Bifunctional Protein Research Articles

Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

Precise Modulation of Protein Degradation by Smart PROTACs.

Proteolysis-targeting chimera (PROTAC)hasemergedas an attractive therapeutic modalityin drug discovery.PROTACs are bifunctional moleculesthat effectivelybridge proteins of interest(POIs)withE3 ubiquitin ligases, such that,the target proteinsaretagged with ubiquitin and subsequently degraded viathe proteasome. Despite significant progressin the field of targeted protein degradation (TPD), the application of conventional PROTAC degradersstill faces significantchallenges,includingsystemic toxicity induced by non-tissue-specific targeting.To address this issue, a variety ofsmart PROTACs that can be activated byspecific stimuli, have been developed for achievingconditional andspatiotemporal modulationof protein levels.Here, on the basis of our contributions, we overview recent advances of smart PROTACs, including tumor microenvironment-, photo-, and X-ray radiation-responsivePROTACs, that enable controllableTPD. The design strategy, case studies, potential applications and challengeswill be focused on.

A dye-decolorizing peroxidase from Vibrio cholerae can demetallate heme

Iron is an essential element for bacterial survival. Bacterial pathogens have therefore developed methods to obtain iron. Vibrio cholerae, the intestinal pathogen that causes cholera, utilizes heme as an iron source. DyP from V. cholerae (VcDyP) is a dye-decolorizing peroxidase. When VcDyP was expressed in Escherichia coli and purified, it was found to contain protoporphyrin IX (PPIX) but not heme, indicating that the protein possesses deferrochelatase activity. Here, we examined the demetallation reaction of VcDyP using fluorescence spectroscopy. Treatment of heme-reconstituted VcDyP with sodium dithionite under anaerobic conditions led to an increase in the fluorescence intensity at 624 nm, suggesting the formation of PPIX. Although the same reaction was conducted using myoglobin, horseradish peroxidase and hemin, no increase in the fluorescence was observed. Therefore, demetallation of heme is specific to VcDyP. This reaction was faster at lower pH, but the amplitudes of the fluorescence increase were larger at pH 6.5–7.5, in clear contrast to the dye-decolorizing activity with the optimal pH of 4.5. In contrast to HutZ from V. cholerae, which is a heme-degrading enzyme that cleaves the heme macrocycle to release iron, VcDyP can remove iron from heme without degradation. To our knowledge, VcDyP is the first enzyme whose demetallation activity has been confirmed at neutral pH. Our results show that VcDyP is a bifunctional protein that degrades anthraquinone dyes and demetallates heme.

Efficacy and Safety of Bintrafusp Alfa, a Bifunctional Fusion Protein Targeting Transforming Growth Factor-β and Programmed Death-Ligand 1, Plus Chemotherapy in Patients With Stage IV NSCLC

IntroductionIn a phase 1 study, bintrafusp alfa was found to have an encouraging clinical activity in patients with previously treated advanced NSCLC. This study evaluated the safety and efficacy of bintrafusp alfa with chemotherapy in patients with stage IV NSCLC regardless of the programmed death-ligand 1 (PD-L1) expression status. MethodsIn this open-label, phase 1b/2 study (NCT03840915), eligible patients were assigned to one of four cohorts. Patients with previously untreated metastatic NSCLC (cohorts A, B, and C) received bintrafusp alfa with chemotherapy as first-line treatment, whereas patients whose disease progressed on previous treatment with programmed cell death protein 1/PD-L1 inhibitors (cohort D) received bintrafusp alfa with chemotherapy as second-line treatment. The primary objective of this study was to evaluate the safety and tolerability of bintrafusp alfa with chemotherapy. ResultsFour serious and one nonserious treatment-emergent adverse events were considered dose-limiting toxicities, none of which were assessed as related to bintrafusp alfa by the investigator. Any-grade bintrafusp alfa-related adverse events occurred in 20.7% of patients in cohorts A+B+C and in 16.7% of patients in cohort D. Keratoacanthoma was the most common transforming growth factor-β inhibition-mediated skin lesion (cohorts A+B+C: 12.1% and cohort D: 8.3%). In cohorts A+B+C, the overall response rate was 48.3%, and in patients with PD-L1 tumor proportion score of more than or equal to 50.0%, it was 71.4%. On the basis of an interim analysis, the data were considered mature, and no further analysis has been planned. ConclusionBintrafusp alfa with chemotherapy was found to have a manageable safety profile and encouraging clinical activity in patients with stage IV NSCLC.

