Antibody 17-1A Research Articles

An Anti-RAGE Chimeric antibody alleviates CCl4-induced liver fibrosis via RAGE/NF-kB pathway in mice

Liver fibrosis is a progressive condition characterized by excessive deposition of extracellular matrix components, leading to organ dysfunction. Chronic inflammation and activation of hepatic stellate cells (HSCs) are two dominant events in all stages of fibrosis development. The receptor for advanced glycation end products (RAGE) pathway is involved in modulating liver injury and fibrosis, and preventing it, or deletion of Ager gene can protect the liver against fibrosis progression. To investigate functions and mechanism of chimeric anti-RAGE monoclonal antibody against liver fibrosis, murine-derived monoclonal anti-RAGE antibodies were used to construct murine-human chimeric antibodies. The properties of the chimeric antibody were characterized, and the biological functions of antibody A5 or its evolved humanized molecule, huA5, were investigated in cell or animal model. The data showed that blocking the RAGE pathway with huA5 robustly reduced liver injury and fibrosis. Furthermore, huA5 significantly suppressed the activation of HSCs and inhibited expression of fibrosis-associated genes, including COL1A1,TIMP1, and ACTA2. huA5 also interfered with RAGE downstream signal transduction and down-regulate both ERK and NF-κB phosphorylation, inhibited the RAGE/NF-kB pathway, leading to reduced expression of pro-inflammatory cytokines and profibrotic markers. Finally, RAGE silencing significantly decreased the expression of activation-related genes in HSCs, inhibiting HSCs proliferation and migration. These results clearly revealed that the anti-RAGE chimeric antibody exerted antifibrotic efficacy in vitro and attenuated liver fibrosis in vivo. HuA5 can be further developed as a lead molecule of drug to treat patients with liver fibrosis.

Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

Atherosclerosis is rare in internal thoracic arteries (ITA) even in patients with severe atherosclerotic coronary artery (ACA) disease. To explore cellular differences, ITA SMC from 3 distinct donors and ACA SMC from 3 distinct donors were grown to sub-confluence and growth arrested for 48 h. Proliferation and thrombospondin-1 (TSP1) production were determined using standard techniques. ITA SMC were larger, grew more slowly and survived more passages than ACA SMC. ACA SMC had a more pronounced proliferative response to 10% serum than ITA SMC. Both ACA SMC and ITA SMC proliferated in response to exogenous TSP1 (12.5 µg/ml and 25 µg/ml) and platelet derived growth factor-BB (PDGF-BB; 20 ng/ml) but TSP1- and PDGF-BB-induced proliferation were partially inhibited by anti-TSP1 antibody A4.1, microRNA-21(miR-21)-3p inhibitors and miR-21-5p inhibitors in each of the 3 ACA SMC lines, but not in any of the ITA SMC lines. PDGF-BB stimulated TSP1 production in ACA SMC but not in ITA SMC but there was no increase in TSP1 levels in conditioned media in either SMC type. In summary, there are significant differences in morphology, proliferative capacity and in responses to TSP1 and PDGF-BB in SMC derived from ITA compared to SMC derived from ACA.

Relationship between Jo-1 B Cell Epitope Profile and Clinical Features of Anti-Synthetase Syndrome.

