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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.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and

To enhance the therapeutic index of T-cell engagers (TCE), we engineered masked, conditionally active TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 [HER2] or epidermal growth factor receptor 2 [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 induces protease-dependent anti-tumor activity and is proteolytically stable in healthy tissues. In non-human primates (NHPs), HER2-XPAT demonstrates a strong safety margin (> 400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT cleavage is low and similar in plasma samples from healthy and diseased humans and NHPs, supporting translatability of stability to human patients. The EGFR-XPAT confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.

Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-DʹD3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.

Antibody-drug conjugates (ADCs) enable cell-specific delivery of small molecules and are validated anti-cancer therapeutics. One factor limiting ADC advancement and broader application is the drug-to-antibody ratio (DAR), which dictates the number of payloads that can be delivered per antibody. With few exceptions, efficacious ADCs with DAR > 4 are inaccessible due to aggregation or rapid clearance in vivo. Here, we report a versatile platform for the generation of homogeneous ADCs with DAR up to 18, combining Cys-engineered THIOMAB antibodies and XTEN polypeptides to give “TXCs”. We show that high-DAR TXCs are stable biochemically and in vivo. We demonstrate that two different cytotoxic TXCs directed toward a tumor xenograft and one TXC targeting Staphylococcus aureus have comparable pharmacokinetics, but significantly enhanced efficacy in vivo versus conventional low-DAR ADCs. Our data suggest that high-DAR TXCs may ultimately offer several advantages versus conventional ADCs, including increased therapeutic index and efficacious delivery of less potent payloads.

Background: TCEs are effective in leukemias, but have been limited in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent cytokine release syndrome (CRS), including step-up dosing and complex designs, have had limited success due to toxicity and immunogenicity. AMX-818, HER2-XPAT, or XTENylated Protease-Activated T Cell Engager, is a prodrug TCE that exploits dysregulated protease activity present in tumors to expand the therapeutic index (TI). The core of the molecule (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core are two unstructured polypeptides (XTEN) that function as universal masks, sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, releasing a potent TCE with short T1/2. Methods: Nonclinical studies were conducted with HER2-XPAT or AMX-818, unmasked HER2-PAT, HER2-NoClvSite (no protease linkers) or the fluorescent labeled counterparts in vitro and in vivo xenografts and NHP. Results: The unmasked HER2-PAT demonstrated potent in vitro T cell cytotoxicity (EC50 1-2pM), target-dependent T cell activation and cytokine production by hPBMCs (human mononuclear cells). HER2-XPAT demonstrated 14,000-fold protection against killing of HER2 tumor cells, and minimal cytotoxicity against cardiomyocytes up to 1uM. In vivo, HER2-XPAT or AMX-818 induced complete tumor regressions (CRs) in HER2+ BT-474 tumors (similar to effect with equimolar doses of HER2-PAT), whereas HER2-NoClvSite lacked efficacy, supporting the need for protease cleavage for activity. HER2-XPAT was also highly efficacious in the HER2 low-expressing HT-55 colorectal model. Labeled XPATs with the same protease cleavage sites in 9 different in vivo xenograft and PDX models demonstrated preferential unmasking into cleaved products in tumors in contrast to healthy organs, with average ~20% of drug in tumor cleaved to the fully active TCE form. In NHPs, AMX-818 caused early T-cell margination as low as 2mg/kg, but has been dose-escalated safely up to 42mg/kg (MTD) with no CRS. Doses of 0.5 to 42 mg/kg demonstrated dose proportional increases in HER2-XPAT exposure, with low to negligible levels of cleavage products present in systemic circulation. Continuous infusion of unmasked HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d but was tolerated at 0.2 mg/kg/d, providing AMX-818 with ~450-fold improvement in safety compared to HER2-PAT. Repeat-dose GLP toxicology studies have demonstrated that the highest dose evaluated (6 mg/kg) was the NOAEL, suggesting that HER2-XPAT has a wide TI based on predicted human PK and predicted efficacious exposures. Conclusions: AMX-818 is a potent, protease-activated prodrug TCE exhibiting preferential tumor unmasking, with minimal systemic unmasking and no CRS at high doses in NHP. AMX-818 represents a promising solution to widen the TI for TCE activity in HER2-high and HER2-low expressing tumors. Citation Format: Milton To, Pete Yeung, Michael Fox, Mikhail Hammond, Fiore Cattaruzza, Ayesha Nazeer, Caitlin Koski, Lucas Liu, Sina Khorsand, Deena Rennerfeldt, Kari Morrissey, Zachary Lange, Ming Dong, Sharon Lam, Mika K. Derynck, Bryan A. Irving, Volker Schellenberger. AMX-818, a novel prodrug HER2-XPAT T-cell engager (TCE) with potent T cell activation, proteolytic cleavage and efficacy in xenograft tumors, and wide safety margins in NHP (Non Human Primate) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P193.

Background: TCEs are effective in leukemias but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent cytokine release syndrome (CRS) including step-up dosing and/or complex molecular designs have been unsuccessful due to toxicity and/or enhanced immunogenicity. HER2-XPAT, or XTENylated Protease-Activated bispecific T-Cell Engager, is a prodrug TCE that exploits the protease activity present in tumors vs healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core, two unstructured polypeptide masks (XTEN) sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a potent TCE with short T1/2, further improving the TI. Methods: Preclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytotoxicity in vitro, anti-tumor efficacy and cleavage in xenograft models, stability in human plasma, and safety in NHPs. Results: The unmasked HER2-PAT demonstrated potent in vitro T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and cytokine production by hPBMCs. HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and exhibited only minimal cytotoxicity against cardiomyocytes at 1μM. In vivo, HER2-XPAT induced complete tumor regressions (CRs) in BT-474 tumors with equimolar dosing as HER2-PAT, whereas HER2-NonClv lacked efficacy, supporting a requirement of protease cleavage. HER2-XPAT was also highly efficacious in the HER2 low-expressing HT-55 colorectal model. Preferential proteolytic cleavage of fluorescent-labeled HER2-XPAT in tumors vs healthy organs was demonstrated in vivo. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD). HER2-XPAT demonstrated early T-cell margination at 2mg/kg but largely spared CRS, cytokines, and major tissue toxicity up to 42mg/kg. In vivo, the PK of HER2-XPAT was comparable to its non-cleavable counterpart, consistent with its ex vivo stability for cleavage in cancer pts plasma for 7 days at 37°C. Continuous infusion of HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d but was tolerated at 0.25mg/kg/d, providing HER2-XPAT with 500-fold protection in tolerated Cmax vs HER2-PAT. Conclusions: HER2-XPAT is a potent, prodrug TCE exhibiting protease-dependent tumor efficacy in mouse xenografts and a wide TI with no CRS in NHPs. XTEN's low immunogenicity has already been demonstrated in humans (growth hormone and FVIII). HER2-XPAT represents a promising solution for HER2-high tumors with potential expansion to HER2 low expressing tumors. IND studies are ongoing. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Caitlin Koski, Mikhail Hammond, Milton To, Pete Yeung, Sharon Lam, Mika K. Derynck, Bryan Irving, Volker Schellenberger. HER2-XPAT, a novel protease-activatable T-cell engager (TCE) with potent T-cell activation and efficacy in solid tumors and large safety margins in non-human primate (NHP) [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 1824.

