Peptide Linker Research Articles

Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

Design and expression of recombinant Xuanwei ham antioxidant peptide in Escherichia coli BL21

Xuanwei Ham Peptide (XHP) is a bioactive compound isolated and extracted from Xuanwei ham. It enhances the oxidative defense system by down-regulating CYP2E1 expression, reducing ROS production, and by activating the Nrf2/HO-1 pathway. In order to solve the problems of low yield, high cost and time-consuming separation process of active peptides from ham. In this study, we designed three peptide sequences derived from Xuanwei ham and expressed them recombinantly. The results showed that the peptides using flexible linker peptide ((GGGGS)2), rigid linker peptide ((EAAAK)2) linkage as well as the head-to-tail tandem linkage were successfully expressed in the E. coli expression system. Peptide monomers were released from the fusion proteins by formic acid cleavage and purified sequentially by Ni-IDA affinity chromatography, membrane filtration and RP-HPLC. The molecular weight of XHP was confirmed to be 717.35 by ESI-MS analysis. The results of the antioxidant activity of XHP showed that the hydroxyl radical scavenging rates of the three recombinant peptides were as high as 79.46%, 87.42%, and 53.60%, respectively. The hydroxyl radical activity of formic acid-cleaved XHP was reduced to 35.31%, but it showed good stability in heat, pH, metal ions and gastrointestinal digestion (GID). The molecular docking results showed that the docking of XHP to Keap1 protein was evaluated by five Molecular dynamics simulation with a maximum binding energy of -8.5 kcal/mol. This study provides references and ideas for the expression of active peptides, which can be widely used in the future can be used in the field of nutraceuticals, functional foods and pharmaceuticals.

Abstract 1351: Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic

Abstract Introduction: Blockade of the CTLA-4 pathway with ipilimumab (IPI), alone and in combination with nivolumab (anti-programmed death-1 antibody), has shown clinical benefit in multiple tumor types. However, not all tumors respond to IPI ± nivolumab, and peripheral effects can lead to immune-related adverse events. To enhance the therapeutic index of CTLA-4-directed therapy, a proprietary Probody® therapeutics technology platform was used to develop a modified version of IPI. BMS-986288 is an anti-CTLA-4 antibody that combines a Probody® therapeutic masking peptide and protease-cleavable linker, designed to localize anti-CTLA-4 activity to the tumor microenvironment, with an NF Fc region hypothesized to enhance antigen-presenting cell (APC)-mediated T-cell priming and regulatory T cell (Treg) modulation via increased binding to the FcγRIIIA (CD16) receptor. Here, we present the characterization of the mechanism of action and pharmacodynamic (PD) response of BMS-986288 in preclinical models. Methods: APC-mediated T-cell priming and antigen-specific responses were evaluated in a superantigen model using peripheral blood mononuclear cells (PBMCs) and human CTLA-4 knock-in (KI) mice. Antitumor activity and immune cell population changes were assessed in an MC38 tumor model implanted in human CTLA-4 KI mice. Intratumoral Treg depletion was further investigated using patient-derived dissociated tumor samples as a physiologically relevant model. Peripheral PD effects and tolerability of BMS-986288 were evaluated in a non-human primate (NHP) Ad5 vaccine model. Results: Protease-cleaved BMS-986288 demonstrated enhanced APC-mediated T-cell priming compared with IPI in superantigen-stimulated PBMCs. Similarly, administration of superantigen staphylococcal enterotoxin B (SEB) peptide elicited an increased antigen-specific T-cell receptor (Vβ8+) T-cell response after treatment with cleaved BMS-986288 vs IPI in human CTLA-4 KI mice. In the MC38 tumor-bearing mouse model, BMS-986288 showed enhanced antitumor activity as compared to IPI, with tumor clearance in all mice. Although BMS-986288 enhanced intratumoral CTLA-4+ Treg depletion in the MC38 model, limited Treg depletion was observed in the patient-derived dissociated tumor model with an NF anti-CTLA-4 antibody without the masking peptide, potentially reflecting differences in CTLA-4 expression on human vs mouse Tregs. BMS-986288 reduced PD effects in the peripheral blood compartment and increased tolerability in NHP relative to IPI. Conclusion: BMS-986288 leverages unique characteristics to differentiate it from IPI, enhancing APC-mediated T-cell priming and anti-tumor activity while also reducing peripheral activity in preclinical models. These data support an ongoing phase 1/2 clinical trial of BMS-986288 (NCT03994601) in patients with advanced solid tumors. Citation Format: Amy Jhatakia, Mohammed Nasser, Anandaroop Mukhopadhyay, Kyeongah Kang, Courtni Newsome, Neha Gupta, Remie Gail Z. Mandawe, Felix Findeisen, Jack A. Lohre, Leslie Leung, Yun Wei, Joshua Dobroff, Karen Price, John Engelhardt, Mark Selby, Alan J. Korman, Nicholas Wilson. Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic [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 1351.