Anti-histidyl-transfer RNA synthetase (Jo-1) antibodies are associated with myositis as well as different extramuscular organ complications comprising the anti-synthetase syndrome. This study aimed to clarify the relationship between anti-Jo-1 epitope recognition patterns and specific clinical features of this syndrome. B cell epitope mapping was performed via enzyme-linked immunosorbent assay in 180 patients who were anti-Jo-1 antibody-positive using overlapping peptides/protein fragments spanning the amino-terminal 151 amino acids of Jo-1 as substrate antigens. Statistical associations with clinical features were assessed through rank-sum, correlation, and cluster analyses. The level of reactivity against subfragments spanning amino acids 1-151 of Jo-1 paralleled that of full-length Jo-1, confirming the immunodominance of this amino-terminal region. The corresponding frequencies of reactivity to peptides 1 (amino acids [aa] 1-21), 3 (aa 27-47), 4 (aa 40-60), 10 (aa 118-138), and 11 (aa 131-151) were 6.1%, 42.5%, 6.8%, 6.7%, and 20.3%. While anti-full-length Jo-1 antibodies were significantly associated with Raynaud phenomenon, anti-fragment A2 (aa 1-60) and A3 (aa 1-90) antibodies were associated with proximal muscle weakness, Raynaud phenomenon, arthritis, and sicca syndrome. Anti-fragment A4 (aa 1-120) and A5 (aa 1-151) antibodies were also associated with sicca syndrome. Peptide 1 (aa 1-21) antibodies were associated with Raynaud phenomenon and dysphagia. Whereas anti-peptide 3 (aa 27-47) antibodies were also linked to Raynaud phenomenon, anti-peptide 9 (aa 105-125) antibodies were associated with mechanic's hands. Autoantibodies targeting different amino-terminal subfragments and/or peptides of Jo-1 were associated with specific clinical features of the anti-synthetase syndrome, demonstrating the biomarker potential of B cell epitope profiling in this disorder.

Kidney disease associated with pre-pubertal obesity initiates pathological alterations in the brain of Dahl salt-sensitive rats

Childhood/pre-pubertal obesity (PPO) is implicated in the development of chronic kidney disease (CKD), and there is evidence that PPO also increases the risk of developing Alzheimer disease (AD) later in life. In the current study, we aim to investigate the correlation between CKD and the development of AD-like pathology in a rat model of CKD associated with PPO. We hypothesize that systemic inflammation due to PPO exacerbates CKD and induces AD-like pathology in the brain at older age. In our preliminary studies, we utilized the Dahl salt sensitive leptin receptor mutant rat (SSLepRmutant), which is a novel model of PPO. 6-week-old Dahl salt-sensitive (SS) (control, n=3) and SSLepRmutant rats (n=5) were monitored for proteinuria every 3 weeks, for a 12-week period. Throughout the study, we observed significantly elevated proteinuria in SSLepRmutant rats (Week-6: 337±81 mg/day, Week-9: 371±147 mg/day, Week-18: 1920±318 mg/day) compared to SS rats (Week-6: 25±7 mg/day, Week-9: 35±39 mg/day, Week-18: 35±33 mg/day). At 18-week, creatinine levels were significantly higher in SSLepRmutant rats (1.5±0.5 mg/dL) versus SS rats (0.4±0.03 mg/dL) indicating CKD. Additionally, we found significant elevation of triglycerides in SSLepRmutant rats (5134±1211 mg/dL) compared to SS rats (79±21 mg/dL). To investigate the brain pathology, we performed immunochemical analysis (dot-blot assay) using A11 antibody to determine the levels of Amyloid-beta oligomers (Aβo). Our results show that there was tendency for Aβo levels to be increased in the brains from obese SSLepRmutant rats compared to SS. Finally, we tested the permeability of the blood-brain barrier to Evans blue dye and observed that the brain of SSLepRmutant rats was more permeable to the dye compared to SS rats as early as 8 weeks of age. Overall, these results indicate that PPO obesity not only increases the susceptibility to develop CKD but also causes Alzheimer-like pathology during the prepubescent stage. NIDDK (DK109133) and AG (AG057842) awarded to Jan M. Williams. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB349: Cysteine restriction enhances antitumor immunity in colorectal cancer

