Half-life Extension Research Articles

Abstract 4076: Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential

Abstract Interleukin-18 (IL-18) possesses a unique combination of innate and acquired immune functions with high potential to be transformative in cancer immunotherapy of solid tumors. In clinical studies, recombinant wild-type IL-18 was limited by rapid upregulation of IL-18 binding protein (IL-18BP), which serves as a negative feedback checkpoint of the IL-18 pathway. As its name implies, IL-18BP tightly binds to IL-18, thereby blocking its ability to activate the IL-18 receptor. We have focused our protein engineering efforts to obtain variants of IL-18 that are resistant to IL-18BP suppression while retaining IL-18 biological activity. To further capitalize on the immunological effects of IL-18, the IL-18BP resistant variants were fused to half-life extension protein scaffolds for enhanced in vivo exposure. Rational protein design and combinatorial approaches were used to generate a pool of IL-18 variants that were screened for potency and resistance to IL-18BP. Several IL-18 variants showed undetectable binding to IL-18BP (up to 1 mM IL-18BP) and displayed a spectrum of potencies relative to recombinant wild-type IL-18. Importantly, these variants retained their full biological activity in the presence of super-physiological levels of IL-18BP, as tested with in vitro cell-based IFN-γ release assays. In vivo pharmacokinetic studies in mice compared several IL-18BP resistant variants fused to a half-life extension scaffold with a non-half-life enhanced IL-18BP resistant variant. The half-lives for the fusion proteins ranged from 15 - 30h, while the non-fused variant showed a half-live of 0.6h. Furthermore, the IL-18 variants displayed a more durable, Th1 immune response compared to a non-half-life enhanced variant. Thus, IL-18 variants with strong resistance to IL-18BP that are fused to a half-life extension protein scaffold displayed enhanced pharmacokinetic and pharmacodynamic properties in preclinical mouse models. These protein engineering modifications will enable us to select an appropriate development candidate with enhanced pharmaceutical properties to achieve the full therapeutic potential of IL-18 to assess in patients with cancer. Citation Format: Jean Chamoun, Clifford DiLea, Kristiana Dreaden, Pinar Gurel, Joshua Heiber, Robert G. Newman, Su-Ping Pearson, Chunhua Wang, Yanchun Zhao, Mark Whitmore. Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4076.

Abstract 4074: Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models

Abstract Systemic administration of proinflammatory cytokines is a promising approach to treat cancer. IL-18 has been shown to promote activation of both innate and adaptive anti-tumor responses in pre-clinical models. Specifically, IL-18 has been shown to impact macrophage suppressive function as well as drive CD4+ T cells towards a TH1 phenotype. However, IL-18 therapies have been hampered by a lack of efficacy due to the suppressive activity of IL-18 binding protein (IL-18BP), which binds to IL-18 and prevents its interaction with its receptor. Several groups have taken the approach of modifying IL-18 to render it resistant to IL-18BP as a potential strategy to enhance anti-tumor activity. To explore the therapeutic benefit of delivering a conditionally-activated IL-18 to the tumor microenvironment, we have developed inducible polypeptides (INDUKINE™ molecules) consisting of mouse IL-18 or IL-18BP resistant IL-18 (BPR IL-18) tethered via protease-sensitive linkers to a high affinity antibody blockade domain, and a half-life extension (HLE) domain which improves exposure in the tumor. IL-18 INDUKINE™ molecules are inactive until reaching the tumor microenvironment, where the linkers are cleaved by intra-tumoral proteases, releasing active IL-18. Intraperitoneal (i.p.) administration of an IL-18BP resistant IL-18 (BPR IL-18) INDUKINE™ molecule led to complete tumor regression in the MC38 tumor model. In contrast, equimolar doses of wild-type IL-18 INDUKINE™ protein were not as efficacious. Moreover, BPR IL-18 INDUKINE treatment led to increased activation and frequencies of NK cells and tumor specific CD8 T cells in MC38 tumors. WTX-518 is a novel INDUKINE™ molecule that is designed to selectively deliver active human BPR IL-18 to the tumor microenvironment. WTX-518 is inducible in vitro, either when tested using a reporter assay or primary immune cells. Importantly, the active BPR IL-18 payload is resistant to IL-18BP in reporter or primary immune cell assays. WTX-518 is also selectively inducible, as incubation ex vivo with various primary human tumors led to the release of active BPR IL-18, while WTX-518 was stable in human serum and after exposure to primary cells from healthy tissues. Pharmacological and efficacy data support continued preclinical development of this innovative and differentiated engineered IL-18 therapy. Citation Format: Kristin R. Morris, Heather Brodkin, Kyriakos Economides, Daniel J. Hicklin, Julie LePrevost, Celesztina Nagy-Domonkos, Christopher Nirschl, Andres Salmeron, Cynthia Seidel-Dugan, Cierra Spencer, Zoe Steuert, William M. Winston. Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4074.