Background TCEs are effective at inducing remissions in hematologic cancers but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent CRS include fractionated or step-up dosing and/or complex molecular designs, but these have been unsuccessful due to toxicity and/or enhanced immunogenicity. Amunix has developed a conditionally active TCE, XPAT or XTENylated Protease-Activated bispecific T-Cell Engager, that exploits the protease activity present in tumors vs. healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFVs targeting CD3 and HER2. Two unstructured polypeptide masks (XTEN) are attached to the core that sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a highly potent TCE with a short T1/2 and further improving the TI. Although checkpoint inhibitors have been ineffective in HER2+ or HR+ breast cancer, TCEs can co-opt T-cells regardless of their antigenic specificity, providing the potential to introduce effective T-cell immunity against tumors expressing HER2. HER2-XPAT, as a tumor protease-activatable prodrug with a wide safety margin, offers the potential to induce effective T-cell cytotoxicity against HER2hi tumors and HR+/HER2 1-2+ tumors. Methods Preclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytolytic activity in vitro, for anti-tumor efficacy in xenograft models, and for stability and safety in NHPs. Results HER2-PAT (cleaved XPAT) demonstrated potent in vitro tumor-directed T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and production of cytokines by PBMCs. HER2-PAT also exhibited cytotoxicity in HR+, HER2 1+ MCF7s at higher doses (EC50 0.13nM). HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and exhibited no cytotoxicity against cardiomyocytes at concentrations up to 1uM. In vivo, HER2-XPAT induced complete tumor regressions in BT-474 tumor models with equimolar dosing to HER2-PAT, whereas non-cleavable HER2-NonClv had no efficacy, supporting tumor protease cleavage requirement for T-cell activity. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD) but was not tolerated at 50mg/kg. HER2-XPAT demonstrated early T-cell margination at 2mg/kg but largely spared CRS, cytokine production, and tissue toxicity at doses up to 42mg/kg. PK profiles of HER2-XPAT and HER2-NonClv were comparable in NHP, indicating minimal systemic cleavage of XPAT, consistent with its ex vivo stability when incubated in the plasma of cancer pts for 7 days at 37°C. Given by continuous infusion, HER2-PAT induced lethal CRS and cytokine spikes at 0.3mg/kg/d, but was tolerated at 0.2mg/kg/d, providing HER2-XPAT with >3000-fold protection in tolerated Cmax versus HER2-PAT, >4 logs over cytotoxicity EC50s for HER2 and HR+ cell lines, and a 20-fold margin of safety over the dose required for pharmacodynamic activity. Conclusions HER2-XPAT is a potent prodrug T-cell engager with promising evidence of activity at low doses while exhibiting minimal CRS and a potentially wide TI based on NHP tolerability at doses up to 42mg/kg. With XTEN’s prior clinical data demonstrating low immunogenicity, the XPAT TCEs provide a promising solution. IND studies are currently ongoing. Additional PK, PD, cytokines, safety, and efficacy data will be presented. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Caitlin Koski, Mikhail Hammond, Angela Henkensiefken, Trang Dao-Pick, Mika K Derynck, Bryan Irving, Volker Schellenberger. Her2-xpat, a novel protease-activatable prodrug t-cell engager (tce), exhibits potent t-cell activation and efficacy in her2 tumors, yielding large predicted safety margins based on non-human primate (nhp) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-11.

Background: TCEs have proven to be effective in creating durable remissions in hematologic malignancies but have been challenging in solid tumors due to on-target, off-tumor toxicity. To circumvent the toxicity, many have tried step-up, fractionated dosing or complex formats, but these have largely been unsuccessful due to toxicity and/or enhanced immunogenicity. To address this challenge, Amunix has developed a conditionally active TCE, the XPAT or XTENylated Protease-Activated T Cell Engager, that exploits the protease activity present in tumors vs. healthy tissue, enabling expansion of the therapeutic index (TI). The core of the XPAT consists of 2 tandem scFVs targeting CD3 and a tumor antigen. Two unstructured and modular polypeptide masks (XTEN) are attached to the core that sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of these XTEN polypeptide masks are then proteolytically released in the tumor microenvironment to unleash a highly potent TCE with a short T1/2, further improving the TI. Methods: HER2-XPAT and EGFR-XPATs were each purified from E.coli expressed from a single plasmid. The activity of both the prodrugs (XPATs) and their protease-activated counterparts (PATs) were characterized for cytotoxicity in vitro and in huPBMC-transduced tumor-bearing mice. Pilot toxicity studies were conducted in NHPs (cyno). Results: Both protease-activated (PATs) forms of EGFR-XPAT and HER2-XPAT demonstrated potent in vitro T cell directed cytotoxicity against tumor cells (EC50s 1-2pM), while the masked XPAT provided 3,000-14,000-fold protection against target cell killing. Intact EGFR-XPAT demonstrated dose-dependent complete tumor responses (CRs) in HT-29 CRC xenografts, with HER2-XPAT demonstrating similar efficacy and CRs in both BT-474 and SK-OV-3 tumors. In cynos, 1 mg/kg of EGFR-XPAT was the MTD and 1.5 mg/kg exceeded the MTD, while the activated PAT was lethal at 0.132mg/kg/day by continuous infusion (with single dose 1 hour bolus of 0.066ug/kg). This indicated a near 2 log protection or more in predicted Cmax of XPAT vs PAT. A HER2-XPAT has been dose escalated up to 21 mg/kg in cynos and was well-tolerated, with the MTD still unreached. At doses starting from 2.5mg/kg of a HER2-XPAT, lymphocyte margination and laboratory abnormalities were observed, indicating evidence of biologic activity well below the tolerated 21 mg/kg dose. An estimated T1/2 of ~4 days was observed. Conclusions: EGFR-XPAT and HER2-XPAT are novel T cell engagers with protease-cleavable XTEN masks with preclinical evidence of potent anti-tumor activity and a wide margin of protection in cynos. With the prior XTEN clinical data of low immunogenicity, the XPAT TCEs provide a promising solution to overcome On-target, Off-tumor toxicity. Additional PD, safety and efficacy data will be presented. Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Mikhail Hammond, Caitlin Koski, Angela Henkensiefken, Mika K. Derynck, Bryan Irving, Volker Schellenberger. HER2-XPAT and EGFR-XPAT: Pro-drug T-cell engagers (TCEs) engineered to address on-target, off-tumor toxicity with potent efficacy in vitro and in vivo and large safety margins in NHP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3376.

