Bifunctional Protein Research Articles

A Novel Bifunctional Fusion Protein (Anti-IL-17A-sST2) Protects against Acute Liver Failure, Modulating the TLR4/MyD88 Pathway and NLRP3 Inflammasome Activation

Acute liver failure (ALF) is a serious inflammatory disorder with high mortality rates, which poses a significant threat to human health. The IL-33/ST2 signal is a crucial regulator in inflammation responses associated with lipopolysaccharide (LPS)-induced macrophages. The IL-17A signaling pathway promotes the release of chemokines and inflammatory cytokines, recruiting neutrophils and T cells under LPS stimulation, thus facilitating inflammatory responses. Here, the potential therapeutic benefits of neutralizing the IL-17A signal and modulating the IL-33/ST2 signal in ALF were investigated. A novel dual-functional fusion protein, anti-IL-17A-sST2, was constructed, which displayed high purity and biological activities. The administration of anti-IL-17A-sST2 resulted in significant anti-inflammatory benefits in ALF mice, amelioration of hepatocyte necrosis and interstitial congestion, and reduction in TNF-α and IL-6. Furthermore, anti-IL-17A-sST2 injection downregulated the expression of TLR4 and NLRP3 as well as important molecules such as MyD88, caspase-1, and IL-1β. The results suggest that anti-IL-17A-sST2 reduced the secretion of inflammatory factors, attenuated the inflammatory response, and protected hepatic function by regulating the TLR4/MyD88 pathway and inhibiting the NLRP3 inflammasome, providing a new therapeutic approach for ALF.

Abstract CT257: Hyperprogressive disease following bintrafusp alfa and DNA damaging chemotherapy in relapsed small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. Current therapies have limited efficacy in SCLC, and despite a highly mutated genome, SCLC is largely unresponsive to immunotherapy. We sought to leverage the immune surveillance triggered by DNA damage in patients with relapsed SCLC using bintrafusp alfa, a bifunctional fusion protein targeting both PD-L1 and TGF-beta. Hyperprogressive disease (HPD), characterized by the rapid acceleration of tumor growth, has been reported in approximately 13% of patients treated with immune checkpoint inhibitors. However, the mechanistic basis is poorly understood. Methods: This is a safety run-in and phase II clinical trial. Cohort 1 enrolled patients with relapsed SCLC across Arms A (bintrafusp alfa 2400 mg q3 wks), B (bintrafusp alfa 2400 mg on D1 plus topotecan 1 mg/m2/day on D1-5 q3 wks), and C (bintrafusp alfa 1200 mg on D1 q2 wks plus temozolomide 200 mg/m2/day on D1-5 q4 wks). The primary endpoint was objective response rate (ORR). Results: Thirty-seven patients enrolled. Grade 3 treatment-related adverse events (TRAE) included transaminitis (27.0%), anemia (24.4%), lymphopenia (10.8%), and maculopapular rash (5.4%). Grade 4 TRAE included thrombocytopenia (8.1%), lymphopenia (5.4%) and transaminitis (5.4%). One patient experienced grade 5 tumor hemorrhage. By RECIST 1.1, partial responses were observed in 5 (13.5%), stable disease in 11 (29.7%), and progressive disease in 21 (56.8%) patients. Importantly, 13 (35.1%) patients developed HPD (≥ 2x increase in tumor growth rate from pre-trial vs on-trial, time to treatment failure ≤ 2 months, and ≥ 50% increase tumor burden). Changes in circulating cell-free DNA (n=20) tumor fraction using DELFI-TF, a mutation-independent, low-coverage whole genome sequencing approach, correlated with treatment responses. Pre-treatment serum from HPD (n=10 vs. 9 non-HPD) patients showed significantly lower levels of inflammatory cytokines (CXCL10, CCL13, CCL8, CCL19, TRAIL) and Granzyme H, and higher levels of anti-inflammatory cytokine IL10. Consistently, pre-treatment tumor transcriptomes of HPD (n=5 vs. 7 non-HPD) patients revealed suppression of IFN-gamma response, allograft rejection, and inflammatory response pathways. Conclusions: Concomitant TGF-beta and PD-L1 blockade is associated with a high frequency of HPD in SCLC patients. cfDNA could be critical for monitoring HPD. Tumor and blood immune signatures may inform the likelihood of HPD, with immune excluded tumors more likely to develop HPD. NCT Number: NCT03554473 Bintrafusp alfa was provided by EMD Serono (CrossRef Funder ID: 10.13039/100004755) Citation Format: Brett Schroeder, Nobuyuki Takahashi, Howard Yang, Renee Donahue, Zachary Skidmore, Alissa Konicki, Michael Nirula, Max Greenberg, George Chrisafis, Yang Zhang, Yo-Ting Tsai, Linda Sciuto, Samantha Nichols, Melissa Abel, Parth Desai, Rajesh Kumar, Christopher Schultz, Danielle Pinkiert, Chante Graham, Ajit Sharma, Justin Malin, Manan Krishnamurthy, Sophie Zhuang, Maxwell Lee, Lorenzo Rinaldi, Jeffrey Schlom, Lalage Wakefield, Anish Thomas. Hyperprogressive disease following bintrafusp alfa and DNA damaging chemotherapy in relapsed small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT257.