Abstract 6343: AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS

Abstract Background: CD3+ T cell engaging bispecific antibodies (TCE) redirect T cells to attack targeted tumor cells by simultaneously binding TAA on cancer cells and CD3 complex on a T cell to form a TCR-independent immune synapse. Multiple TCEs have demonstrated promising therapeutic efficacy in the treatment of hematological malignancies. In contrast, suboptimal efficacy in the treatment of solid tumors and the risk of inducing cytokine release syndrome (CRS) continue to be major challenges. Several factors, such as format, affinity, and valency of the antibody, TAA copy number, and antigen size and epitope, can impact the therapeutic efficacy and safety of a TCE. In this study, we developed a novel “2+1” TCE platform, AnTenGagerTM, which enables conditional T cell binding and activation upon TAA-crosslinking, potent anti-tumor activity, and reduced risk of CRS. Methods: Humanized CD3 antibodies were derived from mouse hybridoma, followed by affinity optimization utilizing Computer Aided Drug Design. The Fab-scFv-Fab format was used as the backbone structure of AnTenGagers. The anti-CD3 scFv sequences and the length of peptide linkers between VH/VL, scFv/CH1, and scFv/CH2 were screened and compared in different preclinical assays. LALA mutations (L234A, L235A) were introduced to abolish the Fc receptor binding capability. In a series of in vitro and/or in vivo models, the efficacy, safety and developability of AnTenGagers targeting multiple TAAs, such as Her2, GD2, CDH6, LILRB4, FLT3 and GPRC5D, were evaluated. Results: A library of humanized parental CD3 antibodies with a broad spectrum of affinities were generated, some of which cross react with monkey CD3. AnTenGagers exhibited limited binding to CD3-positive cells before TAA-crosslinking, suggesting a reduced risk of CRS that is caused by systemic CD3+T cell activation. Four CD3 sequences with varying affinities were utilized to construct AnTenGagers with identical TAA-targeting arm. Compared to clinical benchmarks targeting the same TAA, all AnTenGagers demonstrated substantially lower binding capacity to CD3+ cells in the absence of TAA-crosslinking, while inducing increased cytotoxicity against target-positive tumor cells. In addition, they induced significantly less ex-vivo cytokine release by human PBMC than benchmarks. Notably, all AnTenGagers demonstrated enhanced in vivo efficacy compared to the benchmark, exhibiting complete response (CR) in PBMC humanized MM1S mouse CDX model while the serum concentration of proinflammatory cytokines was substantially lower in groups treated with AnTenGagers. AnTenGagers also demonstrated good developability with high expression yield, good thermostability and high stability under different stress conditions. Conclusions: These results suggested that AnTenGagerTM is a promising platform for developing the next generation of TCEs with enhanced efficacy and safety. Citation Format: Bing Hou, Hui Yuwen, Tengteng Li, Yijing Ren, Ao Sun, Zaoshun Hu, Jay Mei, Bo Shan. AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS [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 6343.