Abstract 4078: WTX-712, a conditionally active IL-21 INDUKINETM molecule, induces a strong anti-tumor phenotype through a differentiated mechanism

Abstract Despite great strides seen recently in the development of immunotherapies such as immune checkpoint inhibitors (ICI), many patients do not respond or acquire resistance, highlighting a need for alternative immunotherapies. As potent immunomodulators, cytokines have been explored as treatments for cancer, but their use has been limited due to toxicity and poor pharmacokinetics (PK). One of these key cytokines, interleukin 21 (IL-21), is a pluripotent cytokine that activates anti-tumor T cell responses, induces B cell activation, and promotes generation and maintenance of germinal centers and tertiary lymphoid structures. IL-21 acts on a broader range of cells and does not induce vascular leak syndrome compared to IL-2, another common-gamma chain family member. Half-life extended IL-21 (IL-21-HLE) drives robust anti-tumor activity in several syngeneic tumor mouse models. Of particular interest, IL-21-HLE showed superior anti-tumor activity compared to IL-2-HLE in the EMT-6 and Renca models, both of which are highly resistant to anti-PD-1/PD-L1 treatment. Although IL-21-HLE and IL-2-HLE induced a similar frequency of tumor infiltrating CD8+ T cells, IL-21-HLE treatment led to higher polyfunctionality in CD8+ T cells than IL-2-HLE treatment. Of note, IL-21-HLE induced greater amounts of effector cytokines, Granzyme B and perforin, than IL-2-HLE. Transcriptomic analysis revealed that IL-21 drives upregulation of a type I IFN signature in the tumor promoting an anti-tumorigenic microenvironment. Clinical activity of IL-21 has been hampered by poor PK and adverse events at dose levels associated with signs of efficacy. To overcome these limitations Werewolf Therapeutics has developed WTX-712, an IL-21 INDUKINETM molecule, which contains wild-type human IL-21, an inactivation domain, and a half-life extension domain tethered together by protease sensitive linkers. In preclinical studies with mouse syngeneic tumor models, WTX-712 is inactive in the periphery, and wild-type IL-21 is selectively released within the tumor, resulting in anti-tumor efficacy with an expanded therapeutic window compared to IL-21-HLE. Anti-tumor efficacy was linked to expansion and activation of tumor infiltrating CD8 T cells with increased polyfunctionality. We will show additional mechanistic data evaluating the effect of WTX-712 dosing on immune populations, including tumor spatial profiling using different models. Citation Format: Jenna M. Sullivan, Pamela A. Aderhold, Heather R. Brodkin, Celesztina Nagy-Domonkos, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Yuka Lewis, Cynthia Seidel-Dugan, William M. Winston, Andres Salmeron. WTX-712, a conditionally active IL-21 INDUKINETM molecule, induces a strong anti-tumor phenotype through a differentiated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4078.