To optimize the action of exogenously administered insulin, we employed XTEN® technology to create insulins with variably sized XTEN amino acid polymers. Recombinant fusions of XTEN polymers linked to insulin lispro with an A21G mutation were prepared in various amino acid lengths. Insulin-XTEN molecules demonstrated 15-fold lower potency in binding and receptor phosphorylation than insulin lispro but did not differ from each other. These insulin-XTEN molecules were equally effective in lowering blood glucose at a 100nmol/kg dose in diabetic Sprague-Dawley rats. Furthermore, the larger insulin-XTEN molecules had a longer duration of glucose lowering. Insulin-XTENs were compared to insulin lispro in rat euglycemic clamp studies, using insulin doses that would elicit steady plasma insulin concentrations and equivalent increases in glucose infusion rate. Insulin-mediated suppression of endogenous glucose production was not significantly different among any of the administered insulins. However, plasma free fatty acids and soleus muscle glucose uptake were significantly decreased in an XTEN size-dependent manner when compared to insulin lispro. Additional studies demonstrated equal hepatic pAkt accumulation in rats treated with insulin lispro or any of the insulin-XTENs, but revealed a significant XTEN size-dependent reduction in skeletal muscle pAkt in rats administered insulin-XTENs compared to insulin lispro. These data suggest a possible XTEN size-dependent regulation of insulin action and that the differing sizes of the XTEN polymer may convey preferential tissue action. In conclusion, XTEN technology may permit “tuning” of the glucodynamic effects of the insulin, leading to an enhanced time extension and improved hepatic and peripheral pharmacodynamic action that could more closely mimic the action of endogenously secreted insulin into the portal circulation. Disclosure M.E. Christe: Employee; Self; Eli Lilly and Company. D. Konkol: None. J. Friedrich: None. J. Jacobs: None. E. Hawkins: Employee; Self; Eli Lilly and Company. J. Moyers: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. C. Zhang: Employee; Self; Eli Lilly and Company. S.D. Kahl: Employee; Self; Eli Lilly and Company. H.E. Baker: None. A.L. Cox: None. R.J. Hansen: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. A. Sperry: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. M. Michael: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. V. Schellenberger: None. D. Baldwin: None. J.M. Beals: Employee; Self; Eli Lilly and Company. A. Korytko: None.

A novel recombinant human growth hormone (rhGH) fusion protein (VRS-317) was designed to minimize receptor-mediated clearance through a reduction in receptor bind- ing without mutations to rhGH by genetically fusing with XTEN amino acid sequences to the N-terminus and the C-terminus of the native hGH sequence. Although in vitro potency of VRS- 317 was reduced approximately 12-fold compared with rhGH, in vivo potency was increased because of the greatly prolonged exposure to the target tissues and organs. VRS-317 was three- fold more potent than daily rhGH in hypophysectomized rats and fivefold more potent than daily rhGH in juvenile monkeys. In juvenile monkeys, a monthly dose of 1.4mg/kg VRS-317 (equivalent to 0.26mg/kg rhGH) caused a sustained pharmacodynamic response for 1 month equivalent to 0.05mg/kg/day rhGH (1.4mg/kg rhGH total over 28 days). In monkeys, VRS-317, having a terminal elimination half-life of approximately 110h, was rapidly and near-completely absorbed, and was well tolerated with no observed adverse effects after every alternate week subcutaneous dosing for 14 weeks. VRS-317 also did not cause lipoatrophy in pig and monkey studies. VRS-317 is currently being studied in GH-deficient patients to confirm the observa- tions in these animal studies. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2744-2754, 2012

A novel β-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (β-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual β-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound β-lactamase activity. SKBR3 cell-binding β-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected β-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted β-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy. © 2007 Wiley-Liss, Inc.

XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis. Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage. This enables one to subject individual phagemid-encoded genes to rapid in vivo evolution. We applied this approach to TEM-1 beta-lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime. After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500-fold increased level of resistance to cefotaxime compared to the starting enzyme. In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme. The same three mutations have been previously observed in TEM-4 beta-lactamase which was discovered in a highly cefotaxime-resistant clinical isolate. The probability of randomly obtaining a beta-lactamase carrying three identical point mutations is less than 10(-10). This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature.

Mutants that secrete increased amounts of enzyme into a selection medium can be efficiently enriched from large populations of mutagenized microorganisms during growth in hollow fibers. Under these conditions, each colony grows in its own microenvironment and cross-feeding between neighboring colonies is limited. We applied the technique to B. subtilis carrying a plasmid-encoded protease gene. The plasmid was subjected to random mutagenesis and clones secreting up to fivefold-increased amounts of enzyme were selected using a medium containing bovine serum albumin as the sole nitrogen source.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.

