Recombinant Fusion Protein Research Articles

Tandem fusion of albumin-binding domains promoted soluble expression and stability of recombinant trichosanthin in vitro and in vivo

Trichosanthin (TCS), as a type 1 ribosome-inactivating protein, has a very high cytoplasmic activity in vitro and can quickly kill cancer cells. However, it is easily filtered and cleared by the kidney, which results in the short half-life and severely limits its application. In this study, we constructed several recombinant proteins by fusing the albumin binding domain mutant ABD035(abbreviated as ABD) to the N- or C-terminus of TCS to endow the recombinant TCS fusion protein with a longer half-life property binding with endogenous human serum albumin (HSA) via ABD to effectively exert its anti-tumor activity in vivo. Pull down, Dynamic light scattering and ELISA assays all showed that TCS fused with two ABD sequences at the C-terminus of TCS, has stronger binding capacity to HSA in vitro than TCS with one ABD. In vivo studies in BALB/C mice were performed and the elimination half-life of TCS-ABD-ABD is about 15-fold longer compared to TCS and anti-tumor activity is about 30% higher than that of TCS alone in BALB/C mouse experiments. Moreover, we found that TCS with two ABDs in tandem have the highest soluble expression level, more than 5 times higher than that of TCS, and the yield of purified protein of TCS-ABD-ABD was as high as 68.9 mg/L culture solution, which was about 7-fold higher than that of TCS. Furthermore, MTT assay showed that the anti-tumor activity of TCS-ABD-ABD was significantly higher than TCS fused with only one ABD sequence, indicating that the repeated ABD sequences facilitated the biological activity of TCS. In this paper, the fusion of the albumin-binding domain in tandem with TCS can effectively improve its stability in vivo and also significantly increase its soluble expression, expanding the application of the albumin-binding domain in the high soluble expression and stability of protein drugs.

Dual Costimulatory and Coinhibitory Targeting with a Hybrid Fusion Protein as an Immunomodulatory Therapy in Lupus Nephritis Mice Models.

Systemic lupus erythematosus is a complex autoimmune disorder mostly mediated by B-cells in which costimulatory signals are involved. This immune dysregulation can cause tissue damage and inflammation of the kidney, resulting in lupus nephritis and chronic renal failure. Given the previous experience reported with CTLA4-Ig as well as recent understanding of the PD-1 pathway in this setting, our group was encouraged to evaluate, in the NZBWF1 model, a human fusion recombinant protein (Hybri) with two domains: CTLA4, blocking the CD28—CD80 costimulatory pathway, and PD-L2, exacerbating the PD-1–PD-L2 coinhibitory pathway. After achieving good results in this model, we decided to validate the therapeutic effect of Hybri in the more severe MRL/lpr model of lupus nephritis. The intraperitoneal administration of Hybri prevented the progression of proteinuria and anti-dsDNA antibodies to levels like those of cyclophosphamide and reduced the histological score, infiltration of B-cells, T-cells, and macrophages and immune deposition in both lupus-prone models. Additionally, Hybri treatment produced changes in both inflammatory-related circulating cytokines and kidney gene expression. To summarize, both in vivo studies revealed that the Hybri effect on costimulatory-coinhibitory pathways may effectively mitigate lupus nephritis, with potential for use as a maintenance therapy.

Genetically modified metallothionein/cellulose composite material as an efficient and environmentally friendly biosorbent for Cd2+ removal

Metallothioneins (MTs) are a class of cysteine-rich metal-binding proteins. Cadmium (Cd) is one of the toxic heavy metal pollutants. In our previous research, the full-length cDNA of MT (Cd specificity) from freshwater crab (Sinopotamon henanense) (ShMT) was cloned and genetically modified to ShMT3 by site-directed mutagenesis to enhance the tolerance for Cd2+, however, it was limited in actual Cd2+ adsorption due to instability. Here, ShMT3-CBM, a novel recombinant fusion protein, was prepared. CBM is a carbohydrate binding module that can specifically bind cellulose while ShMT3 can effectively chelate Cd2+. The biosorbent Cellulose1-ShMT3-CBM was obtained by screening suitable cellulose materials. The selective adsorption experiments showed that Cellulose1-ShMT3-CBM had a preference for Cd2+. In low-concentration Cd2+ solutions, the removal efficiency was >99 %, and the adsorption equilibrium was reached within 15 min. The saturated adsorption capacity of Cellulose1-ShMT3-CBM for Cd2+ is 180.35 ± 4.67 mg/g (Dry Weight). Regeneration experiments showed that adsorption efficiency was maintained after six cycles. The MTT experiment showed that Cellulose1-ShMT3-CBM had low cytotoxicity. Meanwhile, Cellulose1-ShMT3-CBM can preferentially remove Cd2+ in actual water samples and boiler sewage. In this study, an environmentally friendly biosorbent which can adsorb Cd2+ efficiently and quickly was prepared for actual water treatment.

Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG

The recombinant fusion protein HELP-UnaG (HUG) is a bifunctional product that exhibits human elastin-like polypeptide (HELP)-specific thermal behavior, defined as a reverse phase transition, and UnaG-specific bilirubin-dependent fluorescence emission. HUG provides an interesting model to understand how its two domains influence each other’s properties. Turbidimetric, calorimetric, and light scattering measurements were used to determine different parameters for the reverse temperature transition and coacervation behavior. This shows that the UnaG domain has a measurable but limited effect on the thermal properties of HELP. Although the HELP domain decreased the affinity of UnaG for bilirubin, HUG retained the property of displacing bilirubin from bovine serum albumin and thus remains one of the strongest bilirubin-binding proteins known to date. These data demonstrate that HELP can be used to create new bifunctional fusion products that pave the way for expanded technological applications.

Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores.

Here, we describe a stress-tolerant, recyclable, and renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on the Bacillus subtilis spore surface. TIED enables high loading density in the range of 106 to 107 recombinant enzymes per spore, robust catalytic activity of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Furthermore, we demonstrate TIED enzymes to be not only recyclable but also fully renewable after the loss of activity through induction of germination and sporulation, enabling perpetual regeneration of these immobilized biocatalysts.

Evaluation of a novel Echinococcus granulosus recombinant fusion B-EpC1 antigen for the diagnosis of human cystic echinococcosis using indirect ELISA in comparison with a commercial diagnostic ELISA kit

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus sensu lato (s.l.). A large proportion of the patients are asymptomatic at the early and late stages of the disease. CE diagnosis is mainly based on imaging techniques. Laboratory diagnosis including antibody-antigen (recombinant or fusion recombinant) can be used for the diagnosis and follow up of CE and alveolar echinococcosis (AE), but need optimization and standardization. This study aimed to evaluate the efficacy of a recombinant B-EpC1 (rB-EpC1) fusion antigen comprising B1, B2, B4, and EpC1 antigens of E. granulosus using indirect ELISA in comparison with a commercial ELISA kit for the serodiagnosis of CE. The recombinant protein was expressed in the expression host, E. coli BL21, and purified. This recombinant antigen was then evaluated by indirect ELISA and compared to the commercial CE diagnostic kit (Vircell, Spain). The study samples included 124 human sera consisting of 62 sera of patients with CE, and 62 sera of individuals without clinical evidences of CE and specific anti-CE antibodies in routine indirect ELISA. The diagnostic sensitivity and specificity of the indirect rB-EpC1-ELISA test for detection of specific anti-hydatid cyst antibodies in human CE were 95.2% and 96.8%, respectively. Also, the diagnostic sensitivity and specificity of the commercial ELISA test were 96.8% in this study. Initial evaluation of the recombinant fusion antigen (B-EpC1) was promising for the detection of CE by ELISA in clinical settings. Standardization and evaluation of recombinant fusion protein require further studies.

Managing surgery in hemophilia with recombinant factor VIII Fc and factor IX Fc: Data on safety and effectiveness from phase 3 pivotal studies