Strategies for Non-Covalent Attachment of Antibodies to PEGylated Nanoparticles for Targeted Drug Delivery.

Polyethylene glycol (PEG)-modified nanoparticles (NPs) often struggle with reduced effectiveness against metastasis and liquid tumors due to limited tumor cell uptake and therapeutic efficacy. To address this, actively targeted liposomes with enhanced tumor selectivity and internalization are being developed to improve uptake and treatment outcomes. Using bi-functional proteins to functionalize PEGylated NPs and enhance targeted drug delivery through non-covalent attachment methods has emerged as a promising approach. Among these, the one-step and two-step targeting strategies stand out for their simplicity, efficiency, and versatility. The one-step strategy integrates streptavidin-tagged antibodies or bispecific antibodies (bsAbs: PEG/DIG × marker) directly into PEGylated NPs. This method uses the natural interactions between antibodies and PEG for stable, specific binding, allowing the modification of biotin/Fc-binding molecules like protein A, G, or anti-Fc peptide. Simply mixing bsAbs with PEGylated NPs improves tumor targeting and internalization. The two-step strategy involves first accumulating bsAbs (PEG/biotin × tumor marker) on the tumor cell surface, triggering an initial attack via antibody-dependent and complement-dependent cytotoxicity. These bsAbs then capture PEGylated NPs, initiating a second wave of internalization and cytotoxicity. Both strategies aim to enhance the targeting capabilities of PEGylated NPs by enabling specific recognition and binding to disease-specific markers or receptors. This review provides potential pathways for accelerating clinical translation in the development of targeted nanomedicine.

STAT3 and the STAT3‑regulated inhibitor of apoptosis protein survivin as potential therapeutic targets in colorectal cancer (Review).

Colorectal cancer (CRC) is one of the leading types of cancer worldwide. CRC development has been associated with the constitutive activation of signal transducer and activator of transcription 3 (STAT3). STAT3 is a master regulator of inflammation during cancer-associated colitis, and becomes upregulated in CRC. In CRC, STAT3 is activated by IL-6, among other pro-inflammatory cytokines, inducing the expression of target genes that stimulate proliferation, angiogenesis and the inhibition of apoptosis. One of the main STAT3-regulated inhibitors of apoptosis is survivin, which is a bifunctional protein that regulates apoptosis and participates in cell mitosis. Survivin expression is normally limited to foetal tissue; however, survivin is also upregulated in tumours. In silico and experimental analyses have shown that the STAT3 interactome is relevant during CRC progression, and the constitutive STAT3-survivin axis participates in development of the tumour microenvironment and response to therapy. The presence of a STAT3-survivin axis has been documented in CRC cohorts, and the expression of these molecules is associated with poor prognosis and a higher mortality rate in patients with CRC. Thus, STAT3, survivin, and the upstream activators IL-6 and IL-6 receptor, are considered therapeutic targets for CRC. Efforts to develop drugs targeting the STAT3-survivin axis include the evaluation of phytochemical compounds, small molecules and monoclonal antibodies. In the present review, the expression, function and participation of the STAT3-survivin axis in the progression of CRC were investigated. In addition, an update on the pre-clinical and clinical trials evaluating potential treatments targeting the STAT3-survivin axis is presented.

Simultaneously blocking ANGPTL3 and IL-1β for the treatment of atherosclerosis through lipid-lowering and anti-inflammation.