Abstract 4700: Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity

Abstract Antibody-drug conjugates (ADCs) have ushered in a new era of precision cancer therapy, and the caspase-3-cleavable linker has demonstrated its potential as a game-changing addition to the ADC toolkit. The DEVD peptide, specifically cleaved by caspase and lysosomal enzymes, in combination with the KG peptide, provides the necessary flexibility to achieve targeted cancer cell apoptosis. This research explores the key attributes and actions of the linker, aiming to highlight its potential for revolutionizing ADC therapy. The caspase-3-cleavable linker operates through caspase-3 activation during apoptosis. After targeting cancer cells with specific antigens, the ADC is internalized into the cells, and the linker is cleaved by caspase-3, releasing the drug and initiating apoptosis. The specificity of caspase-3 extends the drug’s effect to neighboring cells, even those lacking the target antigen. This cascade effect ensures the continuous activation of ADCs until all cancer cells are eradicated. Firstly, it overcomes tumor heterogeneity by activating the drug outside of the cell. Traditional bystander killing effects rely on internalization followed by drug release and penetration of neighbor cancer cells. However, this linker's unique mechanism ensures that the drug is activated by caspase-3 outside the cell without the need for internalization, effectively targeting all cancer cells within the tumor tissue. This is achieved through the induced phenotype targeting effect (IPTE), which involves caspase-3 cleavage of the linker, inducing a phenotype in the tumor tissue that targets all cancer cells, regardless of their specific markers. Secondly, it maintains therapeutic efficacy even as the target antigen decreases. Current targeted therapies face a challenge as target antigens may diminish during treatment, leading to reduced drug effectiveness. However, the unique mechanism of the linker ensures that the drug continues to be activated as long as there is caspase-3 secretion, resulting in continuous activation of ADCs in the tumor, with a feedback amplification loop that ensures therapeutic efficacy until all cancer cells are eliminated. Lastly, the linker's superior physicochemical properties allow for a maximum drug-to-antibody ratio (DAR). Its hydrophilic nature enables independent drug conjugation, increasing therapeutic potential and mitigating aggregation issues. This innovation addresses the current limitations in ADC conjugation technologies. In summary, the caspase-3-cleavable linker represents a significant leap forward in the field of ADCs. Its unique attributes offer promising prospects for more effective and targeted cancer treatments. This linker is poised to revolutionize the landscape of targeted cancer therapy, bringing us one step closer to the dream of eradicating cancer cells with precision and efficiency. Citation Format: Ha Kyeong Lee, Byoungmo Kim, Hyo Won Chang, Sang Yoon Kim, Youngro Byun. Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity [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 4700.

Abstract 1872: A next generation treatment for Nectin-4 positive cancers - Preclinical characterization of LY4052031, an anti-Nectin-4 antibody, conjugated to a novel camptothecin payload

Abstract Nectin-4 is an immunoglobulin-like antigen overexpressed in a variety of cancers including bladder, breast, NSCLC, and gastric. Normal tissues including skin, salivary glands, bladder and esophagus express only low to medium levels of Nectin-4 making the protein an attractive target for cancer therapies. The approval of enfortumab vedotin-ejfv, an anti-Nectin-4 based antibody-drug conjugate (ADC) with a microtubule inhibitor payload, either as a single agent or in combination with pembrolizumab has been a significant advancement in the treatment of metastatic urothelial cancer. We have developed a novel anti-Nectin-4 ADC with a novel topoisomerase I (TOPO 1) inhibitor drug payload. Topoisomerase inhibitor payload-based ADCs offer potential advantages compared to microtubule payload-based ADCs including a different toxicity profile and the differentiated susceptibility to resistance mechanisms, such as P-gp mediated drug efflux. LY4052031 is a fully human monoclonal anti-Nectin-4 antibody conjugated to a novel TOPO 1 inhibitor by a cleavable peptide linker at a homogeneous drug antibody ratio (DAR) of 8:1. In vitro, both LY4052031 and its unconjugated parental antibody specifically bind to Nectin-4 expressing cell lines and are quickly and efficiently internalized. LY4052031 demonstrated potent cytotoxicity on tumor cells with high and low Nectin-4 expression. Additionally, bystander activity exerted by LY4052031 on antigen negative cells co-cultured with antigen positive cells was greater than that observed with the clinical benchmark enfortumab vedotin-ejfv. In vivo studies in xenografts expressing low to high levels of Nectin-4 showed significant tumor growth inhibition following treatment with LY4052031 as monotherapy. Overall, LY4052031 exhibits specificity, selectivity, potency, and effectiveness as a next generation therapy to treat Nectin-4 positive tumors. An IND submission is planned for 2024. Citation Format: Divya Sagar, Mohan Srinivasan, Kevin Lindquist, Qianxu Guo, Wailin Wong, Maria B. Lebron, Rose Marie Sattler, Jieyu Zhou, Whitney Helms, Jeffrey Boyles, Petra Verdino, Xuan Zhao, Yvonne Mak, Joshua Park, Rikke Holmgaard, Kyla Driscoll, Omar Duramad, Kristin Bedard. A next generation treatment for Nectin-4 positive cancers - Preclinical characterization of LY4052031, an anti-Nectin-4 antibody, conjugated to a novel camptothecin payload [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 1872.