BackgroundSurgical procedures impose hemostatic risk to people with hemophilia, which may be minimized by optimal factor (F) replacement therapy. MethodsThis analysis evaluates the efficacy and safety of extended half‐life factor replacement recombinant FVIII and FIX Fc fusion proteins (rFVIIIFc and rFIXFc) during surgery in phase 3 pivotal (A‐LONG/Kids A‐LONG and B‐LONG/Kids B‐LONG) and extension (ASPIRE and B‐YOND) studies. Dosing regimens were determined by investigators. Injection frequency, dosing, blood loss, transfusions, and hemostatic response were assessed. ResultsForty‐five major (n = 31 subjects) and 90 minor (n = 70 subjects) procedures were performed in hemophilia A; 35 major (n = 22) and 62 minor (n = 37) procedures were performed in hemophilia B. Unilateral knee arthroplasty was the most common major orthopedic procedure (hemophilia A: n = 15/34; hemophilia B: n = 8/24). On the day of surgery, median total dose in adults/adolescents was 81 IU/kg for rFVIIIFc and 144 IU/kg for rFIXFc; most major procedures required ≤2 injections (including loading dose). Through days 1–14, most major procedures had ≤1 injection/day. Hemostasis was rated excellent (rFVIIIFc: n = 39/42; rFIXFc: n = 29/33) or good (n = 3/42; n = 4/33) in evaluable major surgeries, with blood loss comparable with subjects without hemophilia. Most minor procedures in adults/adolescents required one injection on the day of surgery, including median loading dose of 51 IU/kg (rFVIIIFc) and 80 IU/kg (rFIXFc). No major treatment‐related safety concerns were identified. No subjects developed inhibitors or serious vascular thromboembolic events. ConclusionsrFVIIIFc and rFIXFc were efficacious and well tolerated for the management of perioperative hemostasis across a wide spectrum of major and minor surgeries in hemophilia.

Phase I study of aflibercept in combination with docetaxel in Japanese patients with advanced solid malignancies

Angiogenesis is a hallmark of cancer development. This study sought to determine the recommended dose of aflibercept, a recombinant fusion protein targeting VEGF-A, VEGF-B and placental growth factor (PlGF), combined with docetaxel in Japanese patients with advanced solid malignancies. This phase I study was planned to include 12 patients following a 3 + 3 algorithm to determine the maximum tolerated dose of aflibercept combined with docetaxel in patients with metastatic or unresectable solid tumors (trial registration: NCT00545246). Docetaxel (75 mg/m2 every 3 weeks or 60 mg/m2 after protocol amendment) was combined with escalating doses of aflibercept (2, 4 and 6 mg/kg every 4 weeks). Free and VEGF-bound aflibercept were measured to assess free aflibercept in excess of the VEGF-bound form. At the starting dose of the combination, 3 of 6 patients treated experienced febrile neutropenia. After reducing the docetaxel dose to 60 mg/m2 in step 2 and permitting therapeutic granulocyte colony-stimulating factor (G-CSF) use, 2 of 3 patients in both cohorts experienced febrile neutropenia. Five patients (42%) had a partial response and 4 patients had stable disease (33%). Free aflibercept in excess of the VEGF-bound form was not maintained at this dose level. The dose limiting toxicity (DLT) of aflibercept combined with docetaxel was febrile neutropenia, which occurred in 2 of 3 Japanese patients at the lowest aflibercept dose level (2 mg/kg) combined with docetaxel (60 mg/m2) and therapeutic G-CSF use. A recommended dose for further studies was not determined because of the DLT at the starting dose.

Levan-type fructooligosaccharides synthesis by novel levansucrase-inulosucrase fusion enzyme

A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.

Abstract 6180: First-in-human (FIH) phase I studies of SCB-313, a novel TNF-related apoptosis-inducing ligand TRAIL-Trimer™ fusion protein, for treatment of patients (pts) with malignant ascites (MA)