Abstract 4059: Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment

Abstract Background: Interleukin-2 is the first approved immune-oncology (IO) drug to treat multiple metastatic cancers with durable and curative antitumor effects. Yet, its application is restricted due to severe toxicity, short half-life, and a small fraction of response rate. To reduce toxicity and improve efficacy, we developed Vitokine-2, a tumor infiltrating lymphocyte (TIL)-targeting, tumor activable, and precision engineered IL-2 that remains inert systemically but activated in the tumor microenvironment (TME). Methods: Through protein and structure-guided precision engineering, the biological activity of IL-2 was optimized, concealed, guided to exhausted TILs, and activated at TME. The biological activity was determined by measuring proliferation (Ki67) of CD8+ T and NK cells using human peripheral blood mononuclear cells (PBMCs). Pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor efficacy were evaluated in mice, encompassing healthy and multiple syngeneic tumor mouse models. Results: Vitokine-2 was inactive in stimulating CD8+T and NK cell proliferation in vitro. Yet, its biological activity was fully restored following the cleavage and release of the concealing domain. Vitokine-2 displayed a significantly prolonged in vivo pharmacokinetic profile due to half-life extension, concealing and reduction of target sink. Administration of Vitokine-2 in mice resulted in minimal or no detectable activation, proliferation, and expansion of cytotoxic CD8+T and NK cells in the systemic circulation. However, marked CD8+ T cell proliferation and expansion with enhanced expression of granzyme B, a cytotoxic functional biomarker, was observed within the TME. The effect was specific to cytotoxic lymphocytes but not regulatory T cells. Corroborating with the expansion and enhanced functionality of CD8+ T cells in TME, robust antitumor efficacy was observed with Vitokine-2 but not with the non-activable IL-2 counterpart. Furthermore, dose-dependent tumor killing efficacy and complete eradication of tumors were demonstrated in multiple syngeneic tumor models. Vitokine-2 was found to be safe and well tolerated with minimal interferon gamma release and body weight loss even at much higher doses. Conclusion: Vitokine-2 is a TIL-targeting, tumor activable and precision engineered IL-2. It demonstrated robust anti-tumor efficacy with minimal systemic toxicity. It is safe, well tolerated, and permits high dose administration, yielding a widened therapeutic window. Vitokine-2 presents a paradigm-shifting, potentially a first-in-class, highly safe and efficacious IO therapy with an immense potential to address both IO-responsive and unresponsive patient population. Citation Format: Sreerupa V. Challa, Yao-Te Hsieh, Karen Gelinas, Andrea Umana, Wei-chun Weng, Toska Berisha, Cameron Stephens, Yuesheng Li, Jing Xu. Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4059.

A new trivalent recombinant protein for type 2 diabetes mellitus with oral delivery potential: design, expression, and experimental validation

Glucagon-like peptide-1 (GLP-1) receptor agonists are increasingly used in clinical practice for the management of type 2 diabetes mellitus. However, the extremely short half-life of GLP-1 and the need for subcutaneous administration limit its clinical application. Thus, half-life extension and alternative delivery methods are highly desired. DARPin domains with high affinity for human serum albumin (HSA) have been selected for the half-life extension of therapeutic peptides and proteins. In the present study, novel trivalent fusion proteins as long-acting GLP-1 receptor agonists with potential for oral delivery were computationally engineered by incorporating a protease-resistant modified GLP-1, an anti-human serum albumin DARPin, and an approved cell-penetrating peptide (Penetratin, Tat, and Polyarginine) linked either by rigid or flexible linkers. Theoretical studies and molecular dynamics simulation results suggested that mGLP1-DARPin-Pen has acceptable quality and stability. Moreover, the potential affinity of the selected fusion proteins for GLP-1 receptor and human serum albumin was explored by molecular docking. The recombinant construct was cloned into the pET28a vector and expressed in Escherichia coli. SDS-PAGE analysis of the purified fusion protein matched its molecular size and was confirmed by western blot analysis. The results demonstrated that the engineered fusion protein could bind HSA with high affinity. Importantly, insulin secretion assays using a mouse pancreatic β-cell line (β-TC6) revealed that the engineered trivalent fusion protein retained the ability to stimulate cellular insulin secretion. Immunofluorescence microscopy analysis indicated the CPP-dependent cellular uptake of mGLP1-DARPin-Pen. These findings demonstrated that mGLP1-DARPin-Pen is a highly potent oral drug candidate that could be particularly useful in the treatment of type 2 diabetes mellitus. Communicated by Ramaswamy H. Sarma

R2R01: A long-acting single-chain peptide agonist of RXFP1 for renal and cardiovascular diseases.

