Dimerization Of Fusion Proteins Research Articles

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.

424 A phase 1b/2 randomized study of AVB-S6–500 in combination with cabozantinib versus cabozantinib alone in patients with advanced clear cell renal cell carcinoma who have received front-line treatment

BackgroundIn 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. AVB-S6-500 (AVB-500) is 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 mouse ccRCC xenograft models, AVB-500 showed significantly more tumor reduction in combination with pazopanib versus pazopanib alone. In a Ph1b study of AVB-500 plus chemotherapy in platinum-resistant ovarian cancer (NCT03639246), no dose limiting toxicity (DLT) or treatment discontinuation due to adverse events was observed. The recommended phase 2 dose (RP2D) of 15 mg/kg was established by a model-informed drug development (MIDD) approach.MethodsThe P1b portion of this trial is a 3+3 dose escalation study to evaluate safety, pharmacokinetics, and pharmacodynamics of AVB 500 in combination with cabozantinib 60 mg daily. Dose levels of AVB-500 may include 15, 20, and 25 mg/kg every two weeks. The primary objective is to evaluate safety and tolerability. Secondary objectives include identification of the RP2D of AVB-500 and clinical activity. Key eligibility criteria include clear cell histology RCC and at least one prior line of therapy administered in the advanced or metastatic setting.ResultsAs of July 21, 2021, seven patients have received at least one dose of AVB-500 15 mg/kg and cabozantinib, with six patients ongoing treatment. No DLTs were observed. Trough levels at C1D15 were above the minimally efficacious concentration (MEC) identified from MIDD and GAS6 (AXL ligand) levels were suppressed prior to C2D1. Partial responses were observed in 3 of 5 patients (table 1); all patients demonstrated tumor decrease from baseline.Abstract 424 Table 1Preliminary clinical activity in NCT04300140ConclusionsAVB-500 in combination with cabozantanib demonstrates promising preliminary clinical activity and tolerability in patients with ccRCC. AVB-500 15 mg/kg is the presumptive RP2D with C1D15 AVB-500 troughs consistently above MECs observed. Safety, PK/PD and clinical activity will be updated at the time of presentation. (NCT04300140)Ethics ApprovalThis study has obtained ethics approval from WIRB Institutional Review Board®, Protocol ID #20200159, and all subjects provided informed consent prior to taking part in this study.

Biological therapy for dermatological manifestations of inflammatory bowel disease

Ulcerative colitis and Crohn's disease are the two forms of inflammatory bowel disease (IBD). The advent of biological drugs has significantly changed the management of these conditions. Skin manifestations are not uncommon in IBD. Among the reactive lesions (immune-mediated extraintestinal manifestations), erythema nodosum (EN) and pyoderma gangrenosum (PG) are the two major cutaneous ills associated with IBD, while psoriasis is the dermatological comorbidity disease observed more often. In particular, in the last few years, anti-tumor necrosis factor (TNF)-α agents have been successfully used to treat psoriasis, especially these kinds of lesions that may occur during the treatment with biological therapies. The entity of the paradoxical manifestations has been relatively under reported as most lesions are limited and a causal relationship with the treatment is often poorly understood. The reason for this apparent side-effect of the therapy still remains unclear. Although side effects may occur, their clinical benefits are undoubted. This article reviews the therapeutic effects of the two most widely used anti-TNF-α molecules, infliximab (a fusion protein dimer of the human TNF-α receptor) and adalimumab (a fully human monoclonal antibody to TNF-α), for the treatment of the major cutaneous manifestations associated with IBD (EN, PG and psoriasis).

G domain dimerization controls dynamin's assembly-stimulated GTPase activity

Dynamin is an atypical GTPase that catalyzes membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin’s basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0Å resolution crystal structure of a minimal GTPase-GED fusion protein (GG) constructed from human dynamin 1, which has dimerized in the presence of the transition state mimic GDP.AlF4−. The structure reveals dynamin’s catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GG dimer provides new insight into the mechanisms underlying dynamin-catalyzed membrane fission.

