cathepsins, mechanisms of their zymogen activation, regulation of their activities, and functional aspects of these enzymes in neurodegeneration and cancer. Neurodegenerative effects have been evaluated, particularly in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuropsychiatric disorders. Cysteine cathepsins also participate in tumour progression and metastasis through the overexpression and secretion of proteases, which trigger extracellular matrix degradation. To our knowledge, this is the first review to provide an in-depth analysis regarding the roles of cysteine cathepsins B, H, C, and X in neurodegenerative diseases and cancer. Further advances in understanding the functions of cysteine cathepsins in these conditions will result in the development of novel, targeted therapeutic strategies.

The multifaceted roles of tumor-associated proteases and harnessing their activity for prodrug activation.

The role of proteases in cancer was originally thought to be limited to the breakdown of basement membranes and extracellular matrix (ECM), thereby promoting cancer cell invasion into surrounding normal tissues. It is now well understood that proteases play a much more complicated role in all stages of cancer progression and that not only tumor cells, but also stromal cells are an important source of proteases in the tumor microenvironment. Among all the proteolytic enzymes potentially associated with cancer, some proteases have taken on heightened importance due to their significant up-regulation and ability to participate at multiple stages of cancer progression and metastasis. In this review, we discuss some of the advances in understanding of the roles of several key proteases from different classes in the development and progression of cancer and the potential to leverage their upregulated activity for the development of novel targeted treatment strategies.

Crumpled Aluminum Hydroxide Nanostructures as a Microenvironment Dysregulation Agent for Cancer Treatment.

Owing to their unique physicochemical properties, nanomaterials have become a focus of multidisciplinary research efforts including investigations of their interactions with tumor cells and stromal compartment of tumor microenvironment (TME) toward the development of next-generation anticancer therapies. Here, we report that agglomerates of radially assembled Al hydroxide crumpled nanosheets exhibit anticancer activity due to their selective adsorption properties and positive charge. This effect was demonstrated in vitro by decreased proliferation and viability of tumor cells, and further confirmed in two murine cancer models. Moreover, Al hydroxide nanosheets almost completely inhibited the growth of murine melanoma in vivo in combination with a minimally effective dose of doxorubicin. Our direct molecular dynamics simulation demonstrated that Al hydroxide nanosheets can cause significant ion imbalance in the living cell perimembranous space through the selective adsorption of extracellular anionic species. This approach to TME dysregulation could lay the foundation for development of novel anticancer therapy strategies.

A role for stefin B (cystatin B) in inflammation and endotoxemia.

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1ß and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1ß processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1ß, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.

Selective targeting of tumor and stromal cells by a nanocarrier system displaying lipidated cathepsin B inhibitor.

Cathepsin B (CtsB) is a lysosomal cysteine proteinase that is specifically translocated to the extracellular milieu during cancer progression. The development of a lipidated CtsB inhibitor incorporated into the envelope of a liposomal nanocarrier (LNC-NS-629) is described. Ex vivo and in vivo studies confirmed selective targeting and internalization of LNC-NS-629 by tumor and stromal cells, thus validating CtsB targeting as a highly promising approach to cancer diagnosis and treatment.

Isolating Primary Tumor Cells from the MMTV-PyMT Mouse Model and Their Use in Developing an Orthotopic Mouse Model of Breast Cancer.

To study the effect of the immunologically unimpaired microenvironment on tumor progression as well as the efficacy of therapies requiring a functioning immune system, xenograft models are not suitable due to the use of immunodeficient mice. With orthotopic congenic transplantation of tumor cells into mammary tissue, we gain more control and reproducibility regarding tumor growth, while retaining a functioning immune response. Here, we provide a protocol for isolating primary tumor cells from the MMTV-PyMT mouse model and their use in developing an orthotopic mouse model of breast cancer.

Cysteine cathepsins: from structure, function and regulation to new frontiers.

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

The proinflammatory cytokines interleukin-1α and tumor necrosis factor α promote the expression and secretion of proteolytically active cathepsin S from human chondrocytes.

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice.

