Nanobody-Functionalized Cellulose for Capturing SARS-CoV-2

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 253 million people, claiming ∼ 5.1 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, there is a pressing need for cost-effective systems to mitigate the viral spread. Here, we present a generic strategy for capturing SARS-CoV-2 through functionalized cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose to capture viruses from complex fluids in a continuous fashion. By capturing and containing viruses through the Nb-functionalized cellulose, our work may find utilities in virus sampling and filtration towards paper-based diagnostics, environmental tracking of viral spread and reducing viral load from infected individuals. IMPORTANCE The ongoing efforts to address the COVID-19 pandemic center around the development of diagnostics, preventative measures, and therapeutic strategies. In comparison to existing work, we have provided a complementary strategy to capture SARS-CoV-2 by functionalized cellulose materials towards paper-based diagnostics as well as virus filtration in perishable samples. Specifically, we developed a bifunctional fusion protein consisting of both a cellulose-binding domain and a nanobody specific for the receptor-binding domain of SARS-CoV-2. As a proof-of-concept, the fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both wildtype and the D614G mutant variants, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography for binding viruses from complex biological fluids in a highly continuous and cost-effective manner. Such antigen-specific capture can potentially immobilize viruses of interest for viral detection and removal, which contrasts with the common size- or affinity-based filtration devices that bind a broad range of bacteria, viruses, fungi, and cytokines present in blood ( https://clinicaltrials.gov/ct2/show/NCT04413955 ). Additionally, since our work focuses on capturing and concentrating viruses from surfaces and fluids as a means to improve detection, it can serve as an “add-on” technology to complement existing viral detection methods, many of which have been largely focusing on improving the intrinsic sensitivities.

A Novel Bifunctional Fusion Protein, Vunakizumab-IL22, for Protection Against Pulmonary Immune Injury Caused by Influenza Virus

Influenza A virus infection is usually associated with acute lung injury, which is typically characterized by tracheal mucosal barrier damage and an interleukin 17A (IL-17A)-mediated inflammatory response in lung tissues. Although targeting IL-17A has been proven to be beneficial for attenuating inflammation around lung cells, it still has a limited effect on pulmonary tissue recovery after influenza A virus infection. In this research, interleukin 22 (IL-22), a cytokine involved in the repair of the pulmonary mucosal barrier, was fused to the C-terminus of the anti-IL-17A antibody vunakizumab to endow the antibody with a tissue recovery function. The vunakizumab-IL22 (vmab-IL-22) fusion protein exhibits favorable stability and retains the biological activities of both the anti-IL-17A antibody and IL-22 in vitro. Mice infected with lethal H1N1 influenza A virus and treated with vmab-mIL22 showed attenuation of lung index scores and edema when compared to those of mice treated with saline or vmab or mIL22 alone. Our results also illustrate that vmab-mIL22 triggers the upregulation of MUC2 and ZO1, as well as the modulation of cytokines such as IL-1β, HMGB1 and IL-10, indicating the recovery of pulmonary goblet cells and the suppression of excessive inflammation in mice after influenza A virus infection. Moreover, transcriptome profiling analysis suggest the downregulation of fibrosis-related genes and signaling pathways, including genes related to focal adhesion, the inflammatory response pathway, the TGF-β signaling pathway and lung fibrosis upon vmab-mIL22 treatment, which indicates that the probable mechanism of vmab-mIL22 in ameliorating H1N1 influenza A-induced lung injury. Our results reveal that the bifunctional fusion protein vmab-mIL22 can trigger potent therapeutic effects in H1N1-infected mice by enhancing lung tissue recovery and inhibiting pulmonary inflammation, which highlights a potential approach for treating influenza A virus infection by targeting IL-17A and IL-22 simultaneously.

Long-term follow-up of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in advanced squamous cell carcinoma of the head and neck (SCCHN).

