Protease-sensitive Linker Research Articles

In silico evolution of autoinhibitory domains for a PD-L1 antagonist using deep learning models.

There has been considerable progress in the development of computational methods for designing protein-protein interactions, but engineering high-affinity binders without extensive screening and maturation remains challenging. Here, we test a protein design pipeline that uses iterative rounds of deep learning (DL)-based structure prediction (AlphaFold2) and sequence optimization (ProteinMPNN) to design autoinhibitory domains (AiDs) for a PD-L1 antagonist. With the goal of creating an anticancer agent that is inactive until reaching the tumor environment, we sought to create autoinhibited (or masked) forms of the PD-L1 antagonist that can be unmasked by tumor-enriched proteases. Twenty-three de novo designed AiDs, varying in length and topology, were fused to the antagonist with a protease-sensitive linker, and binding to PD-L1 was measured with and without protease treatment. Nine of the fusion proteins demonstrated conditional binding to PD-L1, and the top-performing AiDs were selected for further characterization as single-domain proteins. Without any experimental affinity maturation, four of the AiDs bind to the PD-L1 antagonist with equilibrium dissociation constants (KDs) below 150 nM, with the lowest KD equal to 0.9 nM. Our study demonstrates that DL-based protein modeling can be used to rapidly generate high-affinity protein binders.

Abstract 1817: WTX-613, (JZP898) a selectively activated IFNα INDUKINE™ molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors

Abstract A member of the type-I IFN family, IFNα was also one of the first cytokines tested in the clinic as a therapy for cancer patients, leading to the approval of its use in chronic myelogenous leukemia, lymphoma, and malignant melanoma. Mechanistically, IFNα directly inhibits tumor cell proliferation and induces apoptosis, while simultaneously activating various innate and adaptive immune cell populations. Unfortunately, clinical use of IFNα has been hindered by the adverse events associated with its systemic administration, demonstrating the need for novel approaches to utilize this cytokine in the clinic. WTX-613 is a conditionally activated IFNα prodrug, or INDUKINE™ molecule, which has two identical half-life extension (HLE) domains tethered to IFNα2b via a proprietary tumor protease-sensitive linker. Following systemic administration, WTX-613 circulates as an inactive prodrug that is unable to bind to IFNα receptors in normal tissues due to the steric hinderance of the HLE domains complexed with albumin. However, when WTX-613 enters the tumor, proteases found in the tumor microenvironment (TME) cleave the protease-sensitive linkers, releasing fully active IFNα2b selectively in the tumor. Since human IFNα is not active in mice, a surrogate INDUKINE™ molecule containing mouse IFNα1 (mWTX-613/WW0610) was created. mWTX-613 treatment generated robust anti-tumor immunity in several murine syngeneic tumor models. In the MC38 model, mWTX-613 treatment was more efficacious than equimolar amounts of the recombinant cytokine (mIFNα1) and was accompanied by an increase in the therapeutic window. Mechanistically, systemic treatment with mWTX-613 resulted in prolonged peripheral exposure extending over a week beyond the time of the final dose. mWTX-613 treatment also resulted in a significant increase in the CD8/Treg ratio, and a robust increase in MHC I and MHC II expression by various professional antigen presenting cell populations. In addition, mWTX-613 treatment resulted in a clear and prolonged activation of both tumor infiltrating CD8+ T cells and NK cells. Transcriptionally, systemic treatment with mWTX-613 resulted in prolonged IFNα signaling within the TME, as demonstrated by significant enrichment in multiple Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with an anti-viral immune response. Finally, the combination of mWTX-613 treatment with various checkpoint inhibitor molecules resulted in improved anti-tumor efficacy in syngeneic models. Together, these data support the advancement of this innovative IFNα therapy into clinical testing. Citation Format: Christopher J. Nirschl, Heather R. Brodkin, Celesztina Domonkos, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, William M. Winston, Andres Salmeron. WTX-613, (JZP898) a selectively activated IFNα INDUKINE™ molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1817.

Merging bioresponsive release of insulin-like growth factor I with 3D printable thermogelling hydrogels.