The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half-life of serelaxin. Optimization of fatty acid-acetylated single-chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half-life. Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo-allergic reactions and CD4+ T-cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs. In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half-life. The in vitro assays with LAD2 cells and CD4+ T-cells showed that R2R01 had low potential for pseudo-allergic and immunogenic reactions, respectively. R2R01 is a potent RXFP1 agonist with an extended half-life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.

Six-Year, Real-World Use of Prophylaxis with Recombinant Factor IX-Albumin Fusion Protein (rIX-FP) in Persons with Hemophilia B: A Single-Center Retrospective-Prospective Study.

Background: Extended half-life (EHL) factor IX (FIX) concentrates allow for prophylaxis with prolonged dosing intervals and high bleeding protection in persons with hemophilia B. Long-term real-world studies are lacking. Methods: In a retrospective-prospective study, the six-year use of prophylaxis with the EHL recombinant FIX-albumin fusion protein (rIX-FP) was analyzed, comparing outcomes with previous standard half-life (SHL) FIX in patients already on prophylaxis. Results: Prophylaxis with rIX-FP was prescribed in 15 patients (10 severe, 5 moderate; follow-up: 57 ± 17 months). Based on a pharmacokinetic assessment and clinical needs, the first regimen was 47 ± 7 IU/Kg every 9 ± 2 days. All but one patient remained on rIX-FP prophylaxis, adjusting infusion frequency and/or dose; the last prescribed frequency was ≥10 days in 10/13 patients, being reduced in seven and increased in four vs. the first regimen. The weekly FIX dose was unchanged; FIX trough levels were >5% in all patients. The annual infusion number and FIX IU/Kg significantly decreased (~60%) in eight patients previously on SHL FIX prophylaxis, with similar concentrate costs. Very low bleeding rates (most traumatic bleeds and the last quartile of the infusion interval), improved orthopedic and pain scores, unchanged HEAD-US scores and problem joints, and high treatment adherence (>90%) and satisfaction were registered. Conclusions: Personalized, carefully adjusted rIX-FP regimens contribute to the diffusion and optimization of prophylaxis in persons with severe and moderate hemophilia B, with long-term favorable bleeding, joint, and patient-reported outcomes.

Recombinant factor IX Fc for the treatment of hemophilia B.

Current hemophilia B treatment guidelines recommend routine prophylaxis with factor IX (FIX) replacement products, tailored to maintain plasma activity at levels that will prevent bleeds. However, plasma FIX activity may not be the primary determinant or best indicator of hemostatic efficacy due to its extravascular distribution. FIX replacement therapy has evolved to include extended half-life (EHL) products that provide effective bleed protection when administered at intervals of 7 days or longer. rFIXFc is a recombinant fusion protein with an extended circulation time. rFIXFc has a biodistribution profile consistent with distribution into extravascular space, where it may support hemostasis at sites of vessel injury independent of circulating plasma activity levels. The safety and efficacy of rFIXFc prophylaxis is well established in adults, adolescents and children including previously untreated patients with hemophilia B, with substantial evidence from clinical trials and real-world clinical practice. This review describes the pharmacokinetic characteristics of rFIXFc, summarizes available safety and efficacy data, and evaluates the use of rFIXFc in special populations. Current hemophilia B treatment challenges, including target FIX plasma levels, perioperative use, and management of patients with comorbidities, are discussed together with the potential role of EHL products in the future treatment landscape of hemophilia B.

Reforming solid tumor treatment: the emerging potential of smaller format antibody-drug conjugate.