The lysosomal cysteine proteases cathepsin B (Ctsb) and cathepsin Z (Ctsz, also called cathepsin X/P) have been implicated in cancer pathogenesis. Compensation of Ctsb by Ctsz in Ctsb (-/-) mice has been suggested. To further define the functional interplay of these proteases in the context of cancer, we generated Ctsz null mice, crossed them with Ctsb-deficient mice harboring a transgene for the mammary duct-specific expression of polyoma middle T oncogene (PymT), and analyzed the effects of single and combined Ctsb and Ctsz deficiencies on breast cancer progression. Single Ctsb deficiency resulted in delayed detection of first tumors and reduced tumor burden, whereas Ctsz-deficient mice had only a prolonged tumor-free period. However, only a trend toward reduced metastatic burden without statistical significance was detected in both single mutants. Strikingly, combined loss of Ctsb and Ctsz led to additive effects, resulting in significant and prominent delay of early and advanced tumor development, improved histopathologic tumor grading, as well as a 70% reduction in the number of lung metastases and an 80% reduction in the size of these metastases. We conclude that the double deficiency of Ctsb and Ctsz exerts significant synergistic anticancer effects, whereas the single deficiencies demonstrate at least partial reciprocal compensation.

A New Cathepsin D Targeting Drug Delivery System Based on Immunoliposomes Functionalized with Lipidated Pepstatin A.

Cathepsin D is an aspartic protease and one of the most abundant proteases. It is overexpressed in many cancers and plays an important role in tumor development, progression, and metastasis. While it is a physiologically intracellular protein, cathepsin D is secreted into the extracellular matrix under pathological conditions, making it an appealing target for drug delivery systems. Here, we present the development and evaluation of a new delivery system for tumor targeting based on immunoliposomes functionalized with pepstatin A-a natural peptide inhibitor of cathepsin D. A lipid tail was added to pepstatin A, enabling its incorporation into the liposomal lipid bilayer. The successful targeting of cathepsin D was confirmed using recombinant cathepsin D and in tumor cell lines, showing the feasibility of this targeting approach and its potential for in vivo use in theragnostic applications.

Computational Indicator Approach for Assessment of Nanotoxicity of Two-Dimensional Nanomaterials.

The increasing growth in the development of various novel nanomaterials and their biomedical applications has drawn increasing attention to their biological safety and potential health impact. The most commonly used methods for nanomaterial toxicity assessment are based on laboratory experiments. In recent years, with the aid of computer modeling and data science, several in silico methods for the cytotoxicity prediction of nanomaterials have been developed. An affordable, cost-effective numerical modeling approach thus can reduce the need for in vitro and in vivo testing and predict the properties of designed or developed nanomaterials. We propose here a new in silico method for rapid cytotoxicity assessment of two-dimensional nanomaterials of arbitrary chemical composition by using free energy analysis and molecular dynamics simulations, which can be expressed by a computational indicator of nanotoxicity (CIN2D). We applied this approach to five well-known two-dimensional nanomaterials promising for biomedical applications: graphene, graphene oxide, layered double hydroxide, aloohene, and hexagonal boron nitride nanosheets. The results corroborate the available laboratory biosafety data for these nanomaterials, supporting the applicability of the developed method for predictive nanotoxicity assessment of two-dimensional nanomaterials.

Theranostic Applications of an Ultra-Sensitive T1 and T2 Magnetic Resonance Contrast Agent Based on Cobalt Ferrite Spinel Nanoparticles.

Nano-dimensional materials have become a focus of multiple clinical applications due to their unique physicochemical properties. Magnetic nanoparticles represent an important class of nanomaterials that are widely studied for use as magnetic resonance (MR) contrast and drug delivery agents, especially as they can be detected and manipulated remotely. Using magnetic cobalt ferrite spinel (MCFS) nanoparticles, this study was aimed at developing a multifunctional drug delivery platform with MRI capability for use in cancer treatment. We found that MCFS nanoparticles demonstrated outstanding properties for contrast MRI (r1 = 22.1 s-1mM-1 and r2 = 499 s-1mM-1) that enabled high-resolution T1- and T2-weighted MRI-based signal detection. Furthermore, MCFS nanoparticles were used for the development of a multifunctional targeted drug delivery platform for cancer treatment that is concurrently empowered with the MR contrast properties. Their therapeutic effect in systemic chemotherapy and unique MRI double-contrast properties were confirmed in vivo using a breast cancer mouse tumor model. Our study thus provides an empirical basis for the development of a novel multimodal composite drug delivery system for anticancer therapy combined with noninvasive MRI capability.