3D printing of biomaterials enables spatial control of drug incorporation during automated manufacturing. This study links bioresponsive release of the anabolic biologic, insulin-like growth factor-I (IGF-I) in response to matrix metalloproteinases (MMP) to 3D printing using the block copolymer of poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine) (POx-b-POzi). For that, a chemo-enzymatic synthesis was deployed, ligating IGF-I enzymatically to a protease sensitive linker (PSL), which was conjugated to a POx-b-POzi copolymer. The product was blended with the plain thermogelling POx-b-POzi hydrogel. MMP exposure of the resulting hydrogel triggered bioactive IGF-I release. The bioresponsive IGF-I containing POx-b-POzi hydrogel system was further detailed for shape control and localized incorporation of IGF-I via extrusion 3D printing for future applications in biomedicine and biofabrication.

Abstract 5487: An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer

Abstract Gastric cancer is a worldwide health problem. Concurrent chemoradiotherapy (CRT) represents an important strategy to reduce recurrence and prolong survival for gastric cancer. However, non-targeted cytotoxic drugs capable of enhancing radiosensitization remain the main choice to be combined with radiotherapy, which has limited the anti-tumor efficacy of CRT by off-target side effects. Monomethyl auristatin E (MMAE) is a highly potent anti-tubulin agent and has been reported to act as an effective radiosensitizer recently. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is a cancer/testis antigen highly and exclusively expressed in multiple types of cancers including gastric cancer, representing a promising target for drug delivery. Previously, we identified a KKLC-1-targeting cyclic nonapeptide (1131 peptide) by screening a phage display peptide library. Here, we described a novel peptide-drug conjugate (PDC) 1131-MMAE by connecting the 1131 peptide with MMAE via a protease-sensitive linker for tumor-selective radiosensitization. We demonstrated that 1131-MMAE could be efficiently internalized by gastric cancer cells through KKLC1-mediating endocytosis and exhibited a comparable cytotoxicity profile with free MMAE. 1131-MMAE could arrest the cell cycle at the most radiosensitive G2/M phase in a dose-dependent manner and selectively enhance the radiation response of KK-LC-1 positive gastric cancer cells by increasing DNA double-strand break as indicated by γH2Ax immunofluorescence staining. Furthermore,1131-MMAE was able to inhibit tumor growth as a monotherapy at 2mg/kg with a good safety profile in vivo. Increased tumor control was achieved when 1131-MMAE was combined with radiotherapy at a relatively low dose of 0.3mg/kg. Overall, we proposed an MMAE-based PDC targeting a novel tumor marker KK-LC-1 and demonstrated its tumor-selective radiosensitization in gastric cancer models for the first time. This anti-KKLC1 PDC may help to promote the clinical progress in precision chemoradiotherapy for multiple types of cancer. Citation Format: Xiaotong Chen, Fangcen Liu, Xiaoxiao Yu, Lin Li, Jiayao Yan, Baorui Liu. An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5487.

Abstract 2054: WTX-124 is a novel IL-2 pro-drug that is conditionally activated in tumors and drives anti-tumor immunity by activating tumor infiltrating CD8+ T cells

Abstract Despite the success of immune checkpoint inhibitors (ICI), clinical responses to ICI treatment are still limited to a fraction of patients within specific indications, demonstrating a significant unmet clinical need for novel immunotherapies. In cancer, preclinical and clinical studies have demonstrated the promise of cytokine therapy to stimulate anti-tumor immunity. One of these key cytokines, interleukin-2 (IL-2), is approved for clinical use in metastatic melanoma and renal cell carcinoma. Unfortunately, IL-2 has a poor pharmacokinetic (PK) profile and is linked to serious toxicities, such as vascular leak syndrome (VLS), which limit its clinical utility. To address these limitations, we designed WTX-124, an IL-2 pro-drug (IL-2 INDUKINE™ protein) that takes advantage of dysregulated protease activity in the tumor microenvironment (TME) to selectively release active IL-2 in the tumor after systemic administration. Peripheral inactivation is achieved by linking the cytokine to an inactivation domain using a tumor protease-sensitive linker. WTX-124 is also engineered with a half-life extension element to improve its PK profile and maintain longer exposure in the tumor. Once WTX-124 reaches the tumor, tumor-associated proteases cleave the linkers and release fully active IL-2. Treatment with WTX-124 resulted in the complete rejection of MC38 tumors in a manner dependent on proteolytic processing of the INDUKINE™ protein, as a non-cleavable version of WTX-124 lacked anti-tumor activity. In addition, tumor rejection was dependent on CD8+ T cells, as CD8+ T cell depletion during WTX-124 treatment resulted in a loss of tumor control. Unlike free IL-2, WTX-124 was well tolerated and did not induce VLS in mice, demonstrating the efficacy of the inactivation domain at restricting peripheral toxicity. Due to its enhanced anti-tumor activity and decreased toxicity profile, the therapeutic window of WTX-124 was greatly improved compared to that of native IL-2. In addition, a WTX-124 variant using attenuated (non-α) IL-2 was not active in our efficacy model. Mechanistically, WTX-124 treatment drove the expansion and activation of tumor infiltrating CD8+ T cells and NK cells. Furthermore, WTX-124 treatment resulted in selective activation of the tumor infiltrating CD8+ T cells, with limited evidence of peripheral T cell activation in the blood, spleen, and lymph nodes of tumor bearing animals. In a less immunogenic model, B16F10, WTX-124 also induced CD8+ T cell and NK cell activation and revealed combinatorial anti-tumor activity with αPD-1 therapy. Lastly, in a sample set of over 100 primary human tumor samples, WTX-124 was broadly processed across many indications and disease stages, while remaining intact in the presence of healthy human primary cells from various tissues. Together, these data support the clinical development of WTX-124. Citation Format: Christopher J. Nirschl, Heather R. Brodkin, Daniel J. Hicklin, Nesreen Ismail, Kristin Morris, Andres Salmeron, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, Jenna M. Sullivan, William M. Winston. WTX-124 is a novel IL-2 pro-drug that is conditionally activated in tumors and drives anti-tumor immunity by activating tumor infiltrating CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2054.