Introduction: Prophylactic treatment for hemophilia has demonstrated clear benefit over on demand treatment in hemophilic patients. Alprolix (rFIXFc) is the first of a new generation of long acting clotting factors we made to reduce the frequency of intravenous infusions (Powell, NEJM, 2013). Nevertheless, intravenous injections can be particularly challenging for young patients and patients with limited venous access and are time consuming. We are developing a novel rFIXFc fusion protein, using XTEN recombinant technology, which is suitable for prophylactic subcutaneous dosing in hemophilia B. In the current studies we explore the in vivo bleeding efficacy of subcutaneously dosed rFIXFc-XTEN in hemophilia B mouse model. Methods: recombinant rFIXFc-XTEN was produced in HEK293 cells and affinity purified. The subcutaneous PK profile of rFIXFc-XTEN was compared to that of intravenously dosed rFIXFc in hemophilia B mice. The prolonged and acute bleeding efficacies were evaluated in HemB mouse tail-vein transection (TVT) and tail-clip bleeding models. Results and conclusions: Subcutaneous dosing of rFIXFc-XTEN shows a tmax around 20 hours post dosing in mice, and improved plasma activity levels compared to similar (IU/kg) intravenously dosed rFIX or rFIXFc. Using the TVT bleeding model in HemB mice we show that at 72 hours post dosing, subcutaneously dosed rFIXFc-XTEN has improved in vivo efficacy compared to intravenously dosed rFIXFc at all tested doses. Similarly, acute efficacy testing in the HemB mouse tail-clip bleeding model showed improved efficacy of intravenously dosed rFIXFc-XTEN compared to rFIXFc. These data support the potential of once weekly or less frequent subcutaneous prophylactic dosing of rFIXFc-XTEN in humans. Disclosures Liu: biogen: Employment, Equity Ownership. van der Flier:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment, Equity Ownership. Simpson:Biogen: Employment, Equity Ownership. Kurt:Biogen: Employment, Equity Ownership. Schellenberger:Amunix: Employment, Equity Ownership. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder.

The substrate spectrum of Vsr DNA mismatch endonuclease of Escherichia coli K-12 was investigated using fluorescence-labelled oligonucleotide substrates and a DNA sequencer for detection and quantification of substrates and reaction products. Fourteen substrates were found to be processed by the enzyme, which differ in one or two positions from the canonical pentanucleotide sequence CTA/TGG (T mismatched to G). Relative second-order rate constants of these substrates were determined in groups of four by multiple substrate kinetics and compared to the underresentation of the corresponding pentanucleotides in the E. coli K-12 genome. The high quality of correlation further establishes active mutagenesis by VSP repair as a significant driving force of the evolution of the E. coli K-12 genome and provides clues to its possible selective value.

The S' subsite specificity of four homologous serine proteases, rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni, was studied by measuring acyl-transfer reactions to 100 pentapeptide nucleophiles. Peptides of the general structures H-Xaa-Ala-Ala-Ala-Ala-NH2, H-Ala-Xaa-Ala-Ala-Ala-NH2, and H-Ala-Ala-Xaa-Ala-Ala-NH2 were synthesized, where Xaa is D-Ala, Cit, and all natural amino acids except Cys. The variable residues of these nucleophiles occupy the P'1, P'2, and P'3 positions in acyl-transfer reactions. The P'1 and P'2 residues were found to influence the efficiency of the nucleophiles by more than 2 orders of magnitude, whereas the S'3 subsite shows a lower specificity in all four enzymes. We synthesized consensus peptides of the general structure H-aa1-aa2-aa3-Ala-Ala-NH2, in which two or three positions were occupied by amino acids that showed the highest specificity in the first series of nucleophiles. Peptides with optimal amino acid residues in the P'2 and P'3 positions show a very high efficiency in chymotrypsin- and trypsin-catalyzed reactions. Otherwise, large specific side chains in the P'1 and P'3 positions of the nucleophiles show less than additive binding contributions due to steric hindrance. Comparison of chymotrypsin-catalyzed acyl-transfer reactions to nucleophiles of the structures H-Xaa-Leu-Arg-Ala-Ala-NH2 and H-Xaa-Ala-Ala-Ala-Ala-NH2 reveals a significantly different P'1 specificity for both series which confirms steric hindrance between large P'1 and P'3 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple approaches for screening large sets of compounds for a specific function are of growing interest. The use of substrate mixtures to characterize the specificity of enzymes has been limited so far to compounds with similar kinetic parameters, because the data were analyzed by applying the kinetics of two competing substrates. In this study we introduce a statistical method for the analysis of reactions with many competing substrates which makes use of the specific features of multiple substrate kinetics. It is assumed that the relative concentrations of all substrates in a mixture can be monitored by high-performance liquid chromatography or a similar technique. Relative second-order rate constants, i.e., kcat/KM values, can be calculated for all substrates in the mixture from the resulting data set. The calculation uses the fact that there is a relationship between the concentrations of all pairs of substrates in the mixture. As a result, the precision of the calculated parameters is increased and the range of kinetic constants that can be obtained from one experiment is considerably expanded. Simulations demonstrate that the precision in the kinetic parameters increases with the number of substrates in the mixture. In fact, estimation of ratios of rate constants can be improved (or made possible) for substrates with order of magnitude differences in reactivity by adding "dummy" substrates with intermediate reactivities, even though the rate constants for dummy substrates are themselves of no intrinsic interest.

In spite of the enormous progress in the synthesis of peptides and proteins using commercial peptide synthesizers and the immense technological possibilities of recombinant DNA technology, a CN ligase is an indispensable tool for the racemization-free fragment condensation of peptides. Since activation of the C-terminal α-carboxyl group of a peptide segment could cause partial racemization, chemical condensations of peptide fragments are prone to racemization. For the synthesis of the huge number of peptides and proteins, however, nature has only developed the ribosomal peptidyltransferase, which exhibits its full catalytic function independent of the side-chain functions of the amino acids being coupled. However, its function requires coordination with numerous other ribosomal factors. Besides the limited possibilities of using multienzyme complexes of bacterial peptide synthesis systems, the only alternatives to peptidyltransferase are proteases, which, based on their in vivo function as hydrolases, cannot act as ideal ligases. However, by exploiting the intrinsic reversibility of hydrolytic reactions and by adjusting appropriate physicochemical reaction parameters, the protease acitivity can be used in the direction of ligation. Undoubtedly, the course of kinetically controlled, serine and cysteine protease-catalyzed reactions can be more efficiently influenced than the equilibrium-controlled protease-catalyzed synthesis. This article describes the influence of the enzyme specificity on the efficiency of kinetically controlled synthesis and points the way toward a broad exploitation of serine and cysteine proteases for the catalysis of CN bond formation.

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters α and β for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH2 from Mal-L-Phe-L-AlaOMe and L-LeuNH2. The experimental results demonstrate that this theory adequately describes the real process. © 1992 John Wiley & Sons, Inc.