In recent years, substantial therapeutic efficacy of antibody-drug conjugates (ADCs) has been validated through approvals of 16 ADCs for the treatment of malignant tumors. However, realization of the maximum clinical use of ADCs requires surmounting extant challenges, mainly the limitations in tumor penetration capabilities when targeting solid tumors. To resolve the hurdle of suboptimal tumor penetration, miniaturized antibody fragments with engineered formats have been harnessed for ADC assembly. By virtue of their reduced molecular sizes, antibody fragment-drug conjugates hold considerable promise for efficacious delivery of cytotoxic agents, thus conferring superior therapeutic outcomes. This review will focus on current advancements in novel ADC development utilizing smaller antibody formats from ~6 to 80kDa, with particular emphasis on single-domain antibodies, which have been widely applied in novel ADC design. Additionally, strategies to optimize clinical translation are discussed, including half-life extension, acceleration of internalization, and reduction of immunogenic potential.

Efanesoctocog alfa: the renaissance of Factor VIII replacement therapy.

Efanesoctocog alfa (Altuviiio,TM Sanofi-SOBI) is a B domain-deleted single-chain Factor VIII (FVIII) connected to D'D3 domain of von Willebrand Factor (vWF). Its ingenious design allows efanesoctocog alfa to operate independently of endogenous vWF and results in an outstanding 3-4 times longer half-life compared to standard and extended half-life (EHL) FVIII products. The prolonged half-life ensures sustained high levels of factor activity, maintaining normal to near-normal ranges for the majority of the week, facilitating the convenience of once-weekly administration. Efanesoctocog alfa received regulatory approval in 2023 for application in both adults and children with inherited hemophilia A in the United States and Japan. Its sanctioned use encompasses both prophylaxis and 'on demand' treatment for bleeding episodes. The European Medicines Agency (EMA) is currently undertaking a comprehensive review of Altuviiio. TM This comprehensive review focuses on the immunological profile of efanesoctocog alfa, a highly sophisticated new class of EHL FVIII molecule. The integration of the vWF D'D3 domain, XTEN polypeptides, and potential regulatory T-cell epitopes within various segments of efanesoctocog alfa collectively serves as a mitigating factor against the development of a neutralizing T-cell-mediated immune response. We hypothesize that such distinctive attribute may significantly reduce the risk of neutralizing antibodies, particularly in previously untreated patients. The discussion extends beyond regulatory approval to encompass the preclinical and clinical development of efanesoctocog alfa, including considerations for laboratory monitoring. The review also highlights areas that warrant further investigation to deepen our understanding of this groundbreaking therapeutic agent.

Progress in von Willebrand Disease Treatment: Evolution towards Newer Therapies.

von Willebrand disease (VWD) is a very heterogenous disease, resulting in different phenotypes and different degrees of bleeding severity. Established therapies (i.e., desmopressin, antifibrinolytic agents, hormone therapy for heavy menstrual bleeding, and von Willebrand factor [VWF] concentrates) may work in some subtypes, but not in all patients. In recent years, progress has been made in improving the diagnosis of VWD subtypes, allowing for more specific therapy. The impact of VWD on women's daily lives has also come to the fore in recent years, with hormone therapy, tranexamic acid, or recombinant VWF as treatment options. New treatment approaches, including the replacement of lacking factor VIII (FVIII) function, may work in those subgroups affected by severe FVIII deficiency. Reducing the clearance of VWF is an alternative treatment pathway; for example, rondaptivon pegol is a VWFA1 domain-binding aptamer which not only improves plasma VWF/FVIII levels, but also corrects platelet counts in thrombocytopenic type 2B VWD patients. These approaches are currently in clinical development, which will be the focus of this review. In addition, half-life extension methods are also important for the improvement of patients' quality of life. Targeting specific mutations may further lead to personalized treatments in the future. Finally, a few randomized controlled trials, although relatively small, have been published in recent years, aiming to achieve a higher level of evidence in future guidelines.