Abstract 2611: Non-specific elimination of tumor-associated neutrophils by antibody-drug conjugates boosts anti-tumor immunity for PD-1 immunotherapy

Abstract Background: Antibody-drug conjugates (ADCs) play a key role in targeted therapy of solid tumors. However, few studies have examined the impact of ADCs on the tumor microenvironment (TME), particularly on innate immune cells. To elucidate this, this study focuses on investigating the comprehensive impacts of ICAM1-DXd on the tumor microenvironment, especially on tumor-associated neutrophils (TAN) and its synergistic effect with PD-1 immunotherapy. Methods: Firstly, the efficacy and prognosis of ICAM1-DXd, a potent ADC for cervical cancer, were determined in pre-clinical tumor models, providing crucial transcriptomic and biological function information by using RNA-seq. Then, pathological studies were performed to evaluate the abundance and location of TAN (Ly6G+/MPO+) in TME post-ADC treatment. The interaction between TAN and ICAM1-DXd and subsequent impacts on adaptive immune cells (e.g., CD8+ cells) was examined by flow cytometry (FACS), confocal fluorescent imaging, and western blot techniques (WB). Eventually, the synergistic effect of ICAM1-DXd and PD-1 monoclonal antibody were investigated in an immunocompetent animal model. Results: ICAM1-DXd potently inhibits cervical tumor growth in preclinical models, and RNA-seq analysis revealed that ICAM1-DXd treatment induces a strong down-regulation of neutrophil activation, especially for Ly6G+/MPO+ TANs, a subtype with pro-tumor function. IHC and FACS results confirmed that pro-tumor TANs were efficiently eliminated in TME by ICAM1-DXd treatment via non-specific phagocytosis in comparison with peripheral blood neutrophils. ICAM1-DXd-phagocytised TANs also elicit a strong bystander killing effect by cleaving the quadra peptide linker of ICAM1-DXD by cathepsin B. This ADC-mediated TAN elimination effectively ameliorates immunosuppressive TME and works in synergy with PD-1 immunotherapy in preclinical models. Conclusion: In preclinical models of cervical cancer, ICAM1-DXd not only directly ablates cancer cells via antigen recognition but also non-specifically kills tumor-promoting TANs, resulting in an improved tumor immune microenvironment, providing a shred of mechanistic evidence supporting ADC and PD-1 combination therapy. Citation Format: Shili Yao, Lu Sun, Xiu Zhu, Hanfei Xie, Tong Yang, Peng Guo, Huarong Tang, Tao Zhu. Non-specific elimination of tumor-associated neutrophils by antibody-drug conjugates boosts anti-tumor immunity for PD-1 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 2611.

Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [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 6330.