Abstract CT184: First-in-human trial of TIGIT inhibitor M6223 as monotherapy or in combination with bintrafusp alfa (BA) in patients (pts) with advanced solid unresectable tumors

Abstract Introduction: M6223 is an intravenous (IV), fully human, antagonistic, anti-TIGIT antibody with an Fc-mediated effector region. In preclinical studies, M6223 combined with BA (a bifunctional fusion protein that simultaneously targets the TGF-β and PD-(L)1 pathways) enhanced antitumor efficacy compared to either agent alone. Methods: This first-in-human, dose escalation study of M6223 as monotherapy (M6223; Part 1A) or in combination with BA (M6223+BA; Part 1B) included pts with advanced solid tumors (aged ≥18 years, ECOG PS ≤1) (NCT04457778). In Part 1A, pts received M6223 at 10 mg, 30 mg, 100 mg, 300 mg, 900 mg, 1600 mg, 2400 mg (all Q2W) or M6223 2400 mg Q3W. In Part 1B, pts received M6223 at 300 mg, 900 mg, or 1600 mg, all in combination with BA 1200 mg (both Q2W, IV). Dose escalation decisions by the Safety Monitoring Committee were assisted by a Bayesian 2-parameter logistic regression model. Primary objectives were safety, tolerability, maximum tolerated dose (MTD), and recommended dose for expansion (RDE). Additional objectives included pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity. Results: At final analysis, 40 pts (21 male, age range 24-79 years) had received M6223 (Q2W: n=32; Q3W: n=8), and 18 pts (7 male, age range 34-80 years) had received M6223+BA. Overall, two dose-limiting toxicities were observed: a grade 3 adrenal insufficiency (M6223, 900 mg) and a grade 3 anemia (M6223+BA, 300 mg; unrelated to M6223). In the M6223 group, grade ≥3 TEAEs were observed in 14 (35.0%) pts and M6223-related grade ≥3 TEAEs in 2 (5.0%) pts. In the M6223+BA group, grade ≥3 TEAEs were observed in 14 (77.8%) pts and M6223-related grade ≥3 TEAEs in 4 (22.2%) pts. No MTD was identified. RDEs were 1600 mg Q2W or 2400 mg Q3W for M6223 monotherapy, and 1600 mg+1200 mg Q2W for M6223+BA. Half-life for the two monotherapy RDEs were 9.0 and 14.6 days, respectively, with moderate accumulation at the selected RDEs. Linear PK was observed for M6223 at 100-2400 mg Q2W and at 2400 mg Q3W; co-administration with BA did not change the PK profile of M6223. PD analyses in blood showed full and sustained TIGIT target occupancy, and depletion of suppressive Tregs at M6223 ≥900 mg. Paired biopsies in the M6223 group (900 mg Q2W, 1600 mg Q2W, 2400 mg Q3W; n=12) showed a trend of decrease in TIGIT+ and increase in CD226+ cells. Median overall survival was 7.6 months (95% CI: 4.9, 12.0) and median progression-free survival was 1.4 months (95% CI: 1.3, 1.8). No pt achieved an objective response (per RECIST v1.1); stable disease as the best response was observed in 13 (32.5%) pts in M6223 and 5 (27.8%) pts in M6223+BA. Conclusion: M6223 monotherapy and in combination with BA had a manageable safety profile, and RDEs for both mono- and combination therapy were defined. Further evaluation of M6223 is ongoing in combination with PD-L1 inhibitor avelumab (JAVELIN Bladder Medley; NCT05327530). Citation Format: Aung Naing, Meredith McKean, Anthony Tolcher, Anja Victor, Ping Hu, Keyvan Tadjalli Mehr, Thomas Kitzing, Daniel Holland, Emilia Richter, Lillian Siu. First-in-human trial of TIGIT inhibitor M6223 as monotherapy or in combination with bintrafusp alfa (BA) in patients (pts) with advanced solid unresectable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT184.