Increasing the in vivo residence times of protein therapeutics could decrease their dosing frequencies. We show that genetic fusion of an unstructured recombinant polypeptide of 864 amino acids, called XTEN, to a peptide or protein provides an apparently generic approach to extend plasma half-life. Allometric scaling suggests that a fusion of XTEN to the exenatide peptide should increase exenatide half-life in humans from 2.4 h to a projected time of 139 h. We confirmed the biological activity of the exenatide-XTEN fusion in mice. As extended stability might exacerbate undesirable side effects in some cases, we show that truncating the XTEN sequence can regulate plasma half-life. XTEN lacks hydrophobic amino acid residues that often contribute to immunogenicity and complicate manufacture. Based on data on XTEN fusions to exenatide, glucagon, GFP and human growth hormone, we expect that XTEN will enable dosing of otherwise rapidly cleared protein drugs at up to monthly intervals in humans.

Trotz des enormen Fortschritts bei der Synthese von Peptiden und Proteinen mit kommerziellen Peptidsynthesizern und der grosen biotechnologischen Moglichkeiten der DNA-Rekombinationstechnik ist zur racemisierungssicheren Verknupfung von Peptidsegmenten eine C-N-Ligase ein wichtiges Hilfsmittel. Da die Aktivierung der α-Carboxyfunktion eines Peptidsegmentes mit dem partiellen Verlust der Enantiomerenreinheit verbunden sein kann, bergen chemische Verknupfungen von Peptidsegmenten Racemisierungsgefahren. Die Natur hat zur Synthese der riesigen Sequenzvielfalt von Peptiden und Proteinen mit der Peptidyltransferase nur ein einziges Enzym entwickelt, das unabhangig vom Charakter der Seitenkettenfunktionen der proteinogenen Aminosaurebausteine seine volle katalytische Funktion entfaltet. Jedoch ist die Peptidyltransferase nur als integraler Bestandteil des Ribosoms in einer koordinierten Interaktion mit anderen ribosomalen Faktoren wirksam, d.h. fur die enzymatische Synthese nicht zu verwenden. Alternativ bieten sich fur enzymatische Peptidsynthesen, wenn man von den eingeschrankten Moglichkeiten der Nutzung von Multienzymkomplexen der bakteriellen Peptidsynthese absieht, nur noch die Proteasen an, die aber aufgrund ihrer in-vivo-Funktion als Hydrolasen keine idealen C-N-Ligasen sein konnen. Erst gezielte thermodynamische und reaktionskinetische Manipulationen ermoglichen die Umkehrung der Proteasewirkung und damit eine Programmierung in Richtung Ligation. Ohne Zweifel ist eine synthesebegunstigende Einflusnahme bei einer durch Serin- und Cysteinproteasen katalysierten kinetisch kontrollierten Reaktionsfuhrung variabler und effizienter zu realisieren als bei gleichgewichtskontrollierten Synthesen. Dieser Beitrag beschreibt den Einflus der Enzymspezifitat auf die Effizienz von kinetisch kontrollierten Synthesen. Es werden Schlusfolgerungen fur eine breitere Nutzung von Serin- und Cysteinproteasen zur Katalyse von C-N-Verknupfungen abgeleitet.

In spite of the enormous progress in the synthesis of peptides and proteins using commercial peptide synthesizers and the immense technological possibilities of recombinant DNA technology, a CN ligase is an indispensable tool for the racemization-free fragment condensation of peptides. Since activation of the C-terminal α-carboxyl group of a peptide segment could cause partial racemization, chemical condensations of peptide fragments are prone to racemization. For the synthesis of the huge number of peptides and proteins, however, nature has only developed the ribosomal peptidyltransferase, which exhibits its full catalytic function independent of the side-chain functions of the amino acids being coupled. However, its function requires coordination with numerous other ribosomal factors. Besides the limited possibilities of using multienzyme complexes of bacterial peptide synthesis systems, the only alternatives to peptidyltransferase are proteases, which, based on their in vivo function as hydrolases, cannot act as ideal ligases. However, by exploiting the intrinsic reversibility of hydrolytic reactions and by adjusting appropriate physicochemical reaction parameters, the protease acitivity can be used in the direction of ligation. Undoubtedly, the course of kinetically controlled, serine and cysteine protease-catalyzed reactions can be more efficiently influenced than the equilibrium-controlled protease-catalyzed synthesis. This article describes the influence of the enzyme specificity on the efficiency of kinetically controlled synthesis and points the way toward a broad exploitation of serine and cysteine proteases for the catalysis of CN bond formation.

We investigated the alpha-chymotrypsin-catalyzed acyl transfer to a series of glycine oligomers. It could be established that the electrostatic interactions between the carboxylate group of the nucleophiles and the S'-subsites of the enzyme fall off with the length of the nucleophile molecule. Additional negatively charged residues in the nucleophile lead to a considerable reduction of the acyl transfer efficiency. An arginine residue in P'1- or P'3-position, but not in P'2-position, makes favourable interactions with the appropriate S'-subsites of the enzyme.

Increase in KC1 concentration from 0.1 m to 3 m enhances the chymotrypsin-catalyzed acyl-transfer to a series of dipeptides, H2N—CH(R1)C(O)NHCH(R2)C(O)O, by factors between 15 and 44. The observed positive salt effect seems to be a screening of the unfavorable electrostatic interaction between the dipeptide carboxyl group and a negatively charged area in the S' subsite on the enzyme surface. The effect is of practical use in preparative peptide synthesis—in 3 m KCl solutions the analytical yields of tripeptides of up to 99 per cent have been obtained.