Abstract 1716: WTX-330, a conditionally activated IL-12 INDUKINE, releases IL-12 selectively in the tumor microenvironment to activate anti-tumor immune responses and induce regressions in mouse tumor models

Abstract Systemic therapy with proinflammatory immune modulators is a promising approach to treat cancer by activating innate and adaptive anti-tumor immunity. However, dose-limiting toxicities such as inflammation, cytokine release syndrome, and tissue damage rendered the strategy impractical for cytokines such as the cytotoxic T and NK cell activating interleukin 12 (IL-12). WTX-330 is a novel systemically delivered therapy with targeted intra-tumoral activation to deliver wild-type IL-12 in the tumor microenvironment identified using the Predator™ discovery platform. WTX-330 is a member of a class of inducible polypeptides (INDUKINE™ therapies) designed to be an inactive IL-12 pro-drug with a half-life extension (HLE) domain to support infrequent systemic administration. The INDUKINE™ polypeptide is kept inactive in the periphery via high affinity antibody blockade tethered to IL-12 via a tumor protease-sensitive linker. Cleavage of the same linker also removes the HLE domain producing a PK profile similar to wild-type IL-12. WTX-330 was selected as a lead molecule due to its beneficial in vitro profile. The mouse MC38 syngeneic tumor model was used to demonstrate strong anti-tumor activity of lead mouse INDUKINE™ polypeptides. Intraperitoneal (i.p.) administration of mouse WTX-330 led to complete regression of all MC38 tumors at dose levels which only moderately induced systemic IFNγ and were well tolerated by the mice. Similar efficacy was observed in CT26, B16F10, and EMT-6 syngeneic models. Importantly, wild-type IL-12, while active in mouse tumor models, was not tolerated by the mice after i.p. injection at equimolar amounts compared to the IL-12 INDUKINE™ polypeptide. Mouse WTX-330 strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in MC38 tumors supporting a mechanism of action as described for wild-type IL-12. In non-human primates, WTX-330 was well tolerated at exposure levels which vastly exceeded the levels needed for efficacy in mice. In addition, plasma levels of free IL-12 after dosing with WTX-330 were very low compared to tolerated levels after treatment with wild-type IL-12. Ex vivo incubation of WTX-330 with numerous primary human tumors led to the rapid release of IL-12, while WTX-330 proved to be stable in human serum and normal tissues. Pharmacological and translational data obtained so far clearly support continued preclinical development with the goal of moving this innovative and differentiated engineered IL-12 therapy into human clinical testing. Citation Format: Philipp Steiner, Heather Brodkin, Dan Hicklin, Nesreen Ismail, Kristin Morris, Christopher Nirschl, Andres Salmeron, Cindy Seidel-Dugan, Zoe Steuert, Jenna Sullivan, William Winston. WTX-330, a conditionally activated IL-12 INDUKINE, releases IL-12 selectively in the tumor microenvironment to activate anti-tumor immune responses and induce regressions in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1716.