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

BackgroundRiboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described.ResultsThrough mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content.ConclusionsOur study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

Abstract 2717: Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy

Abstract Many cancer cells upregulate the expression of programmed death ligand 1 (PD-L1) to surpass T-cell mediated immune response. Immune checkpoint blockers targeting PD-L1 or its receptor PD1 have been shown to suppress the inhibitory signals and enhance T cell function. As a new immunotherapy, immune checkpoint blockers have been used to treat multiple cancers. However, most clinically available blockers only engage with one type of immune cell: the T cells. Natural killer (NK) cells, on the other hand, play a significant role in anti-cancer immune responses. It will represent an advancement in anti-cancer immunotherapy if NK cells are engaged with immune checkpoint blockers. In this study, we designed a unique bifunctional fusion gene, PDL1sFv/MICAe, consisting of the variable regions of an anti-PD-L1 antibody and the extracellular domain of stress protein MICA and hypothesized that fusion protein PDL1sFv/MICAe will simultaneously bind to PD-L1 on tumor cells to block the inhibitory signal and extend T cells activity, and to NKG2D to activate NK Cells. cDNA sequence of fusion gene PDL1sFv/MICAe was designed based on the variable region of an established clinically used antibody, Atezolizumab, and the extracellular domain of MICA with linkers in between. After a computational confirmation of its structure and binding affinities, the fusion gene was synthesized and cloned into an expressing vector, and the fusion protein was produced for evaluation. Western blotting and fluorescent microscopic analyses confirmed that the protein binds to PD-L1 of human tumor cells, including MCF7 and MDAMB231, and NKG2D of KHYG1 cells. Preliminary results also show that the fusion protein enhances NK cell cytotoxicity against tumor cells. Studies on confirming the fusion protein’s activities on other tumor cells, NK cell IFN-γ production, T cell function, etc., are underway. In summary, fusion protein PDL1sFv/MICAe represents an improved version of the immune checkpoint blockers and could treat cancer patients quickly and effectively. Citation Format: Junyi Li, Yanzhang Wei. Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2717.

Abstract 2473: A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells

Abstract High-dose IL-2 (HD IL-2) induces complete and durable responses in RCC and melanoma but the treatment is associated with severe toxicities. PD-(L)1 blockade represents a recent breakthrough in immunotherapy with solid tumors, but only 20-30% of patients can benefit from the therapy and most responders eventually develop resistance. In more recent clinical studies with small number of subjects, HD IL-2 has shown promising responses in patients who progressed on anti-PD-1 therapy and combination therapy of HD-IL-2 and PD-1 blockade has resulted in high rate of objective response. In an attempt to maximize the clinical benefit of these immunotherapies to a broader patient population with deeper response and in the meantime to minimize the severe toxicity of HD IL-2, we have generated a novel bifunctional PD-1-proIL-2v fusion protein harnessing the mechanisms of PD-1 blockade and PD-1-cis directed tumor conditional IL-2R activation. PD-1-proIL-2v shows dose dependent inhibition of PD-1 immune checkpoint pathway measured by GM-CSF and IFNγ production in an MLR assay. Protease activated PD-1-proIL-2v, 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 10-20 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PD1-proIL-2v 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 PD-1-proIL-2v treated mice showed dramatically 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 “fresh” effector cells, respectively. Activation of the IL-2 procytokine in PD-1-proIL-2v was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PD-1-proIL-2v demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. In conclusion, our preclinical data demonstrated that PD-1-proIL-2v 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 advanced solid tumors. Citation Format: Zijuan Li, Feifei Zhang, Ze Zhang, Harry Zhou. A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2473.