Blood lipid levels play a critical role in the progression of atherosclerosis. However, even with adequate lipid reduction, significant residual cardiovascular risk remains. Therefore, it is necessary to seek novel therapeutic strategies for atherosclerosis that can not only lower lipid levels but also inhibit inflammation simultaneously. The fusion protein FD03-IL-1Ra was designed by linking the Angiopoietin-like 3 (ANGPTL3) nanobody and human interleukin-1 receptor antagonist (IL-1Ra) sequences to a mutated human immunoglobulin gamma 1 (IgG1) Fc. This construct was transfected into HEK293 cells for expression. The purity and thermal stability of the fusion protein were assessed using SDS-PAGE, SEC-HPLC, and differential scanning calorimetry. Binding affinities of the fusion protein to ANGPTL3 and IL-1 receptor were measured using Biacore T200. The biological activity of the fusion protein was validated through in vitro experiments. The therapeutic efficacy of the fusion protein was evaluated in an ApoE-/- mouse model of atherosclerosis, including serum lipid level determination, histological analysis of aorta and aortic sinus sections, and detection of inflammatory and oxidative stress markers. ImageJ software was utilized for quantitative image analysis. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. The FD03-IL-1Ra fusion protein was successfully expressed, with no polymer formation detected, and it demonstrated good thermal and conformational stability. High affinity for both murine and human ANGPTL3 was exhibited by FD03-IL-1Ra, and it was able to antagonize hANGPTL3's inhibition of LPL activity. FD03-IL-1Ra also showed high affinity for both murine and human IL-1R, inhibiting IL-6 expression in A549 cells induced by IL-1β stimulation, as well as suppressing IL-1β-induced activity inhibition in A375.S2 cells. Our study revealed that the fusion protein effectively lowered serum lipid levels and alleviated inflammatory responses in mice. Furthermore, the fusion protein enhanced plaque stability by increasing collagen content within atherosclerotic plaques. These findings highlighted the potential of bifunctional interleukin-1 receptor antagonist and ANGPTL3 antibody fusion proteins for ameliorating the progression of atherosclerosis, presenting a promising novel therapeutic approach targeting both inflammation and lipid levels.

Targeting IL-33 reprograms the tumor microenvironment and potentiates antitumor response to anti-PD-L1 immunotherapy

BackgroundThe main challenge against patients with cancer to derive benefits from immune checkpoint inhibitors targeting PD-1/PD-L1 appears to be the immunosuppressive tumor microenvironment (TME), in which IL-33/ST2 signal fulfills critical...

Structural insights into BirA from Haemophilus influenzae, a bifunctional protein as a biotin protein ligase and a transcriptional repressor

Biotin is an essential coenzyme involved in various metabolic processes across all known organisms, with biotinylation being crucial for the activity of carboxylases. BirA from Haemophilus influenzae is a bifunctional protein that acts as a biotin protein ligase and a transcriptional repressor. This study reveals the crystal structures of Hin BirA in both its apo- and holo-(biotinyl-5′-AMP bound) forms. As a class II BirA, it consists of three domains: N-terminal DNA binding domain, central catalytic domain, and C-terminal SH3-like domain. The structural analysis shows that the biotin-binding loop forms an ordered structure upon biotinyl-5′-AMP binding. This facilitates its interaction with the ligand and promotes protein dimerization. Comparative studies with other BirA homologs from different organisms indicate that the residues responsible for binding biotinyl-5′-AMP are highly conserved. This study also utilized AlphaFold2 to model the potential heterodimeric interaction between Hin BirA and biotin carboxyl carrier protein, thereby providing insights into the structural basis for biotinylation. These findings enhance our understanding of the structural and functional characteristics of Hin BirA, highlighting its potential as a target for novel antibiotics that disrupt the bacterial biotin synthesis pathways.

Efficacy and safety of bintrafusp alfa in 2 phase I expansion cohorts with advanced HCC.