Abstract 3137: Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​

Abstract Placental alkaline phosphatases, ALPP and ALPPL2, are highly homologous, membrane-bound proteins involved in fetal development. Despite having limited expression in normal tissues, they are highly differentiated in tumor cells making them ideal targets for antibody drug conjugate (ADC) development. AT2604 is an ADC that consists of a humanized IgG1 anti-ALPP/ALPPL2 antibody that displays specificity over other human alkaline phosphatases and is cross reactive to NHP ALPPL2 but not to murine ALPPL2. The anti-ALPP/ALPPL2 antibody shows high target binding affinity and internalization into ALPP and ALPPL2 positive cells.​ AT2604 utilizes AxcynCYSTM technology for site-specific conjugation to achieve a highly homogeneous (>97%) DAR4 ADC product. The payload is the clinically validated microtubule disrupting agent monomethyl auristatin E (MMAE) conjugated to the antibody via a protease-cleavable peptide linker approved for ADC use. When evaluated in mouse xenograft models of gastric (NCI-N87) and pancreatic (HPAC) cancer, AT2604 displayed strong tumor growth inhibition (>90%) at 1 mg/kg (QW3 × 2 dosing) in both models. Furthermore, a pre-tox study in non-human primates concluded AT2604 has a HNSTD of 10 mg/kg (QW3 × 3 dosing) and did not exhibit any non-reversible adverse effects; implying that AT2604 possesses an improved safety profile over other vedotin-based ADCs. Taken together, the data supports the continued development of AT2604 towards evaluation in human trials.​ Citation Format: Bryan K. Yeung, Xiao Hui Koh, Wei Li Ling, Weiqi Tan, You Xue Wu, Xi Yun Zhang, Hong Yee Tan, Shao Jun Liu, Chen Zhong, Bin Zou. Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​ [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 3137.

Abstract 58: Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies

Abstract The effector functions of CAR T cells can be enhanced through delivery of immunostimulatory cytokines, such as IL-12, IL-18, or IL-23. However, systemic delivery of recombinant cytokines or paracrine secretion by CAR T cells can elicit toxicities due to non-specific targeting of the cytokines. Here, we designed immunocytokines with antibodies specific for the invariant linker peptides that join variable domains in the CAR scFv and immunostimulatory cytokines. The immunocytokines were capable of targeting CAR molecules agnostic of target antigen, demonstrating broad applicability without the need for further genetic engineering of CAR therapies, such as co-expression of a surrogate molecule (tEGFR, etc.). The addition of immunocytokines enhanced CAR T cell cytolytic activity and secretion of type I cytokines, even at low concentrations of immunocytokine. In a stringent Nalm6 xenograft model, the addition of immunocytokines delivering IL-12 enhanced the anti-tumor activity of anti-CD19 CAR T cells and overall survival compared to controls of antibody alone or PBS. This technological development presents the opportunity to specifically enhance the activity of multiple CAR T cell therapies in the absence of non-specific toxicities. Citation Format: J T. Keane, Oula K. Dagher, Fang Liu, Avery D. Posey. Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies [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 58.

Adoption of the 2A Ribosomal Skip Principle to Track Assembled Virions of Pepper Mild Mottle Virus in Nicotiana benthamiana.

The coat protein (CP) is an important structural protein that plays many functional roles during the viral cycle. In this study, the CP of pepper mild mottle virus (PMMoV) was genetically fused to GFP using the foot-and-mouth disease virus peptide 2A linker peptide and the construct (PMMoV-GFP2A) was shown to be infectious. The systemic spread of the virus was monitored by its fluorescence in infected plants. Electron microscopy and immunocolloidal gold labelling confirmed that PMMoV-GFP2A forms rod-shaped particles on which GFP is displayed. Studies of tissue ultrastructure and virion self-assembly confirmed that PMMoV-GFP2A could be used to monitor the real-time dynamic changes of CP location during virus infection. Aggregations of GFP-tagged virions appeared as fluorescent plaques in confocal laser microscopy. Altogether, PMMoV-GFP2A is a useful tool for studying the spatial and temporal changes of PMMoV CP during viral infection.

Design and Synthesis of Novel Squaraine-Based Fluorescent Probe for Far-Red Detection of Chymotrypsin Enzyme.

Chymotrypsin, a crucial enzyme in human digestion, catalyzes the breakdown of milk proteins, underscoring its significance in both health diagnostics and dairy quality assurance. Addressing the critical need for rapid, cost-effective detection methods, we introduce a groundbreaking approach utilizing far-red technology and HOMO-Förster resonance energy transfer (FRET). Our novel probe, SQ-122 PC, features a unique molecular design that includes a squaraine dye (SQ), a peptide linker, and SQ moieties synthesized through solid-phase peptide synthesis. Demonstrating a remarkable quenching efficiency of 93.75% in a tailored H2O:DMSO (7:3) solvent system, our probe exhibits absorption and emission properties within the far-red spectrum, with an unprecedented detection limit of 0.130 nM. Importantly, our method offers unparalleled selectivity towards chymotrypsin, ensuring robust and accurate enzyme detection. This pioneering work underscores the immense potential of far-red-based homo-FRET systems in enabling the sensitive and specific detection of chymotrypsin enzyme activity. By bridging the gap between cutting-edge technology and biomedical diagnostics, our findings herald a new era of enzyme sensing, promising transformative advancements in disease diagnosis and dairy quality control.