Background: A variety of approaches have been used to extend the half-life of human factor IX (FIX) in the circulation, including PEGylation and recombinant fusion of either the Fc domain of IgG or albumin to the C-terminus of FIX. Introduction of XTENs, unstructured hydrophilic polypeptides of defined amino acid composition and low immunogenic potential, represents a promising alternative approach for generating FIX variants with improved pharmacokinetic properties. Moreover, the hydrophilic properties of XTEN have the potential to increase the solubility of FIX and thereby render it suitable for subcutaneous administration, which is currently an unmet need. Because XTEN is introduced into FIX by DNA recombination, the location, length, composition and number of XTEN modifications can be readily varied, and impact of these modifications on the activity and clearance of FIX can be evaluated. Aim: To identify sites in FIX that can accommodate the introduction of XTENs without abrogating FIX activity and apply this approach to both otherwise non-modified FIX and a recombinant FIX-Fc fusion protein. Methods: The highly active FIX Padua variant (R338L) was used as a scaffold to counter FIX activity loss due to reduced activity caused by the introduction of XTENs. XTEN insertion within the Gla domain was avoided due to the essential role of the Gla domain in anchoring FIX to phospholipid surfaces and subendothelial type IV collagen. XTEN insertion sites were selected by analysis of available FIX structures in the Protein Data Bank in conjunction with the following criteria: 1) calculated accessible surface area, 2) solvent accessibility assessed by hydrogen/deuterium exchange mass spectrometry (H/DX-MS), 3) exclusion of sites within defined secondary structural elements, 4) preference for positions with significant inter-species protein sequence variability, and 5) exclusion of sites proximal to known hemophilia B mutations. A 42-residue XTEN element (AE-42) was inserted at sites selected by using these criteria or fused at the C-terminus of FIX. FIX activities of these variants were evaluated in conditioned medium of transfected HEK293 cells. Longer XTENs (AE-72, -144 and -288) were then similarly tested at sites shown to be permissive for AE-42 insertion. Finally, based on results obtained with these single XTEN variants, FIX variants with multiple XTEN insertions of varying lengths and at representative sites were evaluated for FIX activity. Results: Based on the criteria described above, a total of 33 sites in FIX were selected and evaluated by insertion of AE-42. Of these, two in EGF domain 2 (EGF2), one in the EGF2-activation peptide (AP) linker region, four in the AP, and four in the catalytic domain, including the C terminus, were identified as permissive sites by FIX activity assay. Only sites in AP and sites at or close to the C-terminus of FIX tolerated longer XTENs (AE-144, AE-288 or AE-864). FIX activity detected in conditioned medium inversely correlated with the length of XTEN introduced. Four representative sites in FIX were selected to generate a combinatorial library of 79 FIX variants. Three groups, FIX with a single XTEN, FIX with dual XTENs and FIX-Fc with a single XTEN insertion, showed detectable activity, while combination of insertion/fusion at three or more sites abolished FIX activity. Conclusions: Several permissive sites for XTEN insertion are present in FIX and select combinations of XTEN insertions variants retain FIX activity. Active FIX-XTEN variants identified here are candidates for pharmacokinetic characterization in hemophilia B mice. Disclosures Liu: Biogen: Employment. van der Flier:Biogen: Employment. Bardan:Biogen: Employment. Ding:Amunix: Employment. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

INTRODUCTION Prophylactic treatment for hemophilia B patients is the therapy of choice to improve quality of life and minimize annual bleeding rates and damage to joints. A new generation of extended half-life (EHL) FIX replacement products has been generated to improve patient care by reducing treatment burden. The factors include the currently marketed rFIXFc, as well as rFIX attached to PEG or albumin. The latter two are in clinical trials. All FIX preparations are administered by intravenous dosing, which can be particularly challenging for young patients and patients with limited venous access; these difficulties are reduced, but not eliminated, by the less frequent dosing achieved with EHL rFIX therapies. In the current study, we evaluate an XTEN-recombinant protein technology in order to develop EHL rFIX-XTEN molecules that are suitable for both acute treatment, as well as prophylactic subcutaneous dosing in hemophilia B, and could potentially further reduce the burden of treatment. XTEN are unstructured polypeptide sequences that consist of a limited set of natural amino acids (Pro, Ala, Gly, Glu, Ser, Thr). Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. MATERIALS AND METHODS Recombinant FIX and FIXFc molecules were expressed as the natural Arg338Leu (R388L, Padua) variant with improved activity. XTEN polypeptides are fused to either the C-terminus of rFIX or inserted into the EGF2 domain or activation peptide (AP) domain of rFIX or rFIXFc. The fusion proteins were prepared by transient expression in human HEK293 cells followed by affinity purification. Hemophilia B (HemB) mice were dosed by either intravenous or subcutaneous injection with a single bolus of 50 or 200 IU/kg of the rFIX- or rFIXFc-fusion proteins. Plasma activity levels were determined over time using a FIX activated partial thromboplastin time assay (aPTT). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.2.1 (Pharsight). RESULTS Insertion of XTEN sequences with increasing length (42, 72, 144 or 288 amino acids long) at either C-terminus of rFIX-R338L or in the AP domain showed a size dependent increase in plasma recovery up to 60% following intravenous bolus dosing. Combinations of XTEN insertions in the EGF2 or AP domain with Fc-mediated half-life extension in rFIXFc-R338L, extended the half-life as well as increased the plasma recovery. The AUC/D for rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 were 8.5 and 14.5 fold improved when compared to intravenously dosed rFIX, respectively. Following a subcutaneous dose of either rFIXFc-AP-XTEN.72 or rFIX-CT-XTEN.288, we observed 28 and 40-fold improved AUC/D; 15- and 30-fold improved Cmax/D and 3-fold increased bioavailability. When compared to rFIXFc the improvement in pharmacokinetic parameters was 6- and 9-fold improved AUC/D; 3- and 10-fold Cmax/D and 1.5- and 2-fold improved bioavailability for FIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288, respectively. Taken together, subcutaneous dosing of rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 in HemB mice showed improved AUC/D when compared to intravenous dosing of rFIXFc. CONCLUSIONS rFIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288 show greatly improved subcutaneous pharmacokinetics in HemB mice compared to both rFIX and rFIXFc. These promising preclinical subcutaneous dosing data in HemB mice suggests the potential of once weekly or every two weeks prophylactic subcutaneous dosing of FIX-XTEN molecules in patients. In addition, the molecules have potential for acute treatment by intravenous dosing. Further studies are ongoing to address the efficacy and allometric scaling in preclinical animal models. Disclosures van der Flier: Biogen: Employment. Liu:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership, Honoraria, Research Funding. Mercury:Biogen: Employment. Ismail:Biogen: Employment. Seth-Chhabra:Biogen: Employment, Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Light:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