Abstract 1723: WTX-124 is an IL-2 pro-drug conditionally activated in tumors and able to induce complete regressions in mouse tumor models

Abstract Cancer Immunotherapy has established itself as the fourth pillar of cancer treatment thanks to the clinical success of checkpoint inhibitors. Despite the durable responses achieved by some patients using these new therapies, the proportion of responders remains low and restricted to some cancer types. Preclinical and clinical studies have demonstrated the promise of cytokine therapy to increase antitumor immunity, however systemic toxicity and poor pharmacokinetic profiles have limited their clinical application. One of these key cytokines, interleukin-2 (IL-2), is approved for clinical use in metastatic melanoma and renal cell carcinoma. Unfortunately, this treatment is linked to serious toxicities which limits its utility. To address these limitations, our approach takes advantage of the dysregulated protease tumor microenvironment (TME) to activate an IL-2 pro-drug (IL-2 INDUKINETM) only at the desired site of activity. Peripheral inactivation is achieved by linking the cytokine to an inactivation domain using a tumor protease-sensitive linker. The INDUKINE is also engineered with a half-life extension element to improve tumor exposure. Once the IL-2 INDUKINETM reaches the tumor, tumor-associated proteases cleave the linker and release the active cytokine. This work presents data summarizing the discovery, biochemical, cellular, and in vivo activity of our lead IL-2 INDUKINETM WTX-124 and reviews the mechanism of action of these molecules. Treatment with WTX-124 results in complete regression of tumors driven by the expansion and activation of CD8+ T cells and NK cells which increases the production of effector cytokines in the tumor. Activity is dependent on the proteolytic processing of the INDUKINE as a non-cleavable control INDUKINE lacks efficacy. PK analysis in mouse models demonstrates a favorable accumulation of free IL-2 in tumors compared to plasma. The animals that rejected the tumors upon IL-2 INDUKINETM treatment were protected against follow up re-challenge with the same tumor cell line. In vitro proteolytic activation by human tumor samples will also be presented. The tumor-selective activation of WTX-124 with reduced peripheral toxicities supports its further clinical development. Citation Format: Andres Salmeron, Heather Brodkin, Daniel Hicklin, Nesreen Ismail, Kristin Morris, Christopher Nirschl, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, Jenna Sullivan, William Winston. WTX-124 is an IL-2 pro-drug conditionally activated in tumors and able to induce complete regressions in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1723.

Engineering a Chemically Defined Hydrogel Bioink for Direct Bioprinting of Microvasculature.

Vascularizing printed tissues is a critical challenge in bioprinting. While protein-based hydrogel bioinks have been successfully used to bioprint microvasculature, their compositions are ill-defined and subject to batch variation. Few studies have focused on engineering proangiogenic bioinks with defined properties to direct endogenous microvascular network formation after printing. Here, a peptide-functionalized alginate hydrogel bioink with defined mechanical, rheological, and biochemical properties is developed for direct bioprinting of microvascularized tissues. An integrin-binding peptide (RGD) and a vascular endothelial growth factor-mimetic peptide with a protease-sensitive linker are conjugated onto a biodegradable alginate to synergistically promote vascular morphogenesis and capillary-scale endothelial tube formation. Partial ionic crosslinking before printing converts the otherwise unprintable hydrogel into a viscoelastic bioink with excellent printability and cytocompatibility. We use the bioink to fabricate a compartmentalized vascularized tissue construct, wherein we observe pericyte-endothelial cell colocalization and angiogenic sprouting across a tissue interface, accompanied by deposition of fibronectin and collagen in vascular and tissue components, respectively. This study provides a tunable and translational "off-the-shelf" hydrogel bioink with defined composition for vascularized bioprinting.

Engineering a protease-based and site-specific PEGylation-based strategy for the controlled release of exenatide.