Simultaneous inhibition of the TGF-β and programmed cell death 1 ligand 1 pathways provides a potential novel treatment approach. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-βRII (a TGF-β "trap") fused to a human IgG1 monoclonal antibody blocking programmed cell death 1 ligand 1, was evaluated in patients with advanced HCC. In this global, open-label, phase I study (NCT02517398), patients with programmed cell death 1 ligand 1-unselected HCC who failed or were intolerant to ≥1 line of sorafenib received bintrafusp alfa 1200mg every 2 weeks in a dose-escalation (n = 38) or dose-expansion (n = 68) cohort until confirmed progression, unacceptable toxicity, or trial withdrawal. The primary endpoint was the best overall response per Response Evaluation Criteria in Solid Tumors version 1.1 by an independent review committee. Secondary endpoints included investigator-assessed best overall response, safety, and pharmacokinetics. Median follow-up times (range) were 41.4 (39.8-44.2) and 38.6 (33.5-39.7) months in the dose-escalation and dose-expansion cohorts, respectively. The objective response rate was below the prespecified 20% objective response rate threshold set to evaluate the efficacy of bintrafusp alfa in both cohorts (10.5% and 8.8%, respectively). Median overall survival and progression-free survival, respectively, were 13.8 and 1.5 months in the dose-escalation cohort and 13.5 and 1.4 months in the dose-expansion cohort. Treatment-related adverse events occurred in 78.9% and 64.7% of patients in the respective cohorts (grade ≥3 in 18.4% and 25.0% of patients). Bintrafusp alfa showed moderate clinical activity and a safety profile consistent with previous studies of bintrafusp alfa in patients with advanced HCC.

In silico-driven identification and experimental confirmation of antifungal proteins (AFPs) against Candidaalbicans

Mycoses infect millions of people annually across the world. The most common mycosis agent, Candida albicans is responsible for a great deal of illness and death. C. albicans infection is becoming more widespread and the current antifungals polyenes, triazoles, and echinocandins are less efficient against it. Investigating antifungal peptides (AFPs) as therapeutic is gaining momentum. Therefore, we used MALDI-TOF/MS analysis to identify AFPs and protein-protein docking to analyze their interactions with the C. albicans target protein. Some microorganisms with strong antifungal action against C. albicans were selected for the isolation of AFPs. Using MALDI-TOF/MS, we identified 3 AFPs Chitin binding protein (ACW83017.1; Bacillus licheniformis), the bifunctional protein GlmU (BBQ13478.1; Stenotrophomonas maltophilia), and zinc metalloproteinase aureolysin (BBA25172.1; Staphylococcus aureus). These AFPs showed robust interactions with C. albicans target protein Sap5. We deciphered some important residues in identified APFs and highlighted interaction with Sap5 through hydrogen bonds, protein-protein interactions, and salt bridges using protein-protein docking and MD simulations. The three discovered AFPs-Sap5 complexes exhibit different levels of stability, as seen by the RMSD analysis and interaction patterns. Among protein-protein interactions, the remarkable stability of the BBQ25172.1-2QZX complex highlights the role of salt bridges and hydrogen bonds. Identified AFPs could be further studied for developing successful antifungal candidates and peptide-based new antifungal therapeutic strategies as fresh insights into addressing antifungal resistance also.

Bintrafusp Alfa for Recurrent or Metastatic Cervical Cancer After Platinum Failure

Cervical cancer is a common and lethal cancer worldwide. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (or transforming growth factor β trap) fused via a flexible linker to the C-terminus of each heavy chain of an immunoglobulin G1 antibody blocking programmed cell death 1 ligand 1. To evaluate the safety and response rates of bintrafusp alfa in patients with recurrent or metastatic cervical cancer. This phase 2 nonrandomized controlled trial evaluated bintrafusp alfa monotherapy in patients with recurrent or metastatic cervical cancer with disease progression during or after platinum-based chemotherapy. Data were collected from March 2020 to February 2022. Patients received bintrafusp alfa, 1200 mg, intravenously once every 2 weeks. The primary end point was confirmed objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 by an independent review committee. At data cutoff, 146 of 203 screened patients received 1 or more doses of bintrafusp alfa; of these, the median (range) age was 53 (24-79) years. The study met its primary end point of a 95% CI above the objective response rate benchmark of 15%, with a confirmed objective response rate of 21.9% (95% CI, 15.5-29.5) per the independent review committee. Of these patients, 19 (59.4%) had a durable response of 6 months or more. At data cutoff, responses were ongoing in 13 of 32 responders (40.6%). The most common treatment-related adverse events were anemia (25 [17.1%]), rash (21 [14.4%]), hypothyroidism (15 [10.3%]), and pruritus (15 [10.3%]). Any-cause adverse events of special interest included anemia (82[56.2%]), bleeding events (81 [55.5%]), and immune-related adverse events (49 [33.6%]). This phase 2 nonrandomized controlled trial of bintrafusp alfa met its primary end point, which may support the potential of a bispecific therapy targeting transforming growth factor β and programmed cell death 1 ligand 1 in patients with recurrent or metastatic cervical cancer. ClinicalTrials.gov Identifier: NCT04246489.