Abstract Background: SCB-313 is a recombinant human TRAIL-Trimer™ fusion protein engineered using a stabilized trimeric form of the TRAIL-protein. Binding of SCB-313 to the death receptors 4 and 5 leads to the activation of the extrinsic apoptosis pathway. Method: We performed a pooled analysis of two phase 1 studies conducted in Australia and China (NCT03443674 and NCT04051112, respectively). SCB-313 was given by intraperitoneal infusion 4 times at day D1, D4, D8, D11 in Australia study and D1, D8, D9, D10 in China study. Five dose cohorts: 10 mg, 20 mg, 40 mg, 80 mg, and 160 mg were evaluated using a sequential dose escalation design (accelerated titration dosing combined with 3+3 design) in pts with MA. The primary objective for both studies was safety and tolerability. Secondary objectives included PK/PD and efficacy. Results: 7 pts were enrolled in Australia (by data cutoff Mar 5th, 2021) and 12 pts in China (by data cutoff May 21st, 2021). 16 pts completed DLT observation period. Median age 57.6y, ECOG 1-2, 36.8% male. Colon cancer, n=5; ovarian cancer, n=4; breast cancer, n=3; gastric cancer, n=2; other cancers, n=5. Treatment-emergent adverse events (TEAE) were reported in 19 pts (100.0%). Most AEs were mild to moderate in severity (Grade 1 or 2). There were 9 (47.4%) pts who experienced Grade 3 AEs and no Grade 4 or 5 AEs were reported. No SCB-313 related SAEs or AEs leading to study discontinuations or death were reported. No DLT occurred. The most common TEAE included abdominal pain, pyrexia, abdominal distension. In Australia study, median (min, max) ascites flow rate of pts with MA (n=7) was 800 (400, 1091) ml/24h at baseline and 163.8 (-767, 412), 137.7 (-796.6, 392.6), -91.5 (-844.8, 517.5) and 120 (-47.92, 1105) ml/24h post first, second, third and the fourth SCB-313 administration respectively. In China study, the median flow rate at baseline (n=11) and post each subsequent SCB-313 dosing were respectively 723.1 (108.2, 1175), 12.1 (-335.1, 965.9), 84.2 (-442.5, 882), -13.9 (-404.4, 431.8) and -57.85 (-446.4, 2028) ml/24h. Estimated half-life of SCB-313 in peritoneal fluid after 1st injection is 3-4 hours (min, max: 1.85, 5.05). Cmax and AUC showed a nonlinear dose-dependent increase and near complete elimination from the peritoneal fluid by 24 hrs. In Australia study, apoptosis specific serum CK-18 concentrations increased following the first dose and remained above baseline for 5/7 pts. Conclusion: Although the MTD was not defined by these studies, SCB-313 therapy presents an acceptable safety profile at all tested dose levels. Measurable ascites flow rate decrease were observed at all dose- levels. These data support further development of SCB-313 for pts with MA. Citation Format: Ye Guo, Aflah Roohullah, Junli Xue, Wei Zhao, Morteza Aghmesheh, David Martin, Yu Zhou, Chao Gao, Yixuan Yang, Derek-Zhen Xu, Jin Li. First-in-human (FIH) phase I studies of SCB-313, a novel TNF-related apoptosis-inducing ligand TRAIL-Trimer™ fusion protein, for treatment of patients (pts) with malignant ascites (MA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6180.

Abstract 4223: Cis-activation of PD-1+ effector T cells with dual-targeting immunocytokines generated using a novel chemical conjugation platform

Abstract Immunocytokines (IC) provide the opportunity for targeted delivery of cytokines as payloads to tissues and cells to improve safety and efficacy of cytokine-based therapies. ICs have gained significant interest in recent years and several antibody-cytokine fusion proteins have entered clinical trials. Compared to recombinant fusion proteins, we have developed an entirely different approach to IC generation based on the site-specific, chemical conjugation of synthetic cytokines to antibodies. Bright Peak generates enhanced and conjugatable cytokines using a novel protein engineering platform based on solid-phase peptide synthesis and subsequent chemical ligation of protein segments. Our synthetic cytokines can then be readily chemically conjugated to specific lysine residues in the Fc region of an existing IgG1, IgG2 or IgG4 antibody without the need for prior antibody engineering. Chemical conjugation of cytokine payloads is rapid, enabling the flexible generation of ICs based on different antibodies and payloads within weeks. We applied our technology to more than 10 antibodies and found that neither antigen binding nor payload potency and selectivity are affected by chemical conjugation. Importantly, binding of the Fc domain of ICs to Fc gamma receptors or FcRn is not significantly affected. We are initially focusing on the development of PD-1-targeted ICs to achieve dual-targeting of PD-1+ effector T cells (cis-signaling). Using several anti-PD-1 antibodies and various synthetic IL-2 variants as payloads, we created ICs with different drug-antibody ratios and explored alternative conjugation sites within the Fc region. Resulting PD-1/IL-2 ICs are highly active showing significantly enhanced potency due to avidity resulting from binding of the cytokine to PD-1+ effector T cells in cis. Our PD-1/IL-2 ICs induce strong pharmacodynamic effects in vivo, and we are currently optimizing the pharmacological profiles of PD-1-targeted ICs for clinical application. In addition, we are actively exploring cis-signaling ICs targeting different surface receptors and immune cells. Citation Format: Jean-Philippe Carralot, Matilde Arévalo Ruiz, Robert Tam, Eric Armentani, Vijaya R. Pattabiraman, Bertolt Kreft. Cis-activation of PD-1+ effector T cells with dual-targeting immunocytokines generated using a novel chemical conjugation platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4223.