Abstract 3996: Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy

Abstract Background: Success of Chimeric Antigen Receptor (CAR) T cells therapy for solid tumors are limited by the suppressive solid tumor microenvironment (TME), T cell exhaustion, lack of persistence, and poor trafficking to tumors. Strategies to improve this include therapeutic combinations, such as checkpoint inhibition (CPI) or cytokine support. However, CPI therapy has largely failed because of refractory and resistant tumors and cytokine administration can lead to lethal toxicities. To this end, we propose that enabling CAR T cells to secrete bi-functional fusion proteins consisting of cytokine modifiers (ie: IL12, IL15, or TGFβ-trap) combined with checkpoint inhibition (αPDL1 scFv) can provide tumor PDL1 sequestered cytokine activity and local tumor-immune modulation to boost solid tumor CAR T cell efficacy, enhance CPI impacts, and safely improve durable outcomes. Methods: Mouse T cells dual transduced to express surface CAR targeting prostate or ovarian solid tumors in addition to secretable cytokine fused to an αPDL1 scFv were generated. In vitro, the PDL1 blockade capacity and tumor-surface cytokine presentation of supernatants from dual transduced CAR T cells was assessed on PDL1 induced tumor cells. Further, secreted fusion protein transduced CAR T cells were assessed for function, phenotype, and cytokine release in a repetitive tumor rechallenge assay. In vivo, mouse CAR T cells dual transduced with fusion proteins and appropriate controls were assessed for anti-tumor efficacy, survival kinetics, and toxicity in immunocompetent solid tumor mouse models of prostate cancer and intraperitoneal disseminated ovarian cancer. Results: CAR T cells were shown to be successfully dual transduced, and secreted αPDL1-cytokine fusion proteins exhibiting functional PDL1 binding characteristics in vitro. CAR T cells engineered with αPDL1-IL12 fusion protein had greater anti-tumor activity and CAR specific expansion compared to other tested fusion proteins in in vitro co-culture assays. In a syngeneic prostate tumor model, mice receiving CAR T cells with αPDL1-IL12 fusion T cells out competed other cytokine fusion combinations. In a syngeneic ovarian tumor model we safely achieved 100% curative response rate with CAR secreting αPDL1-IL12 fusion in contrast to CAR with αPDL1mutIL12 control along with tumor regional PDL1 blockade on myeloid subsets within the TME. Conclusions: Our findings suggest that CAR T cells engineered to secrete αPD-L1-IL12 fusion protein show improved tumor control, less systemic toxicity, and enhanced expansion which promotes eradication of disease in two independent in vivo tumor models with two unique solid tumor CAR targets. We believe this strategy has the potential to improve solid tumor CAR T-cell efficacy and enhance durable innate immune responses. Citation Format: Lea Christian, John P. Murad, Lupita Lopez, Anthony Park, Jason Yang, Eric Lee, Candi Trac, Stephen Forman, Saul J. Priceman. Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3996.

Abstract 6179: Machine learning (ML)-spatial quantification of the tumor microenvironment (TME) identifies differences associated with response to bintrafusp alfa (BA) vs pembrolizumab (PEM) treatment in the Phase 3 INTR@PID Lung 037 study

Abstract Background: ML digital pathology models can accurately quantify predictive biomarkers like PD-L1.1 We combined tissue conserving ML assessment of the TME in hematoxylin and eosin (H&E)-stained whole slide images (WSI) with RNAseq for high-dimensional TME quantification for biomarker discovery. The orthogonal methods were applied to a retrospective analysis of a Phase 3 trial directly comparing BA, 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 PD-L1, and the anti-PD-1 PEM. Methods: Non-small cell lung cancer ML models previously trained to exhaustively quantify cell types and tissue regions in H&E-stained tissue were refined and deployed on 273 WSI from the INTR@PID Lung 037 study/NCT03631706. Human interpretable features (HIFs) describing the complex spatial organization of the TME were extracted from model predictions. HIFs were selected for analysis using K-means consensus clustering and describe the frequency of lymphocytes in cancer epithelium (cTILs); macrophages, fibroblasts, and granulocytes in stroma; and the abundance of cancer cells relative to all other cells in tumor. Bulk RNAseq was performed for 186 samples yielding 177 WSI-RNAseq pairs. Spearman correlation between HIFs and gene expression or signature was computed. Top gene associations were analyzed via gene-set enrichment analysis. HIFs were used for immunophenotyping (immune excluded, inflamed, and desert) via prespecified cutoffs on tumor-infiltrating lymphocyte abundance, and for biomarker discovery via Cox modeling of PFS and OS. Results: Gene expression and signatures were associated with HIFs quantifying immune spatial abundance in TME (e.g., cTILs vs. immune checkpoint signature2). Improved PFS and OS were associated with an immune-inflamed phenotype for PEM (PFS, HR=0.36, p=0.007, 95% CI 0.17-0.75; OS, HR=0.37, p=0.065, 95% CI 0.13-1.06). In interaction with BA, stromal macrophages were associated with improved survival (PFS, HR=0.72, p=0.062, 95% CI 0.53-1.02; OS, HR=0.62, p=0.058, 95% CI 0.38-1.02) and an increased likelihood of treatment response (odds ratio=1.61, p=0.015). PFS and OS were nearly identical between arms in immune-excluded cases (PEM vs. BA PFS, HR=0.93, p=0.73, 95% CI 0.63-1.39; OS, HR=1.09, p=0.77, 95% CI 0.62-1.92. Conclusion: Together HIFs and immunophenotyping enabled response prediction to BA and PEM. PEM response was greater for immune-inflamed tumors, while BA response was associated with macrophage infiltration of stroma. ML-based spatial analysis of the TME shows promise for immunotherapy biomarker discovery and validation. 1V Baxi, et al. Modern Pathology. 2022; 35(1):23-32. 2S Mariathasan, et al. Nature. 2018; 554(7693):544-548. Citation Format: John Abel, Andreas Machl, Aslihan Gerhold-Ay, Limin Yu, Darpan Sanghavi, Ben Trotter, Neel Patel, Ylaine Gerardin, Ramprakash Srinivasan, Sergine Brutus, Thomas Mrowiec. Machine learning (ML)-spatial quantification of the tumor microenvironment (TME) identifies differences associated with response to bintrafusp alfa (BA) vs pembrolizumab (PEM) treatment in the Phase 3 INTR@PID Lung 037 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6179.