A bifunctional protein RANbody based on nanobody facilitates dual-mode immunoassay of Staphylococcal enterotoxin B in food samples

Staphylococcal enterotoxin B (SEB), the major virulence factor of Staphylococcus aureus (S. aureus), brings about grievous food poisoning by infiltrating the food supply chain at diverse phases. ELISA based on traditional antibody and single signal analysis is slightly less specific and stable in SEB detection. Here, we innovatively reported an immunoassay based on a bifunctional protein reconstructed by nanobody, greatly enhancing the specificity of ELISA. Meanwhile, this is complemented by the second signal response, quenching effect of oxidative chromogenic substances on fluorochrome, to heighten the accuracy with dual mode. Under optimal conditions, the dual-mode immunoassay showed a wide linear range from 0.31 to 2500 ng/mL and a lower limit of detection (LOD) with 0.12 ng/mL for colorimetric mode and 0.24 ng/mL for fluorescence mode. There was no nonspecific binding occurred under the interference of S. aureus and other enterotoxins, demonstrating the splendid specificity. Moreover, the proposed assay was successfully employed in detecting SEB in food samples with satisfying practicability. This work integrated the nanobody modification technology with a dual signal sensing strategy, providing a reference for the development of a more specific and feasible platform for food safety.

HcN57, A Novel Unusual Acidic Silk-Like Matrix Protein from Hyriopsis cumingii, Participates in Framework Construction and Nacre Nucleation During Nacreous Layer Formation.

In the classic molecular model of nacreous layer formation, unusual acidic matrix proteins rich in aspartic acid (Asp) residues are essential for nacre nucleation due to their great affinity for binding calcium. However, the acidic matrix proteins discovered in the nacreous layer so far have been weakly acidic with a high proportion of glutamate. In the present study, several silk-like matrix proteins, including the novel matrix protein HcN57, were identified in the ethylenediaminetetraacetic acid-soluble extracts of the nacreous layer of Hyriopsis cumingii. HcN57 is a highly repetitive protein that consists of a high proportion of alanine (Ala, 34.4%), glycine (Gly, 22.5%), and serine (Ser, 11.4%). It forms poly Ala blocks, GlynX repeats, an Ala-Gly repeat, and a Ser-Ala-rich region, exhibiting significant similarity to silk proteins found in spider species. The expression of HcN57 was specifically located in the dorsal epithelial cells of the mantle pallium and mantle center. Notably, expression of HcN57 was relatively high during nacreous layer regeneration and pearl nacre deposition, suggesting HcN57 is a silk matrix protein in the nacreous layer. Importantly, HcN57 also contains a certain content of Asp residues, making it an unusual acidic matrix protein present in the nacreous layer. These Asp residues are mainly distributed in three large hydrophilic acidic regions, which showed inhibitory activity against aragonite deposition and morphological regulation of calcite in vitro. Moreover, HcN57-dsRNA injection resulted in failure of nacre nucleation in vivo. Taken together, our results show that HcN57 is a bifunctional silk protein with poly Ala blocks and Gly-rich regions that serve as space fillers within the chitinous framework to prevent crystallization at unnecessary nucleation sites and Asp-rich regions that create a calcium ion supersaturated microenvironment for nucleation in the center of nacre tablets. These observations contribute to a better understanding of the mechanism by which silk proteins regulate framework construction and nacre nucleation during nacreous layer formation.