The effect of BCG vaccination and risk factors for latent tuberculosis infection among college freshmen in China

ObjectivesIn this study, we aimed to verify whether Bacillus Calmette–Guérin (BCG) can protect first-year college students against Mycobacterium tuberculosis (MTB) infection by the recombinant fusion protein ESAT6-CFP10 skin test (ECST) or the tuberculin skin test (TST). MethodsWe conducted a cross-sectional study to assess risk factors for latent tuberculosis infection (LTBI). Vaccine effectiveness of BCG against LTBI, measured by ECST and TST separately, was assessed using multivariable logistic regression. ResultsA total of 7351 college freshmen accepted ECST, whereas 7228 accepted TST. A total of 263 (3.58%) tested positive with ECST and 581 (8.04%) tested positive with TST. BCG was significantly associated with LTBI (ECST: adjusted odds ratio (aOR) = 0.26; 95% CI 0.09 to 0.73; TST: aOR = 0.25; 95% CI 0.13 to 0.49). The BCG protective effect on freshmen living in rural areas (ECST: aOR = 0.16; 95% CI 0.04 to 0.55; TST: aOR = 0.12; 95% CI 0.04 to 0.33) is better than that of freshmen living in cities (ECST: aOR = 0.53; 95% CI 0.07 to 4.03; TST: aOR = 0.44; 95% CI 0.17 to 1.16). ConclusionsProtection against LTBI was strongly associated with BCG vaccination. A novel skin test (ECST) may underestimate the protective effects of BCG in college freshmen. BCG has better protection in areas with a slightly higher incidence of LTBI.

P75NTR Ectodomain Ameliorates Cognitive Deficits and Pathologies in a Rapid Eye Movement Sleep Deprivation Mice Model

The neurotrophin receptor p75 (p75NTR) is a circadian rhythm regulator and mediates cognitive deficits induced by sleep deprivation (SD). The soluble extracellular domain of p75NTR (p75ECD) has been shown to exert a neuroprotective function in Alzheimer’s disease (AD) and depression animal models. Nevertheless, the role of p75ECD in SD-induced cognitive dysfunction is unclear. In the present study we administrated p75ECD-Fc (10, 3 mg/kg), a recombinant fusion protein of human p75ECD and fragment C of immunoglobulin IgG1, to treat mice via intraperitoneal injection. The results revealed that peripheral supplementation of high-dose p75ECD-Fc (10 mg/kg) recovered the balance between Aβ and p75ECD in the hippocampus and rescued the cognitive deficits in SD mice. We also found that p75ECD-Fc ameliorated other pathologies induced by SD, including neuronal apoptosis, synaptic plasticity impairment and neuroinflammation. The current study suggests that p75ECD-Fc is a potential candidate for SD and peripheral supplementation of p75ECD-Fc may be a prospective preventive measure for cognitive decline in SD.

Extensive involvement of CD40 and CD154 costimulators in multiple T cell-mediated responses in a perciform fish Larimichthys crocea

CD40 and CD154 are well-characterized costimulatory molecules involved in adaptive humoral immunity in humans and other mammals. These two costimulatory molecules were found to be originated from teleost fish during vertebrate evolution. However, the functionality of fish CD40 and CD154 remains to be explored. In this study, we identified the CD40 and CD154 homologs (LcCD40 and LcCD154) from large yellow croaker (Larimichthys crocea), a marine species of the perciform fish family. The LcCD40 and LcCD154 share conserved structural features to their mammalian counterparts, and are widely expressed in immune-relevant tissues and leukocytes at different transcriptional levels. Immunofluorescence staining and FCM analysis showed that LcCD40 and LcCD154 proteins are distributed on MHC-II+ APCs and CD4-2+ T cells, and are significantly upregulated in response to antigen stimulation. Co-IP assay exhibited strong association between LcCD40 and LcCD154 proteins. Blockade of LcCD154 with anti-LcCD154 antibody (Ab) or recombinant soluble LcCD40-Ig fusion protein remarkably decreased the MHC-II+ APC-initiated CD4+ T cell response upon Aeromonas hydrophila stimulation, and alloreactive T cell activation as examined by mixed lymphocyte reaction (MLR). These findings highlight the costimulatory role of LcCD40 and LcCD154 in T cell activities in Larimichthys crocea. Thus, the CD40 and CD154 costimulators may extensively participate in the regulation of multiple T cell-mediated immune responses in teleost fish. It is anticipated that this study would provide a cross-species understanding of the evolutionary history of CD40 and CD154 costimulatory signals from fish to mammals.