44Sc production from enriched 47TiO2 targets with a medical cyclotron

44Sc is a β+-emitter which has been extensively studied for nuclear medicine applications. Its promising decay characteristics [t1/2 = 3.97 h, Eβ+ = 632 keV (94.3%), Eγ = 1157 keV (99.9%); 1499 keV (0.91%)] make it highly attractive for clinical PET imaging, offering an alternative to the widely used 68Ga [t1/2 = 67.7 min, Eβ+ = 836 keV (87.7%)]. Notably, its nearly fourfold longer half-life opens avenues for applications with biomolecules having extended biological half-lives and enables the centralized distribution of 44Sc radiopharmaceuticals. An additional advantage of employing 44Sc as a diagnostic radioisotope lies in its counterpart, the β−-emitter 47Sc, which is currently under investigation for targeted radiotherapy. Together, they form an ideal theranostic pair, providing a comprehensive solution for both diagnostic imaging and therapeutic applications in nuclear medicine.At the Bern medical cyclotron, a study to optimize the production of scandium radioisotopes is currently ongoing. In this context, proton irradiation of titanium targets has been investigated, exploiting the reactions 47Ti(p,α)44Sc and 50Ti(p,α)47Sc. This approach enables the production of Sc radioisotopes within a single PET medical cyclotron facility, employing identical chemical procedures for target preparation and post-irradiation processing.In this paper, we report on cross-section measurements of the 47Ti(p,α)44Sc nuclear reaction using 95.7% enriched 47TiO2 targets. On the basis of the obtained results, the production yield and purity were calculated to assess the optimal irradiation conditions. Production tests were performed to confirm these findings.

P911 Development and Characterization of SPY002, a Novel Extended Half-life Monoclonal Antibody Drug Candidate Targeting TL1A for the Treatment of IBD

Abstract Background Tumor necrosis factor (TNF)-like ligand 1A (TL1A) mediates a broad spectrum of pro-inflammatory and fibrotic effects and is implicated in the pathogenesis of several immunologic disorders. Blockade of the interaction of TL1A with its cognate receptor, DR3, has been shown to ameliorate disease activity patients with Crohn’s disease (CD) and ulcerative colitis (UC). SPY002 is a novel, extended half-life fully human IgG1 monoclonal antibody (mAb) that binds soluble TL1A with high affinity and specificity, and potently inhibits TL1A-mediated signaling. Methods SPY002 was evaluated in multiple in vitro and ex vivo assays compared to other clinical anti-TL1A mAbs (PRA023/MK-7240, RVT-3101, and TEV-48574). Binding affinity to soluble TL1A was determined by surface plasmon resonance (SPR) and binding to membrane-bound TL1A was confirmed by flow cytometry (FACS). Competitive blockade of TL1A binding to cell-surface DR3 was determined by FACS, and competition against the decoy receptor, DcR3, was evaluated by ELISA. The binding epitope of SPY002 was determined using cryogenic electron microscopy. TF-1 cell apoptosis and a primary human whole blood-based assay measuring IFNg secretion were used to assess functional blockade of exogenously added TL1A. Half-life extension was measured via pharmacokinetic analysis in both Tg276 transgenic mice (hemizygous for human FcRn) and cynomolgus monkeys given a single bolus of SPY002 by intravenous and/or subcutaneous administration. Results SPY002 binds specifically to monomeric and trimeric TL1A and not to related TNF super family proteins TNF, FasL, TRAIL, or LIGHT. SPY002 demonstrates sub-nanomolar affinity for soluble TL1A and binds to TL1A over-expressed on the surface of human embryonic kidney (HEK) cells. SPY002 potently inhibits both TL1A-induced apoptosis of TF-1 cells and TL1A-induced secretion of IFNg in human whole blood, with IC50 values comparable to those of RVT-3101 and TEV-48574, and a substantially lower IC50 than that of PRA023/MK-7240. The half-life of SPY002 is significantly extended in Tg276 mice compared to RVT-3101. In cynomolgus monkeys, the half-life of SPY002 is at least 18 days, compared to an observed half-life of 9-12 days for RVT-3101 and PRA023/MK-7240. Conclusion SPY002 exhibits high selectivity and affinity for TL1A, demonstrates effective blockade of the TL1A interaction with DR3, and potently inhibits downstream cellular signaling. With an extended half-life in NHP, SPY002 demonstrates therapeutic potential for effective and safe treatment of CD and UC with the advantage of infrequent SC dosing. Further preclinical and clinical studies are warranted to demonstrate this potential.