Insights into the association of the Chlamydia trachomatis type III secretion chaperone complex, Scc4:Scc1, from sequential expression in Escherichia coli

Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and Chlamydia. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in CT's developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.

Effect of a novel anti-PD-1-proIL-2 bifunctional fusion protein on potent anti-tumor activity via PD-1 checkpoint inhibition and conditional IL-2R agonism.

e14697 Background: HD IL-2 was approved by FDA for metastatic melanoma (mM) and renal cell carcinoma (mRCC) but the treatment is associated with severe toxicities. PD-1 blockade has improved the overall survival, but only in 20-30% of cancer patients and many of them experience frequent relapses. Interestingly, HD IL-2 therapy displayed durable anti-tumor activity in mM and mRCC patients who progressed on anti-PD-1. Furthermore, combination therapy of IL-2 and pembrolizumab in mRCC has shown durable 70% response rate, while the ORR of monotherapy is 20% and 33% for IL-2 and pembrolizumab, respectively. Thus, combining IL-2 receptor activation and PD-1 blockade may represent a promising strategy in the post PD-1 era to overcome the resistance and improve the clinical efficacy. PTX-912 is a novel bifunctional PD-1-proIL-2v fusion protein designed to simultaneously harness PD-1 blockade and PD-1-cis directed IL-2R agonism conditionally in TME without the peripheral toxicities associated with IL-2. Methods: The biological functions were demonstrated in human PBMC assays. Anti-tumor efficacy and biomarker studies were evaluated in mouse / human PBMC xenograft models. Tox studies were performed in NHP. Results: PTX-912 shows dose-dependent inhibition of PD-1 pathway measured by GM-CSF and IFNgamma production in an MLR assay. Protease activated PTX-912, but not the intact drug, stimulates the phosphorylation of STAT5 of NK and T cells, the proliferation of CD8 T cells as well as the cytokine production in human whole blood. The potency of pSTAT5 by protease activated product is enhanced by > 10 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PTX-912 demonstrated high potency of tumor inhibition at as low as 0.1 mg/kg dose. In contrast, non-targeted proIL-2v at molar equivalent or higher dose showed minimal antitumor effect in either monotherapy or combination with anti-PD-1. Analysis of PTX-912 treated mice showed increased tumor infiltrating CD4+ and CD8+ T cells, including PD-1+Tcf-1+ and PD-1+ IL-18Ra+TIM3- CD8+ T cells, referred to “stem-like” progenitor effector cells and “better” effector cells, respectively. Activation of the IL-2v in PTX-912 was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PTX-912 demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. Conclusions: Our preclinical data demonstrated that PTX-912 effectively stimulated the expansion and reinvigoration of antigen specific T cells in the tumor tissue resulting in strong anti-tumor efficacy while it did not cause significant peripheral toxicity. Phase 1 clinical trial of PD-1-proIL-2v is planned to start in early 2024 to evaluate the safety, pharmacokinetics and anti-tumor activity in patients with advanced solid tumors.

Neoadjuvant SHR-1701 with or without chemotherapy in unresectable stage III NSCLC (TRAILBLAZER): Efficacy, safety and feasibility of surgical conversion outcomes from a proof-of-concept, phase 2 trial.