Rigid linker peptides improve the stability and anti-inflammation effect of human serum albumin and α-melanocyte-stimulating hormone fusion proteins.

The anti-inflammatory effect of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS) has been reported for 40 years. However, the short half-life of α-MSH limits its clinical applications. The previous study has shown that a fusion protein comprising protein transduction domain (PTD), human serum albumin (HSA), and α-MSH extends the half-life of α-MSH, but its anti-inflammatory effect is not satisfactory. In this study, optimization of the structures of fusion proteins was attempted by changing the linker peptide between HSA and α-MSH. The optimization resulted in the improvement of various important characteristics, especially the stability and anti-inflammatory bioactivity, which are important features in protein medicines. Compared to the original linker peptide L0, the 5-amino-acid rigid linker peptide L6 (PAPAP) is the best option for further investigation due to its higher expression (increased by 6.27%), improved purification recovery (increased by 60.8%), excellent thermal stability (Tm=83.5°C) and better inhibition in NF-κB expression (increased by 81.5%). From this study, the significance of the design of linker peptides in the study of structure-activity relationship of fusion proteins was proved.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

Chimeric nanobody-decorated liposomes by self-assembly.

Liposomes as drug vehicles have advantages, such as payload protection, tunable carrying capacity and improved biodistribution. However, due to the dysfunction of targeting moieties and payload loss during preparation, immunoliposomes have yet to be favoured in commercial manufacturing. Here we report a chemical modification-free biophysical approach for producing immunoliposomes in one step through the self-assembly of a chimeric nanobody (cNB) into liposome bilayers. cNB consists of a nanobody against human epidermal growth factor receptor 2 (HER2), a flexible peptide linker and a hydrophobic single transmembrane domain. We determined that 64% of therapeutic compounds can be encapsulated into 100-nm liposomes, and up to 2,500 cNBs can be anchored on liposomal membranes without steric hindrance under facile conditions. Subsequently, we demonstrate that drug-loaded immunoliposomes increase cytotoxicity on HER2-overexpressing cancer cell lines by 10- to 20-fold, inhibit the growth of xenograft tumours by 3.4-fold and improve survival by more than twofold.

Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron.

The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fusion protein consisting of a cleavable linker and a destabilizing domain (DD), which conditionally controls the expression and release of a target protein in a ligand-induced state, allowing the free unmodified protein to perform its function. Insertion of an AGIA epitope at the carboxyl terminus of the linker made space for the DUBs to access the site to assist the cleavage reaction when the amino terminus of the target protein caused steric hindrance. The developed system, termed a cleavable degron using ubiquitin-derived linkers (c-DUB), provides robust and tunable regulation of target proteins in their native forms. The c-DUB system is a useful tool for the regulation of proteins that have terminal sites that are essential for the proper localization and function. In addition, a mechanistic investigation using proximity labeling showed that DUBs associate with the refolded DD to reverse ubiquitination, suggesting a cellular surveillance system for distinguishing the refolded DD from misfolded proteins. The c-DUB method may benefit from this machinery so that DUBs subsequently cleave the neighboring linker.