Abstract Background: Colon cancer is the second leading cause of cancer mortality worldwide, however, there are still issues with multidrug resistance and severe adverse reactions. Previous studies have shown that sulfur-containing amino acids are involved in cancer development, drug resistance, and immune evasion. Here, Our experimental findings suggested that restricting the intake of sulfur-containing amino acids may present a novel strategy for treating colorectal cancer. Methods: We assessed the impact of sulfur-containing amino acid restriction (SAR) on distinct cellular subpopulations within the tumor microenvironment through single-cell sequencing on colon carcinogenesis induced by azoxymethane/dextran sodium sulfate (AOM/DSS). Employing our custom-produced neutralizing antibodies against SLC7A11 to restrict cysteine intake, we examined the therapeutic implications and underlying molecular mechanisms by RNA-seq, Multiplexed immunofluorescence staining and Western blotting. Results: In the AOM/DSS model, SAR led to a slight reduction in mouse body weight compared to the normal diet. However, this dietary restriction significantly enhanced the survival rate of mice and effectively suppressed tumor growth. The single-cell sequencing results indicated a significant increase in the proportion of T cells within the tumor microenvironment in response to SAR. This suggested that restricting the intake of sulfur-containing amino acids may be a promising anti-tumor strategy. Previous studies have indicated high expression of the SLC7A11, a cysteine transporter protein, in various cancers, including colorectal cancer. To further investigate the role of cysteine in colorectal cancer, we developed the neutralizing antibody 1A4 against SLC7A11. Our results indicated that 1A4 markedly inhibited colorectal cancer development in mice. As known, 85% of colorectal cancers are microsatellite stable, which hardly benefit from treatment with immune checkpoint inhibitors (ICIs). Compared to anti-PD-1 and anti-CTLA4, 1A4 significantly inhibited the growth of CT26 in vivo. Multiplexed immunofluorescence staining results demonstrated that 1A4 significantly increased the proportion of CD8+ T cells in colorectal cancer tissues. Conclusion: Limiting cysteine intake significantly inhibits the growth of colorectal cancer, increases the proportion of CD8+ T cells, and enhances anti-tumor immunity. Citation Format: Jichang Li, Kang Xia, Zeruo Yang, Xiaoxue Pan, Xiaojing Yang, Pengyuan Wang, Shanwen Chen. Cysteine restriction enhances antitumor immunity in colorectal 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 LB349.

Characterization of humoral immune responses against SARS-CoV-2 accessory proteins in infected patients and mouse model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, encodes several accessory proteins that have been shown to play crucial roles in regulating the innate immune response. However, their expressions in infected cells and immunogenicity in infected humans and mice are still not fully understood. This study utilized various techniques such as luciferase immunoprecipitation system (LIPS), immunofluorescence ​assay (IFA), and western ​blot (WB) to detect accessory protein-specific antibodies in sera of COVID-19 patients. Specific antibodies to proteins 3a, 3b, 7b, 8 and 9c can be detected by LIPS, but only protein 3a antibody was detected by IFA or WB. Antibodies against proteins 3a and 7b were only detected in ICU patients, which may serve as a marker for predicting disease progression. Further, we investigated the expression of accessory proteins in SARS-CoV-2-infected cells and identified the expressions of proteins 3a, 6, 7a, 8, and 9b. We also analyzed their ability to induce antibodies in immunized mice and found that only proteins 3a, 6, 7a, 8, 9b and 9c were able to induce measurable antibody productions, but these antibodies lacked neutralizing activities and did not protect mice from SARS-CoV-2 infection. Our findings validate the expression of SARS-CoV-2 accessory proteins and elucidate their humoral immune response, providing a basis for protein detection assays and their role in pathogenesis.

Augmented mannose-binding lectin levels in primary membranous nephropathy: A pilot study.

There is evidence to suggest that M-type phospholipase A2 (PLA2R) antibodies activate the mannose-binding lectin (MBL) cascade, resulting in glomerular damage and proteinuria in patients with primary membranous nephropathy (PMN). Furthermore, there are few reports indicating that aberrant MBL activation is associated with endothelial dysfunction and accelerated atherosclerosis. While PMN is a common cause of adult nephrotic syndrome, and patients are at increased risk of cardiovascular disease (CVD), there is a lack of research that explores the factors that contribute to this condition. This study aims to determine the MBL levels in PMN and their relation to the clinical activity and endothelial dysfunction in PMN. The MBL levels of 22 biopsy-confirmed PMN patients were assessed at baseline and after 6 months of immunosuppressive therapy. In order to evaluate endothelial dysfunction in PMN patients, flow-mediated vasodilation (FMD) was measured at baseline and after treatment. A total of 22 healthy controls were included in this study to measure MBL levels and FMD. A significant difference was observed between MBL levels in PMN patients and healthy controls (p < .01). MBL levels decreased significantly after immunosuppressive therapy (p = .04). The baseline MBL levels and FMD levels exhibited a strong correlation (Spearman correlation coefficient [ρ] = 0.51: p = .01). In conclusion, the study signals the activation of the MBL cascade and its association with endothelial dysfunction in PMN patients.