8082 Background: Consolidation immunotherapy after chemoradiation is the standard of care for unresectable stage III NSCLC. Addition of immunotherapy-based induction treatment may further improve outcome, partly by enabling surgery. We conducted a proof-of-concept, phase 2 trial to assess neoadjuvant SHR-1701 (an anti-PD-L1/TGF-β bifunctional fusion protein) with or without chemotherapy, followed by surgery or radiotherapy in unresectable stage III NSCLC and without EGFR/ ALK alterations. Methods: Patients with MDT-assessed surgically unresectable disease were given 3 cycles of neoadjuvant SHR-1701 (30 mg/kg Q3W) with or without chemotherapy (paclitaxel 175 mg/m2 Q3W + carboplatin AUC = 5 Q3W).Patients with PD-L1 TPS < 50% received combination therapy (arm A) and those with PD-L1 TPS ≥50% were randomized (1:1) to receive combination therapy (arm B) or SHR-1701 alone (arm C). After reassessment by MDT, patients received either surgery or definitive radiotherapy (60 Gy/30 fractions) plus concurrent cisplatin (30 mg/m2 QW), all followed by consolidation SHR-1701 for 16 cycles. The primary endpoints were post-induction objective response rate (ORR) and 18-month event-free survival (EFS) rate. Arm A+B (patients receiving neoadjuvant chemoimmunotherapy) was the primary analysis cohort. Results: Between Nov. 20, 2020 and Jan. 27, 2022, 107 patients were enrolled (arm A/B/C, n = 88/9/10). Median follow-up was 22.2 months. In arm A+B, both primary endpoints were met, with a post-induction ORR of 58% (95% CI 47-68) and an 18-month EFS rate of 56.6% (95% CI 45.2-66.5). In arm C, the post-induction ORR was 40% (95% CI 12-74) and 18-month EFS rate was 77.1% (95% CI 34.5-93.9). 24-month overall survival rate was 78.3% (95% CI 68.0-85.7) in arm A+B and 100% in arm C. Overall, 27 (25% of 107; arm A+B/C, n = 24/3) patients underwent surgery; all achieved R0 resection. Among them, 12 (44%) MPR and seven (26%) pCR were recorded. The 18-month EFS rate was 74.1% (95% CI 53.2-86.7) in resected patients and 57.3% (43.0-69.3) in those receiving radiotherapy. Across arms, all grade ≥3 TRAEs with frequency ≥10% were hematological toxicities. No new safety signals were identified. Conclusions: NeoadjuvantSHR-1701 with chemotherapy, followed by surgery or radiotherapy, showed promising anti-tumor activity with a tolerable safety profile in unresectable stage III NSCLC. We provided first evidence that surgical conversion was feasible in a notable proportion of patients, and was associated with better survival outcomes. Clinical trial information: NCT04580498 .

A phase I/II open label, multicenter study to assess the safety, tolerability, pharmacokinetics, and efficacy of HB0028 in patients with advanced solid tumors.

2528 Background: HB0028 is a bifunctional fusion protein consisting of an anti-PD-L1 IgG1 single domain antibody and TGF-β RII receptor extracellular domain (TGFβRII-ECD). This is the first dose escalation and expansion phase I/II study to evaluate the safety and preliminary anti-tumor activity of HB0028 in advanced solid tumors. Methods: In the phase I portion of the study, the patients (pts) with advanced solid tumor were enrolled, and an accelerated titration followed by a standard 3+3 design was applied to dose escalation. The objectives were toxicity evaluation, maximum tolerable dose (MTD), and recommended phase 2 dose (RP2D). Eligible pts with advanced solid cancers of any histologic subtype, and ECOG performance status ≤ 1 were treated with HB0028 (0.3mg, 1mg, 3mg, 10mg, 20mg, Q3W), until unacceptable toxicity or disease progression. Radiologic tumor assessments (by RECIST v1.1) were performed every 6 weeks while on treatment. Results: 16 pts [12 females and 4 males; ECOG 0/1, 9/7; median age 52 years (range 36 – 60)] were enrolled in phase I (0.3 mg/kg [n = 1], 1 mg/kg [n = 3], 3 mg/kg [n = 3], 10 mg/kg [n = 3], 20 mg/kg [n = 6]). Median number of prior lines of systemic therapy was 2 (range 1-4). 8(50%) pts received and progressed upon prior anti-PD-1/L1 therapy. All pts had measurable metastatic disease. No DLT and Death occurred. MTD was not reached. As of DEC 25,2023, among 16 patients' safety data are evaluable for toxicity. Any grade treatment‐related adverse events (TRAEs) occurred in 15 subjects (93.8%), with 2 subjects (12.5%) reported with ≥ Grade 3 (G3 Anemia and G3 γ-glutamyltransferase). The most common TRAEs (all grades) were AST increased (n = 4, 25%), anemia (n = 7, 43.8%) and infusion-related response (n = 5, 31.3%). Two SAEs (G2 AST increased and G3 Neoplastic fevers) were reported, only AST increased was considered as possibly related to study drug, but recovered shortly. The efficacy data cutoff date of DEC 25,2023, 16 patients are evaluable for radiologic response. 1 subject with cervical cancer in HB0028 20 mg/kg group had a partial response (PR), 3 subjects (liver cancer, lung cancer, left submandibular gland cancer) had stable disease (SD) and 12 subjects had progressive disease (PD). The ORR per RECIST 1.1 by investigator was 6.25% (1/16; 95% CI, 0%-30.2%), and the DCR was 25% (4/16; 95% CI, 7.3%-52.4%). Durable clinical benefit (SD for > 32 weeks) was seen in 1 subject with malignant tumor in the left submandibular gland. To date, 2 responders are still on treatment. Conclusions: HB0028 was generally well tolerated, and therapy provided modest antitumor activity in patients with heavily pretreated advanced solid tumors. Based on these data, a phase II prospective clinical trial is being planned in specific cancer. Clinical trial information: NCT06223308 .