All currently marketed Factor VIII (FVIII) molecules are administered intravenously (IV) for the treatment of hemophilia A (HemA). Conventional FVIII prophylaxis requires a dosing interval of three times per week to every other day. This frequent dosing regimen necessitates repeated venous access and is associated with complications such as secondary infection in children with a venous port/catheter. More recently, extended half-life variants of FVIII have been shown in clinical trials to decrease the dosing interval to twice weekly or less frequent IV dosing, which reduces, but does not eliminate, the burden of treatment. A FVIII molecule with further prolonged half-life and subcutaneous (SQ) delivery potential could significantly relieve the treatment burden for HemA patients and improve the adherence rate to FVIII prophylaxis. Recombinant FVIIIFc-VWF-XTEN has been shown to not bind endogenous VWF, and is able to achieve a 4-fold extension of half-life in hemophilia A mice compared to conventional FVIII, well beyond the approximately 2-fold half-life extension limit demonstrated by other long-acting FVIII variants that bind endogenous VWF. It comprises of two polypeptide chains: 1) a single chain B-domain deleted FVIIIFc-XTEN chain with a XTEN polypeptide inserted at the B-domain region of native FVIII sequence, and 2) a VWF D'D3-XTEN-Fc chain xtend one that n TEN fragemnt o FVIII prophylaxis.ntial rity of the patients depending on the half-life of the FVIII molecule. with a second XTEN polypeptide inserted between D'D3 domain and Fc. The rFVIIIFc-VWF-XTEN protein was produced in HEK293 cells and affinity purified using VIIISelect resin. The pharmacokinetic (PK) profiles of intravenously (IV) and subcutaneously (SQ) administered rFVIIIFc-VWF-XTEN were compared to those of rFVIII in HemA mice. The duration of the in vivo efficacy of rFVIIIFc-VWF-XTEN post-SQ delivery was assessed in a HemA mouse tail vein transection (TVT) bleeding model. After intravenous dosing in HemA mice, we observed a linear PK profile for rFVIIIFc-VWF-XTEN within the therapeutic dose range (25, 50, 100 IU/kg). The half-life of IV-administered rFVIIIFc-VWF-XTEN was about 37 h, which is more than 4-fold longer than that of rFVIII. In addition, animals that received 25 IU/kg of rFVIIIFc-VWF-XTEN treatment retained 5% of normal FVIII activity at 120 h post-dosing, which suggests the potential for full protection from spontaneous bleeding in this animal model. When delivered subcutaneously, the bioavailability of rFVIIIFc-VWF-XTEN was 20%, a significant increase compared to the bioavailability of rFVIII (less than 1%). Starting at 24 h post-dosing, subcutaneous administration of rFVIIIFc-VWF-XTEN achieved plasma FVIII levels that were equal to or greater than those attained with rFVIII delivered intravenously at the same dose. In addition, greater than 5% of normal circulating FVIII level was observed at 96 h post SQ administration of rFVIIIFc-VWF-XTEN with a 100 IU/kg dose, which provided 80% protection on survival in mice subjected to tail vein transection injury. These results suggest that rFVIIIFc-VWF-XTEN could enable less frequent FVIII replacement treatment compared to rFVIII even when administered subcutaneously. The VWF independence of rFVIIIFc-VWF-XTEN enables a 4-fold increase in circulating half-life compared to that of rFVIII. Also, the addition of D'D3 domains and the two XTEN insertions dramatically increases subcutaneous bioavailability to 20%, compared to less than 1% with conventional FVIII. These unique properties of rFVIIIFc-VWF-XTEN make it a potential candidate for both IV and SQ treatments for hemophilia A. Disclosures Drager: Biogen: Employment, Equity Ownership. Patarroyo-White:Biogen: Employment, Equity Ownership. Chao:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Chhabra:Biogen: Employment, Equity Ownership. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Liu:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

The development of inhibitors to replacement plasma factors in hemophilia is an ongoing clinical complication. Bypass therapies, such as recombinant factor VIIa (rFVIIa), have emerged as important alternative on-demand strategies for hemophilic patients with inhibitors to treat spontaneous bleeds and prevent bleeding during surgery. However, general prophylaxis strategies for hemophilia inhibitor patients are lacking. An attractive approach for effective prophylaxis and on-demand treatment includes engineering potent rFVIIa variants with prolonged pharmacokinetics. Because the primary mechanism of action of rFVIIa is thought to occur on the platelet surface, we combined rFVIIa with platelet-targeting and XTEN half-life extension technologies to improve its pharmacokinetic profile and procoagulant activity. Platelet-targeting was achieved by recombinant fusion of an antibody fragment which binds the human alpha IIb integrin. Half-life extension was achieved by fusion of an XTEN polypeptide which increases the hydrodynamic radius, and therefore half-life, of rFVIIa. We have shown that these rFVIIa modifications improve the pharmacokinetics and efficacy of rFVIIa in vivo in humanized alpha IIb transgenic hemophilia A mice. The goal of the current study was to evaluate and compare thrombin generation and fibrin formation kinetics in hemophilic platelet-rich plasma in the presence of platelet-targeted rFVIIa-XTEN or rFVIIa. To achieve this, platelet-rich plasma from normal human donors was treated with an inhibitory factor VIII antibody to model hemophilia A and spiked with doses of platelet-targeted rFVIIa-XTEN or rFVIIa. Reactions were triggered with low tissue factor and recalcification. Thrombin generation (n=9) was monitored by calibrated automated thrombography and fibrin formation (n=7) was monitored optically on a plate reader. Non-linear regression analysis of dose responses was used to determine EC50 values for each parameter for each donor. Platelet-targeted rFVIIa-XTEN increases the rate and peak of thrombin generation with 2- to 6-fold lower EC50 values (peak and rate, respectively) than rFVIIa. These data were confirmed by thrombin generation in platelet-rich plasma from 1 hemophilia A donor and 1 hemophilia B donor which demonstrated similar responses to platelet-targeted rFVIIa-XTEN with 2.5- to 5-fold lower EC50 values (peak and rate, respectively) than rFVIIa. Notably, platelet count-dependent changes in thrombin generation activity were similar between platelet-targeted rFVIIa-XTEN and rFVIIa. Fibrin formation analysis indicated platelet-targeted rFVIIa-XTEN increases the rate and time to plateau of fibrin formation with 5- to 13-fold lower EC50 values (rate and time to plateau, respectively) than rFVIIa. Analysis of fibrin network structure by confocal microscopy indicated platelet-targeted rFVIIa-XTEN increases fibrin network density in platelet-rich plasma clots. Together, these data show platelet-targeted rFVIIa-XTEN has more procoagulant activity than rFVIIa by supporting more thrombin generation and faster fibrin formation and suggest our approach has the potential to be an effective alternative for the treatment and prevention of bleeds in hemophilia patients with inhibitors. Disclosures Aleman: Biogen: Employment, Equity Ownership. Kistanova:Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Peters:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership.