A phase 1b/2 study of batiraxcept (AVB-S6-500) in combination with cabozantinib in patients with advanced or metastatic clear cell renal cell (ccRCC) carcinoma who have received front-line treatment (NCT04300140).

4511 Background: AXL is up-regulated by hypoxia-inducible factor-1 signaling in both VHL-deficient and hypoxic tumor cells and plays a critical role in the metastatic phenotype of ccRCC. Batiraxcept is a recombinant fusion protein containing an extracellular region of human AXL combined with the human immunoglobulin G1 heavy chain (Fc), demonstrating highly potent, specific AXL inhibition. Methods: Batiraxcept at doses of 15 and 20 mg/kg, plus cabozantinib 60 mg daily, was evaluated using a 3+3 dose escalation study design. The primary objective was safety; secondary and exploratory objectives included identification of the recommended phase 2 dose (RP2D), overall response rate (ORR), and duration of response (DOR). Correlation of serum soluble AXL (sAXL)/GAS6 with ORR was evaluated. Key eligibility criteria include previously treated (2L+) ccRCC patients; prior treatment with cabozantanib was not allowed. sAXL/GAS6 was evaluated at baseline. Results: Data as of 4-February-2022, Phase 1b enrolled 26 patients, 16 patients treated with 15 mg/kg and 10 patients with 20 mg/kg dose of batiraxcept. Baseline characteristics: median age 60 (40-81); male 22 (85%); median prior line of therapy 1 (1-5); IMDC risk group of favorable 6 (23%); prior VEGF inhibitor 15 (58%); 100% with prior immunotherapy. At median follow up of 4.9 months, 92% (n=24) patients remained on the study. No dose limiting toxicities were observed at either 15 mg/kg or 20 mg/kg dose. Batiraxcept and cabozantinib related adverse events (AEs) occurred in 17 subjects (65%). Most common related AE include decreased appetite 31% (n=8), diarrhea and fatigue 23% (n=6). Grade 3 related AEs occurred in 4 patients (15%) including diarrhea, thromboembolism, hypertension, small bowel obstruction, and thrombocytopenia (n=1, 4% each) being most common. No grade 4 or 5 related AEs were observed. The ORR was 46% (n=12, partial response [PR]; Table). No patients had primary progressive disease. Among the patients who had baseline sAXL/GAS6 ratio of ≥ 2.3, the ORR was 67% (12/18). Regardless of baseline sAXL/GAS6 ratio, 3-month DOR was 100%; and 6-month progression free survival was 79%. Batiraxcept PK levels were similar across both doses and GAS6 levels suppressed through the dosing period. Conclusions: Batiraxcept plus cabozantinib is well tolerated. The RP2D of batiraxcept was identified as 15 mg/kg. Early efficacy signals were observed including 100% DOR at 3 months. Baseline sAXL/GAS6 may serve as a potential biomarker to enrich the population. Clinical trial information: NCT04300140. [Table: see text]

TTI-621-03: A phase I/II study of TTI-621 in combination with doxorubicin in patients with unresectable or metastatic high-grade leiomyosarcoma (LMS).