Using the commercially available antidiabetic drug exenatide (exendin-4) as a model peptide, we designed a novel exenatide derivative, termed LEX-1, comprising a 12-mer albumin-binding peptide, a protease-sensitive linker and a native exenatide. In addition, site-specific PEGylation was performed using LEX-1 as a lead sequence to generate four exenatide derivatives (LEX-2 to LEX-5). Moreover, we determined the optimal molecular weight of maleimide-derivatized PEG for the site-specific PEGylation of LEX-1 by an in vitro stability assay and an in vivo hypoglycemic efficacy test. As a result, LEX-3 (PEG10 kDa) exerted enhanced proteolytic stability, rational release rate of free exenatide and the best glucose-stabilizing capability compared with others. In addition, the prolonged hypoglycemic effects of LEX-1 and LEX-3 were demonstrated in type 2 diabetic mice by multiple OGTTs and a hypoglycemic duration test. Furthermore, a pharmacokinetic test was conducted using Sprague Dawley (SD) rats; LEX-3 (PEG10 kDa) showed the best circulating t1/2 of ∼119.7 h for exenatide release from LEX-3, suggesting that LEX-3 has the potential to be developed into a once-weekly antidiabetic agent. The consecutive 8 week treatment of both LEX-1 and LEX-3 exhibited enhanced beneficial efficacies on body weight gain, cumulative food intake, % fat and hemoglobin A1c (HbA1c) reduction compared with exenatide treatment. Meanwhile, the chronic administration of LEX-1 and LEX-3 also effectively improved the blood biochemical indexes. Our results indicate the enhanced antidiabetic effects of LEX-1 and LEX-3, and our strategy of PEGylation and albumin conjugation can be applied to other bioactive agents.

Abstract 2788: VB6-845d tumor cell killing elicits biologic features of immunogenic cell death

Abstract VB6-845d is a Targeted Protein Therapeutic, TPT, that comprises a Fab fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to deBouganin via a furin protease sensitive linker. DeBouganin (deB) is a de-immunized variant of bouganin, a ribosome inactivating protein (RIP), that when internalized blocks protein synthesis thereby leading to cell death. While all cytotoxic molecules induce tumor cell killing, only some are capable of inducing biological manifestations indicative of immunogenic cell death (ICD). ICD is characterized by the collective appearance of distinct cellular changes termed damage associated molecular patterns, or DAMPs, which play a role in immune cell activation by triggering pro-inflammatory processes. Key DAMPs necessary for defining ICD are cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein, ATP secretion and release of HMGB1 (high mobility group box 1). To evaluate whether VB6-845d cell killing elicits the hallmark features of ICD, VB6-845d treated tumor cell lines were assessed for the presence of these distinct signaling molecules. In vitro studies showed that VB6-845d cytotoxicity induces the translocation of calreticulin to the cell surface as well as the release of ATP and HMGB1. The expression of other potential immune regulators following VB6-845d treatment was also examined. In summary, the data presented suggests that VB6-845d mediates tumor cell killing by an ICD pathway. The potential cross-priming effect initiated by VB6-845d-induced ICD suggests the use of VB6-845d in combination with immune checkpoint inhibitors may enhance their effectiveness in EpCAM-positive epithelial cancers. Citation Format: Rachelle L. Dillon, Shilpa Chooniedass, Arjune Premsukh, Glen C. MacDonald, Jeannick Cizeau, Gregory P. Adams. VB6-845d tumor cell killing elicits biologic features of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2788.

Masked Chimeric Antigen Receptor for Tumor-Specific Activation

Adoptive cellular therapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) cells is a powerful form of cancer immunotherapy. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. However, they may also display "on-target off-tumor" toxicities, resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise promising therapeutic modality. To minimize such side effects, we have designed an epidermal growth factor receptor (EGFR)-specific masked CAR (mCAR), which consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker. Proteases commonly active in the tumor microenvironment can cleave the linker and disengage the masking peptide, thereby enabling CAR-T cells to recognize target antigens only at the tumor site. Invitro mCAR showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases. Masked CAR-T cells also showed antitumor efficacy invivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs.

Abstract 2963: VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA

Abstract VB7-756 is Targeted Protein Therapeutic (TPT) comprised of a de-immunized form of bouganin (deBouganin), a potent, plant-derived, type I ribosome-inactivating protein (RIP), genetically linked to the C6.5 anti-Her2 diabody via a furin protease sensitive linker. To engineer the optimal diabody TPT format, several constructs were generated to assess the best diabody-deBouganin orientation. All constructs were expressed as a soluble protein in E. coli supernatant and compared with respect to expression level, stability and potency. The optimal configuration consisted of deBouganin genetically linked to the N-terminus of the VH-VL diabody via a furin protease-sensitive linker and was termed VB7-756. VB7-756 potency was analyzed against a panel of breast cancer cell lines with disparate levels of Her-2 expression and compared to that of Trastuzumab chemically linked to either DM1 (T-DM1) or MMAE (T-MMAE). Overall, VB7-756 was more potent than T-DM1 and T-MMAE with high Her-2 expressing tumor cell lines. In contrast to T-DM1, VB7-756 potency was unaffected by the Her2-Her3 dimerization mediated by heregulin. As opposed to T-DM1 and T-MMAE, which showed only minimal cytoxicity, VB7-756 was highly potent in vitro against tumor cells with cancer stem cell properties. To further differentiate the RIP mechanism of action of deBouganin from tubulin inhibitor reagents, tumor cells that escaped T-DM1 and T-MMAE treatment were incubated in the presence of VB7-756. Results revealed that, VB7-756 was cytotoxic against cancer cells surviving T-DM1 or T-MMAE treatment suggesting that deBouganin can overcome mechanisms of resistance developed by small molecule agents. Moreover, VB7-756 was also cytotoxic against tumor initiating cancer cells evading T-DM1 or T-MMAE toxicity by preventing tumorosphere formation. Overall these results demonstrate that deBouganin's distinct MOA could overcome mechanisms of resistance affecting the efficacy of small molecule drugs. Citation Format: Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Joycelyn Entwistle, Gregory P. Adams, Peter J. Hudson, Glen C. MacDonald, Jeannick Cizeau. VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2963.