Final analysis of the BIMES trial, a phase II single arm study assessing efficacy and safety of bintrafusp alfa in previously treated advanced malignant pleural mesothelioma (GECP 20/09).

8084 Background: Transforming growth factor β (TGF-β) is involved in tumor immune evasion and epithelial–mesenchymal transition (EMT). As TGF-β upregulation and EMT are associated with resistance to anti-PD1 therapy, we evaluated the efficacy of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in malignant pleural mesothelioma (MPM). Methods: Patients with advanced MPM who were previously treated with platinum-based chemotherapy were enrolled and received bintrafusp alfa 1200mg/m2 every 2 weeks until progression or for a maximum of two years. The primary objective was to determine the progression-free survival (PFS) by modified RECIST v1.1. assessed by the investigators. The expected median PFS was 4.5 months. Secondary objectives were overall response rate, duration of response, overall survival (OS) and safety. Results: Between October 2021 to March 2023, 47 patients were enrolled. Mean age was 70 years (41-84) and 36 (78.3%) were males. Most patients had epithelioid histology (82.9%) and had received only 1 prior line of therapy (84.8%). With a median follow-up of 11.5 months, 43 patients had disease progression and 24 patients died. The median number of doses administered was 4 (1-21) and reasons for treatment discontinuation were disease progression (82.6%), toxicity (6.5%), investigator decision (4.4%) and death (6.5%) by tumor progression. The median PFS was 1.9 months (95% CI 1.7 – 5.4) and the 6 month-PFS rate was 15.9%. Disease control rate was 34.8% (95% CI 22.2-49.9) consisting of 2 patients with partial response and 13 patients with stable disease as best response. The median OS was 11.9 months (95% CI 4.4 – not reached) and 6, 12 and 18-month OS rate was 65.3%, 46.5% and 26.6%, respectively. No significant differences in OS and PFS were observed based on MPM histological subtype. Grade 3-4 treatment-related adverse events occurred in 16 (34%) patients, anemia being the most common (n = 5); skin toxicity (n = 3); colitis (n = 2); adrenal insufficiency, acute kidney injury, allergic reaction, lipase, and amylase increased (n = 1 each). Conclusions: Bintrafusp alfa did not reach the expected efficacy in patients with advanced malignant pleural mesothelioma previously treated with platinum-based chemotherapy. Clinical trial information: NCT05005429 .

Bifunctional Protein TC1 Mediated One-Pot Strategy for Robust Immobilization of DNA with High Accessibility.

Immobilizing DNA with high accessibility at the interface is attractive but challenging. Current methods often involve multiple chemical reactions and derivatives. In this study, an endonuclease, TC1, is introduced to develop a robust strategy for immobilizing DNA with enhanced accessibility. TC1 enables direct immobilization of DNA onto a solid support through self-catalytic DNA covalent coupling and robust solid adsorption capabilities. This method demonstrates high accessibility to target molecules, supported by the improved sensitivity of DNA hybridization and aptamer-target recognition assays. TC1-mediated DNA immobilization is a one-pot reaction that does not require chemical derivatives, making it promising for the development of high-performance DNA materials and technologies.