TPS11593 Background: Doxorubicin is a standard of care agent for patients with advanced soft tissue sarcoma, with a response rate of around 15%, progression-free survival of 5-7 months and cumulative cardiac toxicity that limits its use. TTI-621 is a recombinant soluble fusion protein that combines the N-terminal portion of human SIRPα (the binding domain for CD47) with the Fc region of human IgG1, generating a decoy receptor for CD47 on the surface of tumor cells that both over-rides CD47-mediated inhibition of phagocytosis and provides a pro-phagocytic stimulation. Many solid tumors express high levels of CD47 which is associated with poor prognosis, thought to be the result of CD47-mediated inhibition of macrophage phagocytosis and escape of immune-mediated clearance. Interruption of the CD47-SIRPα signaling pathway using monoclonal antibodies to CD47 has shown anti-tumor activity in animal models and in some early clinical trials. The combination of doxorubicin with CD47-targeted antibodies results in enhanced anti-tumor activity and increased macrophage-mediated cell killing in animal models and macrophage-mediated phagocytosis of cancer cell lines in vitro, suggesting that combining TTI-621 with doxorubicin might be more effective than doxorubicin alone in tumor types that express CD47 and have high numbers of macrophages, such as LMS. Thus, a Phase 1/2 study was initiated to evaluate this combination in patients with advanced soft tissue sarcoma, including LMS. Methods: TTI-621-03 is a Phase 1/2, open-label study of TTI-621 in combination with doxorubicin in patients with anthracycline-naïve disease. The Phase 1 dose escalation evaluates doses of TTI-621 (0.2 to 2.0 mg/kg) in combination with doxorubicin at 75 mg/m2 in patients with high-grade soft tissue sarcomas. Expansion cohorts will evaluate TTI-621 (0.2 and 2.0 mg/kg) with doxorubicin in patients with LMS, with pathology confirmed at a central laboratory. The primary goals of this study are evaluation of safety of TTI-621 administered in combination with standard-of-care doxorubicin and to further evaluate clinical activity (ORR, PFS, OS), safety, PK and patient-reported quality of life in the LMS subpopulation. The dose escalation portion of the study has been completed without DLT. Enrollment to the expansion portion of the study is underway. Clinical trial information: NCT04996004.

A phase 1b/2 study of batiraxcept (AVB-S6-500) in combination with cabozantinib, cabozantinib and nivolumab, and as monotherapy in patients with advanced or metastatic clear cell renal cell carcinoma (NCT04300140).

TPS4599 Background: In clear cell renal cell carcinoma (ccRCC) the constitutive expression of hypoxia induced factor 1-α leads to increased expression of AXL. AXL overexpression has been associated with the development of resistance to VEGF inhibitors and suppression of the innate immune response through inhibition of macrophage-driven inflammation. Batiraxcept is a recombinant fusion protein dimer containing an extracellular region of human AXL combined with the human immunoglobulin G1 heavy chain (Fc), which demonstrates highly potent, specific AXL inhibition. In preclinical studies using the 786-O, M62, and SN12L1 tumors, batiraxcept monotherapy resulted in a significant reduction of tumor growth compared to control. In healthy volunteer and ovarian cancer clinical studies, batiraxcept was well tolerated with no dose-related adverse events, and a maximum tolerated dose was not reached. Therefore, batiraxcept could be tested as either a monotherapy or in combination with standard of care drugs in patients with metastatic ccRCC. The Phase 1b dose-escalation portion of this study evaluated batiraxcept in combination with standard of care cabozantanib in patients who progressed on or after first line therapy. No DLT was observed at either of two batiraxcept doses evaluated. The recommended Phase 2 dose of batiraxcept has been identified as 15 mg/kg every 2 weeks (q2w) with cabozantinib 60 mg based upon safety, PK/PD, and preliminary efficacy data. Methods: This Phase 2, multi-center, open-label study includes three parts: Part A) batiraxcept 15 mg/kg q2w in combination with cabozantinib 60 mg daily for ccRCC subjects who have progressed on or after one line of therapy, n=25. Part B) batiraxcept 15 mg/kg q2w with cabozantinib 40 mg daily and nivolumab at the investigator’s choice (240 mg q2w or 480 mg q4w) for first line treatment of advanced or metastatic ccRCC subjects, n=20. If no safety signals are observed in the first 6 subjects enrolled, 10 subjects will be enrolled in the first stage of a Simon 2-stage minmax statistical design. If ≥ 6/10 subjects achieve PR or CR, stage 2 will open to enroll up to 20 total subjects. Part C) batiraxcept 15 mg/kg q2w monotherapy for subjects with advanced/metastatic ccRCC ineligible for curative intent therapies, n=10. The primary objective for each arm is objective response rate by RECIST v1.1. Secondary objectives include safety, duration of response, clinical benefit rate, progression free survival by RECIST v1.1, and overall survival. Exploratory objectives include pharmacokinetic and pharmacodynamic assessments. The Phase 2 portion of this Ph1b/2 study is currently enrolling. Clinical trial information: NCT04300140.