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells

The outcome of cell-based therapies can benefit from carefully designed cell carriers. A multifunctional injectable vehicle for the co-delivery of human mesenchymal stem cells (hMSCs) and osteoinductive peptides is proposed, to specifically direct hMSCs osteogenic differentiation. The osteogenic growth peptide (OGP) inspired the design of two peptides, where the bioactive portion of OGP was flanked by a protease-sensitive linker, or its scrambled sequence, to provide faster and slower release rates, respectively. Peptides were fully characterized and chemically grafted to alginate. Both OGP analogs released bioactive fragments in vitro, at different kinetics, which stimulated hMSCs proliferation and osteogenesis. hMSCs-laden OGP–alginate hydrogels were tested at an ectopic site in a xenograft mouse model. After 4weeks, OGP–alginate hydrogels were more degraded and colonized by vascularized connective tissue than the control (without OGP). hMSCs were able to proliferate, migrate outward the hydrogels, produce endogenous extracellular matrix and mineralize it. Moreover, OGP-groups stimulated hMSCs osteogenesis, as compared with the control. Overall, the ability of the proposed platform to direct the fate of transplanted hMSCs in loco was demonstrated, and OGP-releasing hydrogels emerged as a potentially useful system to promote bone regeneration.

Abstract P6-10-01: A randomized phase 2 study of the antibody-drug conjugate CDX-011 in advanced GPNMB-overexpressing breast cancer: The EMERGE study

Abstract Background: GPNMB is an internalizable transmembrane glycoprotein overexpressed in multiple tumor types, including breast cancer (BC). GPNMB promotes BC metastases in a murine model and is a poor prognostic marker in patients (pts) (Rose 2010). CDX-011 (glembatumumab vedotin) is a fully human GPNMB-specific monoclonal antibody conjugated to the potent cellular toxin MMAE via a protease-sensitive peptide linker, designed to cleave upon cellular internalization. In Phase I/II studies of CDX-011 in BC (n = 42), an objective response rate (ORR) (including confirmed and unconfirmed response) of 12% overall and 20% for “triple-negative” (ER/PR/HER2−; TN) pts was observed (Saleh, ASCO 2010). Methods: The Phase II EMERGE study examined the efficacy of CDX-011 by level of GPNMB expression. Refractory BC (2–7 prior cytotoxic regimens in metastatic setting), GPNMB expression in ≥ 5% of tumor or stroma cells (as centrally determined on archival tumor), and no persistent treatment-related toxicity (including neuropathy) of ≥ Grade 2 severity were required for enrollment. Pts were stratified by GPNMB expression pattern (any tumor, low stromal or high stromal) and randomized 2:1 to CDX-011 (1.88 mg/kg IV q 21 days) or “investigator's choice” (IC) single-agent chemotherapy, and treated until progression (PD) or intolerance with IC pt crossover permitted to CDX-011 at PD. Endpoints: ORR (primary), progression-free survival (PFS), safety, pharmacokinetics, pharmacodynamics. Results: 99% of screened tumor samples expressed GPNMB. 122 treated pts (81 CDX-011 vs. 41 IC) had median age = 56 vs. 56 years; median # prior anticancer regimens = 6 vs. 5; visceral disease (liver/lung) = 83% vs. 80%; TN = 33% vs 27%, respectively. To date, 15 IC pts have crossed over to CDX-011, and 14 pts (8 CDX-011, 6 IC) continue on treatment. CDX-011 was well tolerated with less hematologic toxicity than IC; CDX-011-related toxicity included rash (overall = 38%; Grade 1 = 20%; Grade 2 = 15%; Grade 3 = 3%) and neuropathy (overall = 18%; Grade 1 = 8%; Grade 2 = 7%; Grade 3 = 2%). Conclusions: CDX-011 was a well-tolerated and active agent in this heavily pre-treated BC pt population. Preliminary analysis of the EMERGE study demonstrates enhanced activity of CDX-011 in TN as well as high GPNMB expressing BC tumors, confirming that CDX-011 is a targeted agent. This noted promising activity in advanced TN and hGPNMB enriched pt populations warrants further evaluation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-01.