P765 A Novel Monoclonal Antibody Drug Candidate SPY001 Targeting Integrin ɑ4β7 for the Treatment of IBD Demonstrates Prolonged Half-Life in Non-Human Primates

Abstract Background Background: Antagonism of the interaction between the cellular adhesion integrin ɑ4β7 and endothelial ligand mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) has proven to be relatively safe and effective in the treatment for Crohn’s disease (CD) and ulcerative colitis (UC). Additional benefit may be gained from an ɑ4β7 antagonist given via the subcutaneous (SC) route at extended intervals (eg, every 8 to 12 weeks) during maintenance therapy. Methods The humanized monoclonal IgG1 antibody SPY001 binds to the same ɑ4β7 epitope as vedolizumab and includes a YTE modification within the Fc region to increase its serum half-life via increased affinity for the neonatal Fc receptor (FcRn), thereby favoring recycling into the circulation over degradation. Half-life extension was evaluated via pharmacokinetic analysis in Tg276 transgenic mice (hemizygous for human FcRn) following a single intravenous bolus dose of SPY001 (10 mg/kg, 5 mg/kg, 1 mg/kg) and in cynomolgus monkeys following a single bolus dose of SPY001 (150 mg/kg), given by either intravenous (IV) and subcutaneous (SC) administration. PK simulations in humans were based on allometric scaling of SPY001 clearance observed in cynomolgus monkey and conducted in MATLAB. Results In Tg276 mice, the half-life of SPY001 was 10-11 days across all dose cohorts compared to approximately 3 days for vedolizumab. In cynomolgus monkeys, the half-life of SPY001 was 19-23 days following a single IV or SC infusion, a significant increase over the reported half-life of 10 days for vedolizumab in cynomolgus monkeys. Based on allometric scaling of the clearance of SPY001 observed in this study, predictive simulations of SPY001 PK in humans suggested that Q8W-Q12W SC dosing at 300 mg would be able to achieve a C6-wks, induction ≥ 35 mg/mL and Ctrough, ss ≥ 6 mg/mL for simulated patients covering a large range of body weights and albumin concentrations. Conclusion SPY001 is a novel humanized monoclonal IgG1 with an extended half-life over that of vedolizumab in Tg276 mice and cynomolgus monkeys. It offers the potential for effective and safe treatment of CD and UC with the advantage of infrequent SC dosing. Further preclinical and clinical studies are warranted to demonstrate this potential.

Perioperative management in haemophilia with extended half-life factors: a case series

Hemophilia B is a hereditary bleeding disorder characterized by deficient or defective coagulation factor IX, leading to a propensity for recurrent bleeding episodes, particularly in the joints. Management of Hemophilia B in surgical settings poses unique challenges due to the risk of excessive bleeding. This paper presents a series of two cases that demonstrate the effective use of perioperative extended half-life (EHL) factor IX products, specifically N9-GP (Refixia), in the surgical management of Hemophilia B. The cases include total knee replacement and total hip replacement. In each case, early initiation of EHL was observed to maintain adequate factor IX levels and control perioperative bleeding effectively, leading to successful surgical outcomes. These findings support the growing body of evidence suggesting the advantages of EHL perioperative treatment in patients with Hemophilia B, particularly in preparation for surgery. The implementation of EHL factor IX products as part of perioperative management plans may contribute to improved surgical outcomes and overall quality of life in Hemophilia B patients.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Safety and efficacy of damoctocog alfa pegol prophylaxis in patients with severe haemophilia A: Results of an interventional, post-marketing study.