Establishing RTS,S/AS01 as a benchmark for comparison to next-generation malaria vaccines in a mouse model

New strategies are needed to reduce the incidence of malaria, and promising approaches include vaccines targeting the circumsporozoite protein (CSP). To improve upon the malaria vaccine, RTS,S/AS01, it is essential to standardize preclinical assays to measure the potency of next-generation vaccines against this benchmark. We focus on RTS,S/AS01-induced antibody responses and functional activity in conjunction with robust statistical analyses. Transgenic Plasmodium berghei sporozoites containing full-length P. falciparum CSP (tgPb-PfCSP) allow two assessments of efficacy: quantitative reduction in liver infection following intravenous challenge, and sterile protection from mosquito bite challenge. Two or three doses of RTS,S/AS01 were given intramuscularly at 3-week intervals, with challenge 2-weeks after the last vaccination. Minimal inter- and intra-assay variability indicates the reproducibility of the methods. Importantly, the range of this model is suitable for screening more potent vaccines. Levels of induced anti-CSP antibody 2A10 equivalency were also associated with activity: 105 μg/mL (95% CI: 68.8, 141) reduced liver infection by 50%, whereas 285 μg/mL (95% CI: 166, 404) is required for 50% sterile protection from mosquito bite challenge. Additionally, the liver burden model was able to differentiate between protected and non-protected human plasma samples from a controlled human malaria infection study, supporting these models’ relevance and predictive capability. Comparison in animal models of CSP-based vaccine candidates to RTS,S/AS01 is now possible under well controlled conditions. Assessment of the quality of induced antibodies, likely a determinant of durability of protection in humans, should be possible using these methods.

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Annexin A2 antibodies in post-treatment Lyme disease.

Anti-annexin A2 (AA2) antibodies have been described in Lyme arthritis and erythema migrans, although they have not been described in post-treatment Lyme disease (PTLD). Determine whether anti-AA2 antibodies are present among patients with PTLD and determine the clinical relevance of these antibodies. Anti-AA2 levels were tested serially in a longitudinal cohort of 44 patients with acute Lyme disease, 22 with a return to health (EM RTH), and 22 with PTLD. Anti-AA2 antibodies were also assessed in a cross-sectional group of 281 patients with PTLD. Anti-AA2 antibodies were highest after antimicrobial therapy in both the EM RTH and PTLD cohorts. By 6 months, there was no difference between EM RTH and healthy controls. Anti-AA2 antibodies were higher in the cross-sectional PTLD group (79.69 versus 48.22 units, p < 0.0001), though with no difference in total symptom burden. Anti-AA2 persists in PTLD, though did not identify a clinical phenotype.

Molecular engineering of a theranostic molecule that detects Aβ plaques, inhibits Iowa and Dutch mutation Aβ self-aggregation and promotes lysosomal biogenesis for Alzheimer's disease.