Abstract 4080: IBB0979, A novel B7H3/IL-10 immunocytokine of monovalent anti-B7H3 antibody fused with IL-10 homodimer

Abstract Background: This study demonstrates the preclinical evaluation of a novel B7H3/IL-10 immunocytokine IBB0979. IBB0979 was designed to solve the problems of immune cell exhaustion and drug resistance in current immunotherapy. As the tumors progress, the domination of progenitor exhausted T cells (which are the major target cells of current PD-1/L1 based immunotherapy) will be replaced by terminally exhausted T cells (which are the major target cells of IL-10) causing resistance of current PD-1/L1 based therapies. Thus, IL-10 is a potent activator of antigen-specific CD8+ T cells in the tumor microenvironment and can restore the tumor-killing activity of tumor-infiltrating lymphocytes by restoring the oxidative phosphorylation metabolism of terminally exhausted T cells. B7H3 is highly expressed on various tumors but expressed at lower levels in normal tissues and blood vessels, which makes it an ideal target for developing cancer treatment. Also, B7H3 antibody extended the half-life and performed tumor-targeted delivery of IL-10, which improves the safety and efficacy of IL-10. Methods: We designed several structures of B7H3 antibody and IL-10 bifunctional fusion protein. Binding activity to B7H3 and IL-10 receptor was evaluated by both ELISA and FACS. C57BL/6J mice bearing MC38-hB7H3 cell line were used to evaluate the anti-tumor efficacy of IBB0979. Preclinical PK and toxicity study was also performed in cynomolgus monkeys to describe the safety profile of IBB0979. Results: Preclinical study showed that IBB0979 has high affinity to both targets and exhibited potent TGI in C57BL/6J mice bearing MC38-hB7H3 cell line, with TGI of 100% at 0.3mg/kg, 1mg/kg and 3mg/kg. An in vivo study in cynomolgus monkeys showed that after intravenously administered with 1 mg/kg, 2 mg/kg and 6 mg/kg of IBB0979 once a week for 29 days (5 times in total, given on days 1, 8, 15, 22 and 29), MTD was not reached up to 6 mg/kg which is significantly higher than the 10-20 μg/kg tolerated by IL-10 cytokines alone. Conclusion: IBB0979 is the world's first B7H3/IL-10 immunocytokine receiving IND approvals from both the FDA and the NMPA, showing excellent preclinical efficacy and safety profile. The Phase I clinical trial is currently on-going, with the first patient dosed in July 2023 and achieved SD at first efficacy evaluation after 2 cycles of administration. Since B7H3 is overexpressed in a wide range of cancers including lung, prostate, glioma, thyroid, head and neck, rectal, breast, skin, renal cell, and ovarian cancers, IBB0979 has the potential to become a next-generation therapy for resolving T cell exhaustion in cancer patients. Citation Format: Xiaoling Jiang, Chongbing Wu, Zi Chen, Liusong Yin. IBB0979, A novel B7H3/IL-10 immunocytokine of monovalent anti-B7H3 antibody fused with IL-10 homodimer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4080.

Abstract 4501: First reported PIM kinase degraders: Design, profiling & optimization