A comparative study on the effects of human serum albumin and α-melanocyte-stimulating hormone fusion proteins on the anti-neuroinflammatory in the central nervous system of adult mice

The immunomodulatory effects of α-melanocyte stimulating hormone (α-MSH) in the central nervous system (CNS) have been investigated for forty years. The clinical applications of α-MSH are limited due to its short half-life. Our previous study has indicated that the short half-life of α-MSH can be extended by fusion with carrier human serum albumin (HSA) and this fusion protein has also retained the anti-inflammatory effect on the CNS. This improvement is still far from the clinical requirements. Thus, we expected to enhance the half-life and activity of the fusion protein by optimizing the linker peptide to get closer to clinical requirements. In a previous study, we screened out two candidates in vitro experiments with a flexible linker peptide (fusion protein with flexible linker peptide, FPFL) and a rigid linker peptide (fusion protein with rigid linker peptide, FPRL), respectively. However, it was not sure whether the anti-inflammatory effects in vitro could be reproduced in vivo. Our results show that FPRL is the best candidate with a longer half-life compared to the traditional flexible linker peptides. Meanwhile, the ability of FPRL to penetrate the blood-brain barrier (BBB) was enhanced, and the inhibition of TNF-α and IL-6 was improved. We also found that the toxicity of FPRL was decreased. All of the results suggested that trying to choose the rigid linker peptide in some fusion proteins may be a potential choice for improving the unsatisfactory characteristics.

A tumor‐targeting nano‐adjuvant for in situ vaccine based on ultrasound therapy

AbstractUltrasound‐generated antigens combined with TLR7/8 agonists as adjuvants have demonstrated significant anti‐tumor efficacy as an in‐situ vaccine. However, the use of TLR7/8 agonists can cause severe inflammatory responses. In this study, we present a novel tumor‐targeting nano‐adjuvant termed aPDL1‐PLG/R848 NPs, which are composed of aPDL1 antibody, Fc‐III‐4C peptide linker (Fc‐linker) and poly(L‐glutamic acid)‐grafted‐R848. Under ultrasound irradiation, antigen‐presenting cells activate immune mechanisms in vivo under dual stimulation of in situ antigens and immune adjuvants. The strategy inhibits primary tumor growth and induces a strong antigen‐specific immune memory effect to prevent tumor recurrence in vivo. This work offers a safe and potent platform for an in situ cancer vaccine based on ultrasound therapy.

An Anti-CD7 Antibody-Drug Conjugate Target Showing Potent Antitumor Activity for T-Lymphoblastic Leukemia (T-ALL).

Acute T-lymphoblastic leukemia (T-ALL) is a type of leukemia that can occur in both pediatric and adult populations. Compared to acute B-cell lymphoblastic leukemia (B-ALL), patients with T-cell T-ALL have a poorer therapeutic efficacy. In this study, a novel anti-CD7 antibody-drug conjugate (ADC, J87-Dxd) was successfully generated and used for T-ALL treatment. Firstly, to obtain anti-CD7 mAbs, we expressed and purified the CD7 protein extracellular domain. Utilizing hybridoma technology, we obtained three anti-CD7 mAbs (J87, G73 and A15) with a high affinity for CD7. Both the results of immunofluorescence and Biacore assay indicated that J87 (KD = 1.54 × 10-10 M) had the highest affinity among the three anti-CD7 mAbs. In addition, an internalization assay showed the internalization level of J87 to be higher than that of the other two mAbs. Next, we successfully generated the anti-CD7 ADC (J87-Dxd) by conjugating DXd to J87 via a cleavable maleimide-GGFG peptide linker. J87-Dxd also possessed the ability to recognize and bind CD7. Using J87-Dxd to treat T-ALL cells (Jurkat and CCRF-CEM), we observed that J87-Dxd bound to CD7 was internalized into T-ALL cells. Moreover, J87-Dxd treatment significantly induced the apoptosis of Jurkat and CCRF-CEM cells. The IC50 (half-maximal inhibitory concentration) value of J87-Dxd against CCRF-CEM obtained by CCK-8 assay was 6.3 nM. Finally, to assess the antitumor efficacy of a J87-Dxd in vivo, we established T-ALL mouse models and treated mice with J87-Dxd or J87. The results showed that on day 24 after tumor inoculation, all mice treated with J87 or PBS died, whereas the survival rate of mice treated with J87-Dxd was 80%. H&E staining showed no significant organic changes in the heart, liver, spleen, lungs and kidneys of all mice. In summary, we demonstrated that the novel anti-CD7 ADC (J87-Dxd) had a potent and selective effect against CD7-expressing T-All cells both in vitro and in vivo, and could thus be expected to be further developed as a new drug for the treatment of T-ALL or other CD7-expression tumors.