Damoctocog alfa pegol (BAY 94-9027, Jivi® ) is an approved extended half-life factor VIII (FVIII) for treatment of previously treated patients with haemophilia A aged ≥12 years. We report the final results of an interventional, post-marketing study of damoctocog alfa pegol prophylaxis in patients with severe haemophilia A. In this open-label, interventional, post-marketing, phase 4 trial (NCT04085458), previously FVIII-treated patients with severe haemophilia A aged ≥18 years received damoctocog alfa pegol for ≥100 exposure days (EDs). Patients initially received 45IU/kg every 5 days (recommended) or 40IU/kg twice-weekly. At Visit3, patients' doses could be increased, or treatment frequency adapted. The primary endpoint was FVIII inhibitor development (titre ≥.6Bethesda units). Secondary endpoints included anti-polyethylene glycol (PEG) antibody development, treatment-emergent adverse events (AEs) and annualized bleeding rate (ABR). Overall, 36 patients were enrolled; 32 patients received treatment, of whom, 27 completed the study. No patients developed FVIII inhibitors; three tested transiently positive for low-titre anti-PEG antibodies without clinical relevance. Three patients reported study-drug-related AEs of mild or moderate intensity. Two patients discontinued the study due to AEs. No deaths occurred. Most patients (70%) were treated with E5D/E7D regimens. The median (Q1;Q3) total ABR (N=30) was 3.0 (.0;9.0) pre-study and 1.8(.7;5.9) during the study. Damoctocog alfa pegol individualized prophylaxis regimens were well-tolerated with no immunogenicity concerns. ABRs improved following the switch from pre-study prophylaxis to damoctocog alfa pegol prophylaxis. These results support the favourable safety and efficacy profile of damoctocog alfa pegol prophylaxis.

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics.

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.

Impact of tissue penetration and albumin binding on design of T cell targeted bispecific agents

Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodies⁎⁎Humabody® is a registered trademark of Crescendo Biologics Ltd. are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.

Exploring a novel long-acting glucagon-like peptide-1 receptor agonist built on the albumin-binding domain and XTEN scaffolds

In recent years, glucagon-like peptide-1 (GLP-1) has demonstrated considerable potential in the treatment of type 2 diabetes (T2D) and obesity. However, the half-life of naturally occurring GLP-1 is quite short in vivo. Two common strategies employed for half-life extension are the use of the Albumin-binding domain (ABD) and XTEN polypeptide, which operate through different mechanisms. In this study, we designed an innovative GLP-1 receptor agonist with an extended duration of action. This new construct incorporated an albumin binding domain (ABD) and an XTEN sequence (either XTEN144 or XTEN288) as carriers. We referred to these fusion proteins as GLP-ABD-XTEN144 and GLP-ABD-XTEN288. In an E. coli system, the said constructs were efficaciously produced in substantial quantity. It was observed from in vitro studies that the fusion protein GLP-ABD-XTEN144 demonstrated a five times stronger affinity towards human serum albumin (HSA), boasting a binding affinity (Kd) of 5.50 nM. This was in contrast to GLP-ABD-XTEN288, whose Kd value was registered at 27.78 nM. Moreover, GLP-ABD-XTEN144 presented a half-life of 12.9 h in mice, thus exceeding the corresponding value for GLP-ABD-XTEN288, 7.32 h in mice. Both these fusion proteins significantly mitigated non-fasting blood sugar levels and overall food consumption for 48 h subsequent to a one-time injection in mice. Notably, GLP-ABD-XTEN144 exhibited more pronounced hypoglycemic activity and food inhibitory effects than GLP-ABD-XTEN288. The designed GLP-ABD-XTEN144 fusion protein shows promising prospects for clinical application in T2D treatment. Our findings also suggest that ABD and XTEN polypeptides synergistically contribute to half-life extension, further enhancing the pharmacokinetic characteristics of a payload.