Abstract The PIM serine/threonine kinases (PIM1/PIM2/PIM3) are downstream effectors of ABL, JAK2 and Flt-3 oncogenes and are required for tumorigenesis. Overexpression has been reported in hematological and solid tumors, myeloma, lymphoma, leukemia and adenocarcinoma. The first generation of PIM inhibitors to make it to the clinic, including SGI-1776, AZD1208 and PIM447, are known to be pan-PIM inhibitors, while the aim of this project was to identify novel & selective PIM3 inhibitors. To this end, several Hit-finding approaches were employed to rapidly generate novel chemical matter, which were then assessed for their activity against PIM3 using a robust ADP-Glo™ assay. In parallel to this Hit-findings activity, we used AZD1208 scaffold as a starting point to generate PIM3 bifunctional protein degraders and designed a specific screening cascade in order to profile such modalities. The PIM degrader library, synthetized using Automated Robotics Lab, was characterized using ADP-Glo assays. It was then screened on our binding and activity kinase platforms, KINOMEscan® and KinaseProfiler™, and then fully profiled including ternary complex formation with biophysics and in vitro Safety Pharmacology Profiling Panels. One example is the AZD1208 derivate degrader, i.e. EC108154-1, which showed nanomolar range affinity for PIM3 and the E3-ligase CRBN-DDB1, and its affinity range was also confirmed with the ternary complex in SPR and MST-TRIC. This comprehensive screening cascade ensures an in-depth characterization and optimization of protein degraders, with the possibility of monitoring the ternary complex formation with biophysics methods. It is a good illustration of how this combination of tests can be instrumental in selecting the best degraders to progress to the next step of drug discovery. Citation Format: Celine Legros, Paul Ratcliffe, Vanessa Porkolab, Michele Modugno, Olivier Mirguet. First reported PIM kinase degraders: Design, profiling & optimization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4501.

Efficacy, safety, and biomarker analyses of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in patients with advanced non-small cell lung cancer

BackgroundBintrafusp alfa, a first-in-class bifunctional fusion protein targeting transforming growth factor-β (TGF-β) and programmed cell death ligand 1, has demonstrated encouraging efficacy as second-line treatment in patients with non-small cell...

The Mechanism of Inhibition of Mycobacterial (p)ppGpp Synthetases by a Synthetic Analog of Erogorgiaene.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

Bifunctional peptides as alternatives to copper-based formulations to control citrus canker

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial “killer” peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations.Key points• The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri.• Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane.• Mel-CgDEF protected citrus leaves against citrus canker disease.

Structural and functional properties of uridine 5′-monophosphate synthase from Coffea arabica

In higher eukaryotes and plants, the last two sequential steps in the de novo biosynthesis of uridine 5′-monophosphate (UMP) are catalyzed by a bifunctional natural chimeric protein called UMP synthase (UMPS). In higher plants, UMPS consists of two naturally fused enzymes: orotate phosphoribosyltransferase (OPRTase) at N-terminal and orotidine-5′-monophosphate decarboxylase (ODCase) at C-terminal. In this work, we obtained the full functional recombinant protein UMPS from Coffea arabica (CaUMPS) and studied its structure-function relationships. A biochemical and structural characterization of a plant UMPS with its two functional domains is described together with the presentation of the first crystal structure of a plant ODCase at 1.4 Å resolution. The kinetic parameters measured of CaOPRTase and CaODCase domains were comparable to those reported. The crystallographic structure revealed that CaODCase is a dimer that conserves the typical fold observed in other ODCases from prokaryote and eukaryote with a 1-deoxy-ribofuranose-5′-phosphate molecule bound in the active site of one subunit induced a closed conformation. Our results add to the knowledge of one of the key enzymes of the de novo biosynthesis of pyrimidines in plant metabolism and open the door to future applications.

Safety, Pharmacodynamic, and Anti-Tumor Activity of SL-172154 As Monotherapy and in Combination with Azacitidine (AZA) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) Patients (pts)