Pharmacokinetic and pharmacodynamic studies of supaglutide in rats and monkeys

We demonstrated recently that supaglutide, a novel GLP-1 mimetic generated by recombinant fusion protein techniques, exerted hypoglycemic effects in type 2 diabetes db/db mice and spontaneous diabetic monkeys. In this study, we investigated the pharmacokinetics and pharmacodynamics of supaglutide by single subcutaneous and intravenous injection(s) in rats and rhesus monkeys, as well as fourconsecutive subcutaneous injections in monkeys.We found the half-life (t1/2) of supaglutide was 39.7 h and 35.8 h at dosing 0.1 mg/kg upon subcutaneous or intravenous administration respectively, in rhesus monkeys. The plasma supaglutide peaked at 8–10 h, while the plasma drug exposure levels increased with the increase of dose, showing approximately a linear pharmacokinetic characteristic. The elimination kinetics (Ke) were found to be similar between subcutaneous (∼0.025 in rats and ∼0.018 in monkeys) and intravenous administration (0.021 in rats and 0.020 in monkeys), whereas the bioavailability was found to be 31.1% in rats and 63.9% in monkeys. In monkeys, a single dose injection of supaglutide markedly decreased the random blood glucose levels that reaching the maxima effects in 14–16 h, gradually recovered and returned to the baseline level approximately after 72 h. 125I-supaglutide was found mainly distributed in the serum and organs rich in blood supply. Urine was found to be the primary excretion route of supaglutide, following by feces, but mostly not in bile.Our results show that supaglutide possess linear pharmacokinetic characteristics associated with prolonged hypoglycemic effects inanimals,suggestinga potential weekly dosing therapeutic reagent for the treatment of type 2 diabetes and metabolic diseases.

CATANA: an online modelling environment for proteins and nucleic acid nanostructures.

In the last decade, significant advances have been made towards the rational design of proteins, DNA, and other organic nanostructures. The emerging possibility to precisely engineer molecular structures resulted in a wide range of new applications in fields such as biotechnology or medicine. The complexity and size of the artificial molecular systems as well as the number of interactions are greatly increasing and are manifesting the need for computational design support. In addition, a new generation of AI-based structure prediction tools provides researchers with completely new possibilities to generate recombinant proteins and functionalized DNA nanostructures. In this study, we present Catana, a web-based modelling environment suited for proteins and DNA nanostructures. User-friendly features were developed to create and modify recombinant fusion proteins, predict protein structures based on the amino acid sequence, and manipulate DNA origami structures. Moreover, Catana was jointly developed with the novel Unified Nanotechnology Format (UNF). Therefore, it employs a state-of-the-art coarse-grained data model, that is compatible with other established and upcoming applications. A particular focus was put on an effortless data export to allow even inexperienced users to perform in silico evaluations of their designs by means of molecular dynamics simulations. Catana is freely available at http://catana.ait.ac.at/.

Production and optimization of a vasostatin-30 and vasoinhibin fusion protein that inhibits tumor angiogenesis and dissemination of breast cancer cells in a zebrafish model

Angiogenesis plays a key role in tumor growth and metastasis. Current antiangiogenic drugs have shown poor clinical benefit making the development of new antiangiogenic agents a clinical need. In this work, we optimized the production of a recombinant fusion protein derived from vasostatin-30 (VS30) and vasoinhibin (Vi), named VS_VI, using Escherichia coli and the central composite experimental design. The bioactivity of soluble VS_VI was compared to VS30, Vi, and angiostatin using in vitro cell cultures and the zebrafish model. Results showed that 21.3 and 17.5 mg/L of total VS_VI and soluble VS_VI, respectively, were attained at pH 7.0 and 28.5 °C. In vitro studies showed that purified soluble VS_VI decreased by 2-fold the VEGF-induced HUVEC proliferation compared to VS30 and Vi, showing no effect on 3T3 NIH cell proliferation at 10 nM. In vivo using zebrafish, VS_VI significantly reduced intratumoral angiogenesis at 1 mg/kg compared to a control, VS30, Vi, and angiostatin. VS_VI decreased metastatic dissemination of MDA-MB-468 and MDA-MB-231 cells at 1 mg/kg. Meanwhile, vasoinhibin and angiostatin at 1 mg/kg and vasostatin-30 at 10 mg/kg decreased the dissemination of T47D cells. Results showed VS_VI fusion protein impairs tumor angiogenesis and early dissemination of metastatic breast cancer cells, making it a promising agent in breast cancer treatment.