6020 Background: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. A previous report of an expansion cohort from a phase 1 study (NCT02517398) suggested that bintrafusp alfa had a manageable safety profile and early signs of clinical activity in patients with heavily pretreated, advanced SCCHN after a median follow-up of 86.4 weeks. Here we report long-term efficacy and safety for this cohort. Methods: Patients with advanced SCCHN that progressed/recurred after platinum therapy in the recurrent/metastatic setting, or < 6 months after platinum therapy in the locally advanced setting, received bintrafusp alfa 1200 mg every 2 weeks until confirmed progressive disease, unacceptable toxicity, or trial withdrawal. The primary endpoint was confirmed best overall response assessed per RECIST 1.1 assessed by independent review committee (IRC); safety was a secondary endpoint. Results: As of May 15, 2020, 32 patients had received bintrafusp alfa for a median of 2.8 months (range, 0.5-29.9 months), no patient remained on treatment, and median follow-up to data cutoff was 41.7 months (range, 39.8-43.5 months). The objective response rate (ORR; 13%) was unchanged since the previous report; median duration of response (DOR) was increased at 21.4 months (95% CI, 5.5 months to not reached [NR]). While the clinical activity of bintrafusp alfa may be improved in patients with HPV-positive tumors (Table), outcomes were generally similar between PD-L1 subgroups (≥1% vs < 1% tumor cells). The overall safety profile was consistent with the previous report for this cohort, without grade 4 nor 5 treatment-related adverse events (TRAEs); no new TRAEs of grade 3 or that led to discontinuation of bintrafusp alfa were reported. Conclusions: With a median follow-up of over 3 years in patients with heavily pretreated advanced SCCHN, bintrafusp alfa showed sustained clinical activity and 3-year OS of 24.0%, which compares favorably to historical data. Clinical activity appeared to be greater in patients with HPV-positive tumors than those with HPV-negative tumors. The safety profile was manageable and consistent with earlier analysis. Further investigation of bintrafusp alfa in SCCHN and other HPV-associated cancers is ongoing. Clinical trial information: NCT02517398. [Table: see text]

SHR-1701, a bifunctional fusion protein targeting PD-L1 and TGF-β, for advanced NSCLC with EGFR mutations: Data from a multicenter phase 1 study.

9055 Background: Despite the development of targeted therapies for advanced NSCLC harboring EGFR mutations ( EGFR+), acquired resistance remains inevitable. Immune checkpoint inhibitor as monotherapy has limited efficacy. Blockade of the TGF-β pathway which plays a key role in immune suppression may enhance the tumor response to anti-PD-1/PD-L1 antibodies. Here, we assessed SHR-1701, a novel bifunctional fusion protein composed of a mAb against PD-L1 fused with the extracellular domain of TGF-β receptor II, in advanced NSCLC pts including one separate EGFR+ cohort. Methods: This phase 1 study includes a 3+3 dose-escalation and dose-expansion period of pretreated advanced NSCLC and multiple clinical expansion cohorts of different tumor types, genetic aberrations, or prior therapies. During the dose-escalation and dose-expansion period, pathologically confirmed pts received SHR-1701 at 3, 10, or 20 mg/kg Q3W or 20 mg/kg Q2W by intravenous infusion. The primary objectives were to determine the safety profile, maximum tolerated dose (MTD), and recommended phase 2 dose (RP2D) of SHR-1701. In the EGFR+ NSCLC clinical expansion cohort, histologically or cytologically confirmed advanced pts after at least 1L standard EGFR TKI received SHR-1701 at RP2D, and the primary endpoint was objective response rate (ORR). Treatment beyond progression was allowed. Results: During the dose-escalation and dose-expansion period, 30 pts were recruited: all stage IV; 83.3% had ≥2 metastasis sites; 76.7% had received ≥2L prior systemic therapy. One dose-limiting toxicity (immune-mediated pneumonitis) in the 20 mg/kg Q2W group was observed, and the MTD was not reached. Population pharmacokinetics and exposure‐response analysis of SHR-1701 based on this study and another phase 1 study for advanced solid tumors (NCT03710265) demonstrated 30 mg/kg Q3W as the RP2D. In the EGFR+ NSCLC cohort, 27 pts were enrolled: all stage IV; 77.8% had ≥2 metastasis sites; 70.4% had received ≥2L prior systemic therapy; 29.6% had 19-Del, 14.8% 19-Del and T790M, 7.4% 20-ins, 29.6% L858R, 18.5% L858R and T790M. With a median SHR-1701 exposure of 8.7 weeks (range, 3.0-24.0), 4 of the 24 pts who had at least one post-baseline radiographic assessment achieved objective responses, including 3 ongoing confirmed and 1 unconfirmed partial response. ORR was 16.7% (95% CI, 4.7%-37.4%), and disease control rate was 50.0% (95% CI, 29.1%-70.9%). Grade 3 treatment-related adverse events (TRAEs) occurred in 2 (7.4%) pts, including anemia, hypokalemia, and asthenia (1 [3.7%] for each). There were no grade 4 or 5 TRAEs. No pts discontinued treatment due to TRAEs. Conclusions: SHR-1701 monotherapy shows a manageable safety profile and encouraging antitumor activity in advanced EGFR+ NSCLC pts after failure of at least 1L standard EGFR TKI. Further investigation of SHR-1701 combination therapy for EGFR+ NSCLC is warranted. Clinical trial information: NCT03774979.