Extracellular clustering of amyloid-β (Aβ) and an impaired autophagy lysosomal pathway (ALP) are the hallmark features in the early stages of incurable Alzheimer's disease (AD). There is a pressing need to find or develop new small molecules for diagnostics and therapeutics for the early stages of AD. Herein, we report a small molecule, namely F-SLCOOH, which can bind and detect Aβ1-42, Iowa mutation Aβ, Dutch mutation Aβ fibrils and oligomers exhibiting enhanced emission with high affinity. Importantly, F-SLCOOH can readily pass through the blood-brain barrier and shows highly selective binding toward the extracellular Aβ aggregates in real-time in live animal imaging of a 5XFAD mice model. In addition, a high concentration of F-SLCOOH in both brain and plasma of wildtype mice after intraperitoneal administration was found. The ex vivo confocal imaging of hippocampal brain slices indicated excellent colocalization of F-SLCOOH with Aβ positive NU1, 4G8, 6E10 A11 antibodies and THS staining dye, affirming its excellent Aβ specificity and targetability. The molecular docking studies have provided insight into the unique and specific binding of F-SLCOOH with various Aβ species. Importantly, F-SLCOOH exhibits remarkable anti-fibrillation properties against toxic Aβ aggregate formation of Aβ1-42, Iowa mutation Aβ, and Dutch mutation Aβ. F-SLCOOH treatment also exerts high neuroprotective functions and promotes autophagy lysosomal biogenesis in neuronal AD cell models. In summary, the present results suggest that F-SLCOOH is a highly promising theranostic agent for diagnosis and therapeutics of AD.

Hybrid human-porcine factor VIII proteins partially escape the inhibitory effects of anti-factor VIII inhibitor alloantibodies having A2 or C2 domain specificity.

Porcine factor (pF)VIII has low cross-reactivity with anti-human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital haemophilia A patients with inhibitor (PwHA-I) are in progress. Most polyclonal anti-hFVIII inhibitors recognize its A2 and/or C2 domain(s), and recombinant human-porcine hybrid (hp)FVIII proteins may escape neutralization by these inhibitors. To evaluate the ability of hpFVIII to limit the anti-FVIII activity of inhibitor alloantibodies. Three hybrid proteins were created by substituting the hFVIII A2, C2 domain or both with the corresponding domains of pFVIII [termed hp(A2), hp(C2) and hp(A2/C2), respectively]. The reactivity of these hybrids was assessed by one-stage clotting assays (OSA), thrombin generation assays (TGA) and rotational thromboelastometry (ROTEM) by adding them to FVIII-deficient samples. OSA demonstrated that the hybrid proteins avoided neutralization by anti-FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor-antibodies (polyAb) from PwHA-I. In TGA, thrombin generation with hp(A2) and hp(A2/C2) was not attenuated in the presence of patient IgG recognizing anti-A2 domain. In contrast, that with hFVIII and hp(C2) was suppressed by this IgG to levels equivalent to those of FVIII-deficient plasma. With anti-A2/C2 polyAb, the activity of hp(A2/C2) was unaffected. ROTEM demonstrated that the addition of hp(A2) or hp(A2/C2) to anti-A2 polyAb shortened clot times/clot formation times, whilst hFVIII or hp(C2) were ineffective. Similarly with anti-A2/C2 polyAb, hp(A2/C2) restored coagulation potential to a greater extent than hp(A2) and hp(C2). Hybrid FVIII proteins containing porcine FVIII A2 and/or C2 domain(s) could support effective therapy in PwHA-I by avoiding neutralization.

A population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes.

Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.

Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography.

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈1014 vp mL-1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%).

Regulation of von Willebrand factor by ADAMTS13 ameliorates lipopolysaccharide-induced lung injury in mice.

The relationship between von Willebrand factor (VWF) and inflammation has attracted considerable attention in recent years. VWF, which is stored in the Weibel-Palade bodies (WPBs) of endothelial cells (ECs), is released from WPBs in response to inflammatory stimuli and is thought to contribute to inflammation by promoting leukocyte extravasation. In this study, lung injury model mice were produced by intratracheal injection with lipopolysaccharides. The severity of lung inflammation was evaluated in mice with different genotypes (wild-type, Vwf-/-, Adamts13-/-) and mice treated with drugs that inhibit VWF function. Lung inflammation was significantly ameliorated in Vwf-/- mice compared with wild-type mice. Furthermore, inflammation was significantly suppressed in wild-type mice treated with anti-VWF A1 antibody or recombinant human ADAMTS13 compared with the untreated control group. The underlying mechanism appears to be an increased VWF/ADAMTS13 ratio at the site of inflammation and the interaction between blood cell components, such as leukocytes and platelets, and the VWF A1 domain, which promotes leukocyte infiltration into the lung. This study suggested that ADAMTS13 protein and other VWF-targeting agents may be a novel therapeutic option for treatment of pulmonary inflammatory diseases.