Hepatitis B Virus Capsid-like Particles Can Display the Complete, Dimeric Outer Surface Protein C and Stimulate Production of Protective Antibody Responses against Borrelia burgdorferi Infection

Hepatitis B virus capsid-like particles (CLPs), icosahedral assemblies formed by 90 or 120 core protein dimers, hold promise as immune-enhancing vaccine carriers for heterologous antigens. Insertions into the immunodominant c/e1 B cell epitope, a surface-exposed loop, are especially immunogenic. However, display of whole proteins, desirable to induce multispecific and possibly neutralizing antibody responses, can be restrained by an unsuitable structure of the foreign protein and by its propensity to undergo homomeric interactions. Here we analyzed CLP formation by core fusions with two distinct variants of the dimeric outer surface lipoprotein C (OspC) of the Lyme disease agent Borrelia burgdorferi. Although the topology of the termini in the OspC dimer does not match that of the insertion sites in the carrier dimer, both fusions, coreOspCa and coreOspCb, efficiently formed stable CLPs. Electron cryomicroscopy clearly revealed the surface disposition of the OspC domains, possibly with OspC dimerization occurring across different core protein dimers. In mice, both CLP preparations induced high-titered antibody responses against the homologous OspC variant, but with substantial cross-reactivity against the other variant. Importantly, both conferred protection to mice challenged with B. burgdorferi. These data show the principal applicability of hepatitis B virus CLPs for the display of dimeric proteins, demonstrate the presence in OspC of hitherto uncharacterized epitopes, and suggest that OspC, despite its genetic variability, may be a valid vaccine candidate.

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145–170 K protein; this reactivity was lost when the antigen preparation was heated to 100°C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by > 50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.

Interprotein disulfide bonding between F and G glycoproteins of human respiratory syncytial virus

The envelope glycoprotein G, of human respiratory virus was purified by immunoaffinity chromatography using a monoclonal antibody reacting with G glycoprotein. The purified material was analyzed for its protein patterns and by western blot for its reactivity with specific monoclonal antibodies. In addition to the G specific proteins at 90 and 55 kilodalton (kDa) range, high molecular weight species were coeluted with G protein. Three high molecular weight species were noticed: one (140 kDa) reacting with fusion protein (F) monoclonal antibody and two other species (230 and 195 kDa) reacting with both fusion protein and G protein monoclonal antibodies. The protein reacting only with F monoclonal antibody consists of fusion protein dimer. Western blot and two dimensional gel electrophoretic analysis revealed that each of the other two complexes is composed of two moles of F protein and one mole of G protein. These two complexes differ in their molecular sizes depending on whether G is in the form of 90 or 55 kDa. Upon heat denaturation, fusion protein monomer (70 kDa) is released from the complex, leaving the two complexes, consisting of one mole of F protein and one mole of G protein (160 and 125 kDa species respectively). Disulfide-reducing agents are required to break the monomers of F and G complexes. These results provide a direct evidence for the presence of envelope glycoprotein complexes linked by interprotein disulfide bonding. This may have implications on the structural and functional properties of envelope glycoproteins.

Immunovirological studies on human respiratory syncytial virus structural proteins

Immunovirological studies suggest that human respiratory syncytial virus may well be composed of five structural proteins as are other members of the Paramyxoviridae family: the two external membrane glycoproteins H (90 000) and Fo (F1, 49 000; F2, 20 000; disulfide linked), the internal membrane protein M (34 000), the nucleoprotein N (42 000), and a protein (78 000) designated P that could be the equivalent of the polymerase of the morbillivirus and paramyxovirus genus. Neutralizing monoclonal antibodies showed, by immunoprecipitation and immunoblotting, that the fusion protein carries neutralizing epitopes. One monoclonal antibody, which shows a high neutralizing titer, immunoblotted directly with the F1 fragment (49 000) of the fusion protein. Analysis in mice of the immunogenicity of the structural proteins separated on sodium dodecyl sulphate gels indicated that, under our conditions, only the fusion protein dimer Fo and its F1 fragment were capable of inducing neutralizing antibodies.