Background: CD47 blocking antibodies and SIRPα-Fc fusion proteins have demonstrated activity in combination with azacitidine (AZA) in previously untreated (UnTx) HR-MDS and AML pts. SL-172154 (SIRPα-Fc-CD40L), a hexameric, bi-functional fusion protein consisting of SIRPα domains linked to CD40L domains through an inert Fc linker demonstrated improved anti-tumor activity in comparison to naked CD47 blocking antibodies in pre-clinical studies [de Silva et al. Cancer Immunol Res 2020]. SL-172154, as a CD47 inhibitor, requires combination with AZA to enhance pro-phagocytic signals on leukemic stem cells/blasts thereby potentiating macrophage phagocytosis in AML/HR-MDS. Here we report the results from the Phase 1 Dose Escalation. cohorts for SL-172154 monotherapy [SL-mono] and for SL-172154 + AZA combination [SL-AZA] in pts with HR-MDS or AML. Methods: The objectives of the dose escalation cohorts include evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic effects and efficacy per IWG 2006 for MDS and ELN 2017 for AML. SL-172154 was administered IV weekly of a 28-day cycle as monotherapy or with AZA 75 mg/m 2 for 7 days per cycle. mTPI-2 was used for dose escalation with a DLT threshold of 20%. Pts ≥ 18 years old with R/R HR-MDS or R/R AML were eligible for dose escalation cohorts. In addition, UnTx pts with TP53 mutant (TP53m)-MDS were eligible for SL-AZA cohorts. Results: As of May 25, 2023, 37 pts were enrolled (19 in SL-mono; 18 in SL-AZA cohorts): 14 women; median age 70 years [range 44-81]. 27 pts had R/R AML (16 de novo; 11 secondary), 5 had R/R MDS, and 5 were UnTx TP53m-MDS. 23 (85%) of 27 R/R AML pts had adverse genetic abnormalities per 2017 ELN classification, including 9 (33%) with TP53m or del 17. The median number of prior lines of AML therapy was 2 [range 1-4]; 25 (93%) had received venetoclax (VEN) and 23 (85%) had received a hypomethylating agent (HMA). All 5 pts with R/R MDS had previously received HMA and 2 pts were previously treated with VEN. SL-172154 was dose escalated as monotherapy or with AZA as shown in Table 1. Infusion-related reactions (IRRs) were the most common SL-172154-related treatment-emergent AEs (TEAEs) reported in 13 (68%) and 8 (44%) pts in SL-mono and SL-AZA cohorts, respectively. IRRs (51 events) were Grade (G) 1 or 2 except for 3 G3 events (1 each at 6 mg/kg SL-mono and SL-AZA; 1 at 3 mg/kg SL-mono). Other SL-172154-related TEAEs observed in ≥ 3 pts were AST increased (4; 21%), ALT increased (3; 16%) and nausea (3; 17%) in SL-mono cohorts, and nausea (3; 17%) in SL-AZA cohorts. All events of AST/ALT increased were transient. Only one DLT (G3 IRR in the 6 mg/kg SL-AZA cohort) was reported, which required a dose reduction to 3 mg/kg. As of July 10, 2023, in SL-mono cohorts, 1 pt with R/R AML post 7+3, FLAG and VEN/AZA achieved Morphologic Leukemia-Free State (blast reduction from 19% to <5%) after 1 cycle of 6 mg/kg SL-172154 and proceeded to allogeneic hematopoietic cell transplant (allo-HCT) after cycle 2. In 12 evaluable pts with R/R AML receiving SL-AZA, although no objective responses (OR) were observed, relative reduction in bone marrow (BM) blasts from baseline was reported in 2/5 pts at 1 mg SL-AZA (-50%, -75%) and 5/7 pts at 3 mg SL-AZA (ranging from -35% to -90%). One pt underwent an allo-HCT. In 4 evaluable pts with UnTx TP53m-MDS, the ORs were 1 complete remission (CR), 1 marrow CR, and 2 stable disease. 2 of them proceeded to allo-HCT. SL-172154 induced elevations in serum IL-12p40, IP-10, IL-8, IL-10, MIP3α and MCP1: greater response at 3 mg/kg compared to 1 mg/kg while similar between 3 and 6 mg/kg. In the BM, SL-172154 induced a dose-dependent increase in phagocytic cells (CD11b, HLA-DR, CD16, CD36 and CD64) within mature myeloid immune cell compartments (CD45highCD34-). Reduction in leukemic blasts (CD45lowCD34+) was associated with an increase in mature myeloid and phagocytic cell phenotypes (3 mg/kg SL-AZA > 1 mg/kg SL-AZA). Conclusions: SL-172154 was well tolerated up to 3 mg/kg as monotherapy and in combination with AZA. Preliminary efficacy signals were detected in UnTx TP53m-MDS and R/R AML. A response to SL-172154 monotherapy, dose-dependent increases in serum cytokines and accumulation of mature myeloid cells in BM suggest a potential role for CD40 stimulation. Based on the safety, preliminary efficacy and pharmacodynamic activity, 3 mg/kg SL-172154+ AZA is being evaluated in treatment naïve pts with TP53m AML and HR-MDS.

627. Bioinformatic Approach to Design a Plasmodium falciparum PfRipr Multi-Epitope Vaccine Construct

Abstract Background Malaria infections remain an enormous contributor to global fatalities with an estimated death toll of 619,000 in 2021 and 77% of deaths being children aged under 5 years. Transmitted via mosquito, Plasmodium falciparum is the most lethal parasite of its genus but has evaded many treatment and vaccine efforts due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise for malaria vaccine development due to their ease of development and ability to target dominant regions in antigenically variable pathogens. The recently characterized protein P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. Merozoite Invasion Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. Methods Using P. falciparum sequences collected from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes with regard to T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. Putative epitopes were filtered using allele coverage, conservation, antigenicity, and allergenicity. B-Cell Epitope Prediction Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction.​ Epitope Sorting and Filtering Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope).​ Results Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. These epitopes were used to design a multi-epitope-based bloodstage vaccine construct against P. falciparum. MHC Epitopes Partial list of MHC epitopes found in Burkina Faso and Uganda datasets. B-Cell Epitopes B-cell epitopes found in Burkina Faso and Uganda datasets. PfRipr 3D Structure with B-cell Epitopes 3D model of PfRipr predicted via AlphaFold with labeled B-cell epitopes Conclusion To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to P. falciparum sequence datasets collected from other malaria endemic regions to develop vaccines effective against circulating strains. Multi-Epitope Vaccine Construct Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Disclosures All Authors: No reported disclosures