Mechanisms of Antiproliferative Action of Streptococcal Arginine Deiminase Against Jurkat Lymphoblastic Leukemia Cells

Arginine deprivation strategy is considered as a promising trend in cancer therapy. The aim of the study was to investigate the influence of streptococcal arginine deiminase on Jurkat lymphoblastic leukemia cells. The effects of the supernatants of the destroyed streptococci of the original strain expressing arginine deiminase and its isogenic mutant with the inactivated arcA gene were compared. Cell proliferation was evaluated in an MTT-test. The remaining parameters were examined using flow cytometry. The cell cy-cle changes were studied using DAPI dye and anti-cyclin A2 antibodies. The autophagy intensity was assessed using the LysoTracker ™ Green DND-26 reagent. To investigate cell viability DAPI staining was performed. Streptococcal arginine deiminase suppressed proliferative activity Jurkat lymphoblastic leukemia cells, increased the proportion of cells in the G0/G1 phases, reduced S/G2 phases proportion of cells and enhanced autophagy, without declaing viability. Arginine supplementation leveled the effects of the enzyme. The obtained results open up the possibility of using arginine-hydrolyzing activity of the streptococcal enzyme for combined therapy of oncological diseases.

Genomic Surveillance of Rabies Virus in Georgian Canines.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Testing of a novel liposomal anti-Pseudomonasvaccine based on outer-membrane protein F (OprF) of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa is an opportunistic pathogen associated with an increased risk of morbidity and mortality among cystic fibrosis patients and immunocompromised individuals. Current antibiotics in the market are not successful against P. aeruginosa infections due to the pathogen’s ability to cause antibiotic resistance. Therefore, our prime objective is to formulate a liposomal-based anti-Pseudomonas vaccine that can produce long-lasting IgG antibodies and T cell memory. The vaccine we introduced accommodates several B cell epitopes of the outer-membrane protein F (OprF) of P. aeruginosa. The recombinant OprF antigen was conjugated with a Toll-like receptor 2/1 (TH1/TH2) agonist, Pam 3CysSK 4, and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Our results show that the resulting vaccine conjugate can successfully produce high anti-OprF antibody titers in mice. These antibodies can also recognize intact P. aeruginosa bacteria and kill them with the support of rabbit complement and murine macrophage RAW264.7 cells. To further enhance the vaccine efficacy, the liposome was optimized with 2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycerol-3-phospho-(1′-rac-glycerol) (DMPG), Chol, Pam 3CysSK 4and Quillaja Saponaria-derived saponin adjuvant QS21. The mice immunized with the modified vaccine elicit higher antibody titers and increased IgG 2a antibodies. Overall, our liposomal vaccines were found to be highly effective at generating a balanced and robust TH1/TH2 response. Supported by grants from NIH (R01AI148570)

GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

Patients with cancer undergoing chemotherapy experience cachexia with anorexia, body weight loss, and the depletion of skeletal muscles and adipose tissues. Effective treatment strategies for chemotherapy-induced cachexia are scarce. The growth differentiation factor 15 (GDF15)/GDNF family receptor alpha-like (GFRAL)/rearranged during transfection (RET) axis is a critical signalling pathway in chemotherapy-induced cachexia. In this study, we developed a fully human GFRAL antagonist antibody and investigated whether it inhibits the GDF15/GFRAL/RET axis, thereby alleviating chemotherapy-induced cachexia in tumour-bearing mice. Anti-GFRAL antibodies were selected via biopanning, using a human combinatorial antibody phage library. The potent GFRAL antagonist antibody A11 was selected via a reporter cell assay and its inhibitory activity of GDF15-induced signalling was evaluated using western blotting. To investigate the in vivo function of A11, a tumour-bearing mouse model was established by inoculating 8-week-old male C57BL/6 mice with B16F10 cells (n=10-16 mice per group). A11 was administered subcutaneously (10mg/kg) 1day before intraperitoneal treatment with cisplatin (10mg/kg). Animals were assessed for changes in food intake, body weight, and tumour volume. Plasma and key metabolic tissues such as skeletal muscles and adipose tissues were collected for protein and mRNA expression analysis. A11 reduced serum response element-luciferase reporter activity up to 74% (P<0.005) in a dose-dependent manner and blocked RET phosphorylation up to 87% (P=0.0593), AKT phosphorylation up to 28% (P=0.0593) and extracellular signal regulatory kinase phosphorylation up to 75% (P=0.0636). A11 inhibited the action of cisplatin-induced GDF15 on the brainstem and decreased GFRAL-positive neuron population expressing c-Fos in the area postrema and nucleus of the solitary tract by 62% in vivo (P<0.05). In a melanoma mouse model treated with cisplatin, A11 recovered anorexia by 21% (P<0.05) and tumour-free body weight loss by 13% (P<0.05). A11 significantly improved the cisplatin-induced loss of skeletal muscles (quadriceps: 21%, gastrocnemius: 9%, soleus: 13%, P<0.05) and adipose tissues (epididymal white adipose tissue: 37%, inguinal white adipose tissue: 51%, P<0.05). Our study suggests that GFRAL antagonist antibody may alleviate chemotherapy-induced cachexia, providing a novel therapeutic approach for patients with cancer experiencing chemotherapy-induced cachexia.

Abstract 2638: Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers

Abstract Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer. Moreover, PSMA is expressed in the neovasculature that supplies most non-prostatic solid tumors, including carcinomas of the lung, colon, breast, kidney, liver, and pancreas. ADCT-212 is an antibody-drug conjugate composed of the human IgG1 antibody 2A10 directed against human PSMA, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the pyrrolobenzodiazepine (PBD) dimer warhead SG2000 (drug-antibody ratio of ~1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-212 in human cancer cell lines and xenograft models and to determine its tolerability and pharmacokinetic (PK) in the rat. In vitro, ADCT-212 demonstrated potent cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. ADCT-212 was efficiently internalized by PSMA-expressing LNCaP cells. Trafficking to the lysosomes started as early as 30 minutes and peaked at 1-2 hours post treatment. In line with the mechanism of action of the PBD dimer, ADCT-212 produced DNA interstrand cross-links (ICLs) that peaked by 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of DNA ICLs formation for SG2000, the PBD dimer warhead alone, was observed immediately after 2-hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA ICLs. Moreover, ADCT-212 showed indirect bystander killing activity in PSMA-negative PC3 cells incubated with conditioned medium from ADCT-212-treated LNCaP cells. In vivo, ADCT-212 showed strong antitumor activity against CWR22Rv1 and LNCaP prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with heterogeneous PSMA expression, ADCT-212 achieved dose-dependent antitumor activity when administered as single dose at 2, 4 or 6 mg/kg, which resulted in increased survival compared to the control animals. In the LNCaP model, a single dose of ADCT-212 at 5 or 10 mg/kg resulted in strong and specific antitumor activity. Conversely, in the PSMA-negative PC3 xenograft model, ADCT-212 did not show anti-tumor activity compared to the controls, highlighting its target-mediated antitumor activity. ADCT-212 was tolerated as a single 20 mg/kg dose in male rats, with exposure data being indicative of a linear PK profile with a half-life of approximately 12 days. In conclusion, ADCT-212 demonstrated potent and specific in vitro and in vivo anti-tumor activity while it was stable and well tolerated in the rat, warranting further development of ADCT-212 into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Lolke de Haan, Ian Kirby, Narinder Janghra, Veronica Gil, Pedro Alves, Kristina Zaitseva, Meghann Kerr, Ben Leatherdale, Afroze Patel, Marie Thoelke, Shiran Huang, John A Hartley, Patrick H van Berkel. Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2638.