Role of SbmA in the uptake of peptide nucleic acid (PNA)-peptide conjugates in E. coli.

Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-D((KFF)(3)K) peptide retained antibacterial activity in sbmA deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-D((KFF)(3)K), H-(RFR)(4)-Ahx-βAla, H-(R-Ahx-R)(4)-Ahx-βAla, and H-(R-Ahx)(6)-βAla, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA.

Arylamine N-Acetyltransferases: Characterization of the Substrate Specificities and Molecular Interactions of Environmental Arylamines with Human NAT1 and NAT2

Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolism enzymes that catalyze the detoxification of arylamines by N-acetylation and the bioactivation of N-arylhydroxylamines by O-acetylation. Endogenous and recombinant mammalian NATs with high specific activities are difficult to obtain in substantial quantities and in a state of homogeneity. This paper describes the overexpression of human wild-type NAT2 as a dihydrofolate reductase fusion protein containing a TEV protease-sensitive linker. Treatment of the partially purified fusion protein with TEV protease, followed by chromatographic purification, afforded 2.8 mg of homogeneous NAT2 from 2 L of cell culture. The kinetic specificity constants ( k cat/ K m) for N-acetylation of arylamine environmental contaminants, some of which are associated with bladder cancer risk, were determined with NAT2 and NAT1. The NAT1/NAT2 ratio of the specificity constants varied almost 1000-fold for monosubstituted and disubstituted alkylanilines containing methyl and ethyl ring substituents. 2-Alkyl substituents depressed N-acetylation rates but were more detrimental to catalysis by NAT1 than by NAT2. 3-Alkyl groups caused substrates to be preferentially N-acetylated by NAT2, and both 4-methyl- and 4-ethylaniline were better substrates for NAT1 than NAT2. NMR-based models were used to analyze the NAT binding site interactions of the alkylanilines. The selectivity of NAT1 for acetylation of 4-alkylanilines appears to be due to binding of the substituents to V216, which is replaced by S216 in NAT2. The contribution of 3-alkyl substituents to NAT2 substrate selectivity is attributed to multiple bonding interactions with F93, whereas a single bonding interaction occurs with V93 in NAT1. Unfavorable steric clashes between 2-methyl substituents and F125 of NAT1 may account for the selective NAT2-mediated N-acetylation of 2-alkylanilines; F125 is replaced by S125 in NAT2. These results provide insight into the structural basis for the substrate specificity of two NATs that play major roles in the biotransformation of genotoxic environmental arylamines.

Specific Tumor Targeting and Potent Bystander Killing with SGN-35, an Anti-CD30 Antibody Drug Conjugate.