In Silico Prediction of Plasmodium falciparum PfRipr Epitopes as Vaccine Candidates

Abstract Background The global malarial death toll in 2020 was ~627,000, 77% of which occurred in children <5 years of age. Plasmodium falciparum is the most lethal parasite of its genus but has evaded vaccine development due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise due to their ability to target dominant and/or conserved regions in highly antigenically variable pathogens, including malaria. The recently characterized P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. (Figure 1) We sought to analyze P. falciparum sequences from highly endemic areas using bioinformatic approaches that identify candidate PfRipr epitope-based bloodstage vaccine antigens for inclusion in a multiantigen vaccine. Figure 1: Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. (https://doi.org/10.1038/s41564-022-01261-2 , https://creativecommons.org/licenses/by/4.0/) Methods Using two P. falciparum sequence datasets from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes for T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan and NetMHCIIpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. (Figure 2) Putative epitopes were filtered using binding thresholds, allele coverage, conservation, antigenicity, and allergenicity. (Figure 3) Figure 2: Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction. Figure 3: Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope). Results The Burkina Faso PfRipr dataset predicted 1729 unique T-cell and 51 unique B-cell epitopes, and 28 T-cell and 4 B-cell epitopes remained post-filtration. Epitope prediction using the Ugandan dataset predicted 576 unique T-cell and 53 unique B-cell epitopes, and 57 T-cell and 7 B-cell epitopes remained post-filtration. Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. (Tables 1-2, Figure 4) These epitopes were used to design a multi-epitope-based vaccine construct with a Flagellin adjuvant and peptide linkers to separate functional regions for immune processing. (Figure 5) Figure 4: 3D model of PfRipr with labeled B-cell epitopes. Predicted tertiary structure adapted from AlphaFold database. (https://doi.org/10.1038/s41586-021-03819-2, https://doi.org/10.1093/nar/gkab1061, https://creativecommons.org/licenses/by/4.0/) Figure 5: Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Conclusions Immunoinformatic tools employed in sequence identified candidate PfRipr epitopes used to design a bloodstage vaccine construct against P. falciparum. To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to a larger database of diverse P. falciparum sequences collected from other malaria endemic regions to develop vaccines effective against circulating strains.

Nanoscale Bilirubin Analysis in Translational Research and Precision Medicine by the Recombinant Protein HUG.

Bilirubin is a toxicological biomarker for hemolysis and liver diseases. The current automated diazo method used in clinical chemistry has limited applicability in rodent models and cannot be used in small animals relevant to toxicology, microphysiological systems, cell cultures, and kinetic studies. Here, we present a versatile fluorometric method for nanoscale analysis of bilirubin based on its highly specific binding to the recombinant bifunctional protein HELP-UnaG (HUG). The assay is sensitive (LoQ = 1.1 nM), accurate (4.5% relative standard error), and remarkably robust, allowing analysis at pH 7.4-9.5, T = 25-37 °C, in various buffers, and in the presence of 0.4-4 mg × L-1 serum albumin or 30% DMSO. It allows repeated measurements of bilirubinemia in murine models and small animals, fostering the 3Rs principle. The assay determines bilirubin in human plasma with a relative standard error of 6.7% at values that correlate and agree with the standard diazo method. Furthermore, it detects differences in human bilirubinemia related to sex and UGT1A1 polymorphisms, thus demonstrating its suitability for the uniform assessment of bilirubin at the nanoscale in translational and precision medicine.

511MO A phase I study of SHR-1701, a bifunctional fusion protein targeting PD-L1 and TGF-β, in patients with advanced non-small cell lung cancer (NSCLC)

511MO A phase I study of SHR-1701, a bifunctional fusion protein targeting PD-L1 and TGF-β, in patients with advanced non-small cell lung cancer (NSCLC)

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aβ scFvs: Applications for gene therapies

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept invivo study, we evaluated three different anti-amyloid Aβ scFvs (anti-Aβ scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aβ scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.