A Probody T Cell-Engaging Bispecific Antibody Targeting EGFR and CD3 Inhibits Colon Cancer Growth with Limited Toxicity.

T cell-engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are "masked" to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications. SIGNIFICANCE: A conditionally active EGFR-CD3 T cell-engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.

Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody-Drug Conjugate.

Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.

Praluzatamab Ravtansine, a CD166-Targeting Antibody-Drug Conjugate, in Patients with Advanced Solid Tumors: An Open-Label Phase I/II Trial.

PURPOSE: Praluzatamab ravtansine (CX-2009) is a conditionally activated Probody drug conjugate (PDC) comprising an anti-CD166 mAb conjugated to DM4, with a protease-cleavable linker and a peptide mask that limits target engagement in normal tissue and circulation. The tumor microenvironment is enriched for proteases capable of cleaving the linker, thereby releasing the mask, allowing for localized binding of CX-2009 to CD166. CX-2009 was evaluated in a phase I/II clinical trial for patients with advanced solid tumors. PATIENTS AND METHODS: Eligible patients had metastatic cancer receiving ≥2 prior treatments. CX-2009 was administered at escalating doses every 3 weeks (0.25-10 mg/kg) or every 2 weeks (4-6 mg/kg). Primary objective was to determine the safety profile and recommended phase II dose (RP2D). RESULTS: Of 99 patients enrolled, the most prevalent subtype was breast cancer (n = 45). Median number of prior therapies was 5 (range, 1-19). Dose-limiting toxicities were observed at 8 mg/kg every 3 weeks and 6 mg/kg every 2 weeks. On the basis of tolerability, the RP2D was 7 mg/kg every 3 weeks. Tumor regressions were observed at doses ≥4 mg/kg. In the hormone receptor-positive/HER2-nonamplified breast cancer subset (n = 22), 2 patients (9%) had confirmed partial responses, and 10 patients (45%) had stable disease. Imaging with zirconium-labeled CX-2009 confirmed uptake in tumor lesions and shielding of major organs. Activated, unmasked CX-2009 was measurable in 18 of 22 posttreatment biopsies. CONCLUSIONS: CD166 is a novel, ubiquitously expressed target. CX-2009 is the first conditionally activated antibody-drug conjugate to CD166 to demonstrate both translational and clinical activity in a variety of tumor types.

Promising approaches in using magnetic nanoparticles in oncology.

The development of new and effective drug delivery systems for cancer treatment represents one of the significant challenges facing biomedical technology in the last decade. Among the different methods of drug delivery, magnetic drug targeting, by enabling specific delivery of chemotherapeutic agents through the use of magnetic nanoparticles and magnetic field gradient, could be a promising approach. Recently, magnetic nanoparticles have attracted additional attention because of their potential as contrast agents for magnetic resonance imaging and heat mediators for cancer therapy. This review summarizes these approaches in the use of magnetic nanoparticles in biomedical applications and novel methods for their optimization.

Analyzing the Role of Proteases in Breast Cancer Progression and Metastasis Using Primary Cells from Transgenic Oncomice.

It is becoming increasingly evident that progression and metastasis of solid cancers is driven by the interaction of oncogene-transformed cancer cells and non-malignant host cells in the tumor stroma. In this process, the immune system contributes a complex set of highly important pro- and antitumor effects, which are not readily recapitulated by commonly used xenograft cancer models in immunodeficient mice.Therefore, we provide protocols for isolation of primary tumor cells from the MMTV-PymT mouse model for metastasizing breast cancer and their resubmission to congenic immunocompetent mice by orthotopic transplantation into the mammary gland or different routes of injection to induce organ-specific experimental metastasis, including intravenous, intracardiac, and caudal artery injection of tumor cells. Moreover, we describe protocols for sensitive detection and quantification of the metastatic burden.

Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies.

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.

Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.