CD30 is expressed on the surface of Reed Sternberg cells in Hodgkin's Lymphoma (HL) and on anaplastic large cell lymphoma (ALCL) cells. The anti-mitotic agent monomethyl auristatin E (MMAE) has been conjugated to cAC10, the anti-CD30 monoclonal antibody SGN-30, to yield the antibody-drug conjugate SGN-35. SGN-35 is internalized into lysosomes of CD30-positive cells and MMAE is released intracellularly when the protease-sensitive linker is cleaved by lysosomal proteases such as cathepsin B. In mouse xenograft models of HL and ALCL, complete remissions have been observed at well tolerated doses of SGN-35. To characterize further the mode of action in vivo, mice with established CD30-positive xenografts were treated with SGN-35, and the concentration of released MMAE was measured in the tumor and serum. The tumor concentrations of released MMAE achieved were several hundred-fold above the IC50 for MMAE in tumor cell cultures. Peak tumor to serum ratios for released MMAE reached more than 1000:1. In tumors and serum, the peak released MMAE concentrations were achieved 3 days and 1 hour post dose, respectively. As a considerable portion of the tumor volume in HL consists of a CD30-negative cellular infiltrate, the ability of SGN-35 to kill CD30-negative cells in mixed cell populations was investigated. First, cell growth medium from the SGN-35-treated HL cell line L540cy (CD30 positive) was shown to be cytotoxic to the SGN-35-insensitive Ramos cells (CD30 negative). Measurements with mass spectrometry confirmed that MMAE was released into the growth medium of SGN-35-treated L540cy cells, and that the observed concentration of MMAE was consistent with the cytotoxic effect on the Ramos cells. Second, a mixed culture of Karpas 299 cells (ALCL, CD30 positive) and Ramos cells was treated with SGN-35 and examined by FACS. The viability of both cell populations was reduced or eliminated, in contrast to proliferation observed in single Ramos cultures treated with SGN-35 or untreated mixed cultures. These results demonstrate that SGN-35 effectively targets the potent cytotoxic agent MMAE to CD30-positive tumor cells, while free MMAE released by these tumor cells is also available for killing CD30-negative infiltrating bystander cells. SGN-35 is a promising treatment for tumors with homogeneous (ALCL) and heterogeneous (HL) expression of CD30. Clinical trials with SGN-35 are expected to begin by the end of 2006.

Cellobiose Dehydrogenase – A Flavocytochrome from Wood-Degrading, Phytopathogenic and Saprotropic Fungi

Cellobiose dehydrogenase, the only currently known extracellular flavocytochrome, is formed not only by a number of wood-degrading but also by various phytopathogenic fungi. This inducible enzyme participates in early events of lignocellulose degradation, as investigated in several basidiomycete fungi at the transcriptional and translational level. However, its role in the ascomycete fungi is not yet obvious. Comprehensive sequence analysis of CDH-encoding genes and their translational products reveals significant sequence similarities along the entire sequences and also a common domain architecture. All known cellobiose dehydrogenases fall into two related subgroups. Class-I members are represented by sequences from basidiomycetes whereas class-II comprises longer, more complex sequences from ascomycete fungi. Cellobiose dehydrogenase is typically a monomeric protein consisting of two domains joined by a protease-sensitive linker region. Each larger (dehydrogenase) domain is flavin-associated while the smaller (cytochrome) domains are haem-binding. The latter shorter domains are unique sequence motifs for all currently known flavocytochromes. Each cytochrome domain of CDH can bind a single haem b as prosthetic group. The larger dehydrogenase domain belongs to the glucose-methanol-choline (GMC) oxidoreductase superfamily - a widespread flavoprotein evolutionary line. The larger domains can be further divided into a flavin-binding subdomain and a substrate-binding subdomain. In addition, the class-II (but not class-I) proteins can possess a short cellulose-binding module of type 1 at their C-termini. All the cellobiose dehydrogenases oxidise cellobiose, cellodextrins, and lactose to the corresponding lactones using a wide spectrum of different electron acceptors. Their flexible specificity serves as a base for the development of possible biotechnological applications.

Definition of structural elements in Plasmodium vivax and P. knowlesi Duffy-binding domains necessary for erythrocyte invasion.

Plasmodium vivax and P. knowlesi use the Duffy antigen as a receptor to invade human erythrocytes. Duffy-binding ligands belong to a family of erythrocyte-binding proteins that bind erythrocyte receptors to mediate invasion. Receptor-binding domains in erythrocyte-binding proteins lie in conserved cysteine-rich regions called Duffy-binding-like domains. In the present study, we report an analysis of the overall three-dimensional architecture of P. vivax and P. knowlesi Duffy-binding domains based on mild proteolysis and supportive-functional assays. Our proteolysis experiments indicate that these domains are built of two distinct subdomains. The N-terminal region from Cys-1-4 (C1-C4) forms a stable non-functional subdomain. The region spanning C5-C12 forms another subdomain, which is capable of binding Duffy antigen. These subdomains are joined by a protease-sensitive linker. Results from deletion constructs, designed for expression of truncated proteins on COS cell surface, show that regions containing C5-C8 of the Duffy-binding domains are sufficient for the binding receptor. Therefore the central region of Duffy-binding domains, which is flanked by two non-functional regions, is responsible for receptor recognition. Moreover, the minimal Duffy-binding region identified here is capable of folding into a functionally competent module. These studies pave the way for understanding the architecture of Duffy-binding domains and their interactions with host receptors.

Identification of keratinocyte-specific markers using phage display and mass spectrometry.

Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.