INTRODUCTION More than 95% of circulating clotting factor VIII (FVIII) exists in a non-covalent complex with von Willebrand Factor (VWF). While VWF stabilizes and protects FVIII from its clearance pathways, it also subjects FVIII to VWF-mediated clearance. Thus, interaction with VWF imposes a limitation on the extent of FVIII half-life extension achieved by current technologies (Fc fusion, PEGylation etc.). Recombinant FVIIIFc-VWF-XTEN (rFVIIIFc-VWF-XTEN) is a novel fusion protein, consisting of the FVIII binding D'D3 domains of VWF fused to a single chain rFVIIIFc (scFVIIIFc). Appending the domains of VWF to FVIII provides the protection and stability of endogenous VWF, while avoiding the limitation imposed by VWF clearance. Besides D'D3 domains, it also contains two XTEN linkers. XTEN is an unstructured polypeptide consisting of six amino acids repeats (Gly, Ala, Pro, Glu, Ser, Thr). Fusion of XTEN to a protein reduces the rate of clearance and degradation of the fusion protein. In rFVIIIFc-VWF-XTEN, one XTEN linker replaces the B-domain of FVIII and other is attached to the D'D3 domains. In preclinical studies, this protein has shown >4-fold prolonged half-life and similar in vivo acute efficacy compared to rFVIII. In the current study, we examined the impact of various modifications on the in vitro plasma stability of rFVIIIFc-VWF-XTEN protein. MATERIALS AND METHODS rFVIIIFc-VWF-XTEN is a fusion protein which is expressed as a dual chain molecule. One chain expresses the D'D3 domains linked to a Fc monomer through an XTEN linker. This polypeptide is co-expressed with a single chain rFVIIIFc monomer to generate a dimer, via the disulfide-bond between the Fc domains. To assess the in vitro plasma stability, fusion proteins were expressed in HEK293 cells, purified and incubated with plasma from FVIII KO (Hem A) or FVIII/VWF DKO mice, for various time periods at 37 degree centigrade. After the desired incubation time, plasma stability of the recombinant proteins was determined by FVIII chromogenic activity assay. Results and Conclusions rFVIIIFc-VWF-XTEN fusion protein showed significantly enhanced in vitro plasma stability compared to rFVIII. In FVIII KO plasma, rFVIII started losing activity by 4 hours, and by 24 hours it lost more than 80% of its activity. The decline in activity was more pronounced and rapid when rFVIII was incubated with FVIII/VWF DKO plasma, mainly due to the absence of protection provided by VWF. Conversely, in the case of rFVIIIFc-VWF-XTEN, there was no significant drop in activity even after 6 hours (in both FVIII KO and DKO plasma). By 24 hours, only 10-15% activity reduction was observed in FVIII KO plasma and about a 35% decrease in DKO plasma. Further studies were conducted to evaluate various parameters which contributed to the improved stability of this fusion protein. Our results suggest that there are multiple factors which contribute to the overall stability of rFVIII-VWF-XTEN protein. These include: presence of covalently attached D'D3 domains, enhanced stability of single chain FVIII isoform used in the fusion protein and presence of the XTEN linker in the B-domain of FVIII. These data suggest that superior plasma stability of this novel fusion protein might be a contributing factor to its prolonged in vivo half-life and efficacy. Disclosures Seth Chhabra: Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Furcht:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. β-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of β-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. β-Lactamase was found to contain four CD4+ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The β-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of β-lactamase variant with adjuvant. The β-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease-catalyzed peptide synthesis. Here the synthesis of leucine-enkephalin using enzymes for N-terminal amino group protection, peptide bond formation and deprotection is described.

The S′-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P1′ position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.

We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.

We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase-catalyzed processing. An artificial substance P-precursor is produced as a beta-galactosidase (1-459) fusion protein containing nine copies of the decapeptide sequence Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by alpha-chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted converted to substance P by papain-catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase-catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo- and polypeptides.

Objective While the majority of current diabetes treatments focus on reducing blood glucose levels, hypoglycemia represents a significant risk associated with insulin treatment. Glucagon plays a major regulatory role in controlling hypoglycemia in vivo, but its short half-life and hyperglycemic effects prevent its therapeutic use for non-acute applications. The goal of this study was to identify a modified form of glucagon suitable for prophylactic treatment of hypoglycemia without increasing baseline blood glucose levels. Methodology/Principal Findings Through application of the XTEN technology, we report the construction of a glucagon fusion protein with an extended exposure profile (Gcg-XTEN). The in vivo half-life of the construct was tuned to support nightly dosing through design and testing in cynomolgus monkeys. Efficacy of the construct was assessed in beagle dogs using an insulin challenge to induce hypoglycemia. Dose ranging of Gcg-XTEN in fasted beagle dogs demonstrated that the compound was biologically active with a pharmacodynamic profile consistent with the designed half-life. Prophylactic administration of 0.6 nmol/kg Gcg-XTEN to dogs conferred resistance to a hypoglycemic challenge at 6 hours post-dose without affecting baseline blood glucose levels. Consistent with the designed pharmacokinetic profile, hypoglycemia resistance was not observed at 12 hours post-dose. Importantly, the solubility and stability of the glucagon peptide were also significantly improved by fusion to XTEN. Conclusions/Significance The data show that Gcg-XTEN is effective in preventing hypoglycemia without the associated hyperglycemia expected for unmodified glucagon. While the plasma clearance of this Gcg-XTEN has been optimized for overnight dosing, specifically for the treatment of nocturnal hypoglycemia, constructs with significantly longer exposure profiles are feasible. Such constructs may have multiple applications such as allowing for more aggressive insulin treatment regimens, treating hypoglycemia due to insulin-secreting tumors, providing synergistic efficacy in combination therapies with long-acting GLP1 analogs, and as an appetite suppressant for treatment of obesity. The improved physical properties of the Gcg-XTEN molecule may also allow for novel delivery systems not currently possible with native glucagon.

From the literature we collected all available quantitative data on the chymotrypsin-catalyzed hydrolysis of series of amino acid and peptide substrates. Utilizing this data base, we performed calculations on their quantitative structure/activity relationship (QSAR). The substrates were considered to be composed of fragments; log(kcat/Km) values for the substrates resulted from additive contributions of their fragments. Despite the fact that the kinetic constants in the data base were determined by different authors under various reaction conditions, the data are well described by the simple additivity model. Obviously, the intrinsic specificity of chymotrypsin dominates the influence of varying reaction conditions.

Benzyloxycarbonyl-L-proline p-guanidinophenyl ester is an "inverse substrate" for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin-catalyzed acyl-transfer reactions leading to the synthesis of Pro-Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine-containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P(1)'-position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl-enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl-enzyme.