Specific Monoclonal Antibodies Research Articles

Abstract 1876: Development of therapeutic antibodies targeting the GLUT-1 transporter to restrict cancer cell growth

Abstract Background: Upregulated glucose metabolism is one of the strategies cancer cells use to fuel their abnormal cell growth and division. Targeting the cancer-specific glucose transporter GLUT-1 is a promising approach to restrict glucose uptake and challenge the metabolic needs of tumor cells. Antibodies, as opposed to small molecule inhibitors, are an attractive modality to therapeutically target complex muti-pass membrane transporters. Here we report the development of antibodies that very specifically block the function of only GLUT-1. Methods: Monoclonal antibodies against GLUT-1 were generated using virus-like particles. For immunization, chickens were used as they are evolutionarily distant from mammals and can produce antibodies with prolonged CDR3-s in VH, which could facilitate their binding to the limited extracellular region of GLUT-1. Antibody discovery was performed with HybriFree B cell cloning technology followed by functional screens with 2-deoxyglucose uptake interference or cell proliferation measurements. Results: The discovered antibodies specifically bind to GLUT-1 with low nanomolar EC50 values and do not target other glucose transporters. The lead candidate, ICO-33, inhibits glucose uptake and rewires the metabolism of GLUT-1-dependent cancer cells to rely on oxidative phosphorylation. This results in a significantly synergistic cancer proliferation inhibition with ICO-33 and OXPHOS inhibitor combinations in doses that do not inhibit proliferation as single agents. ICO-33 and OXPHOS inhibitor treatment is well-tolerated and demonstrates a drastic tumor growth inhibition in in vivo colorectal and pancreatic cancer models. Conclusions: We describe the discovery of highly specific monoclonal antibodies targeting GLUT-1. We further demonstrate that the restriction of the much-needed glucose uptake by ICO-33 makes cancer cells highly susceptible to OXPHOS inhibitor co-therapy both in vitro and in vivo, validating the antibody as a promising candidate for clinical development. Citation Format: Siret Tahk, Kai Virumae, Korneelia Anton, Paule Hermet, Robin Pau, Mario Plaas, Maiken Abel, Denis Belitškin, Luciano Galdieri, Steven Garner, Jillian Krings, Kaleb Collver, Emily Schultz, Kaitlyne Powers, Alastair King, Francisca Neethling, Anu Planken, Mart Ustav, Joan Terya, Andres Mannik, Mart Ustav. Development of therapeutic antibodies targeting the GLUT-1 transporter to restrict cancer cell growth [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 1876.

Abstract 629: Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells

Abstract Introduction: In patients with acute myeloid leukemia (AML), CD123 has been shown to have high expression on blast cells and leukemic stem cells (LSC) compared with normal hematopoietic stem cells (HSCs) and other more mature CD34+ subsets (Zhou J, World J Stem Cells 2014). LSCs are inherently resistant to standard of care chemotherapeutics and LSC persistence after chemotherapy is associated with disease relapse. VIP943 is an ADC consisting of an anti-CD123 antibody, a unique linker cleaved intracellularly by legumain which is required for release, and activation of a novel kinesin spindle protein inhibitor (KSPi) payload that accumulates inside the cell. Herein, we evaluated the potential of VIP943 to target LSC and progenitor cells. Methods: Cytotoxicity assay: Bone marrow aspirates (BMA) and peripheral blood from previously untreated patients with primary or transformed from myelodysplastic syndrome AML were used in this experiment. Cells were treated with VIP943 (66 nM or 330 nM). After the end of the 48 h or 72 h incubation, the CD123+ cells and the CD34+CD38- LSC were detected by flow cytometry. Characterization of progenitor cell population: Fresh BM samples from healthy volunteers (HVBM) were stained with specific monoclonal antibodies to identify progenitor cell populations. Depletion assay: The effect of VIP943 on HVBM was evaluated by measuring the depletion of CD34+ progenitor cell populations in comparison to the anti-CD33-ADC, gemtuzumab ozogamicin (gem-oz). At the end of the incubation time, red blood cells were lysed; the remaining cells were stained with a cocktail of antibodies to discriminate between progenitor cells and analyzed by flow cytometry. Results: Treatment of patient-derived CD123+ leukemic blasts with VIP943 resulted in a 50% reduction after 48 h and up to 80% at 72 h. A reduction of >70% of CD123+CD34+CD38- LSCs derived from BM aspirates of untreated patients with AML was achieved after incubation with VIP943 up to 72h. In addition, HVBM samples derived from five healthy volunteers were treated with VIP943 or gem-oz in a depletion assay, where gem-oz was toxic to CD34+ cells with an EC50 of 0.16 µM in contrast to VIP943 with an EC50 value of 8.83 µM. Conclusions: In this in vitro analysis of primary samples from patients with AML, activity of VIP943 is observed in AML patient-derived CD123+ blasts as well as chemoresistant LSCs. At anticipated pharmacologic levels, VIP943 shows no adverse effects on HSCs unlike gem-oz suggesting an improved therapeutic index. In the ongoing first-in-human dose-escalation study in subjects with advanced CD123+ hematologic malignancies, VIP943 demonstrates a promising safety profile (NCT06034275). Citation Format: Beatrix Stelte-Ludwig, Tibor Schomber, Melanie M. Frigault, Joseph Birkett, Amy J. Johnson, Anne-Sophie Rebstock, Sebastian Ludwig, Raquel Izumi, Ahmed Hamdy. Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem 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 629.

Abstract 1900: TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer

Abstract Cadherin 17 (CDH17) is a cell-to-cell adhesion protein that is a member of the cadherin superfamily. In normal tissues, expression of this single pass transmembrane protein is restricted to the lateral surfaces of intestinal and pancreatic ductal epithelial cells. However, in cancer, CDH17 is frequently overexpressed in tumors of the colon, stomach, and pancreas. The selective expression of this cell surface protein in these hard-to-treat cancer makes it an attractive target for the development of antibody-based therapeutics. Here, we describe the preclinical development of TORL-3-600, a novel CDH17-targeting antibody drug conjugate (ADC). CDH17 specific monoclonal antibodies were generated using traditional hybridoma technology and splenocytes isolated from mice immunized with cocktails of NIH3T3 cells overexpressing full length hCHD17 plus purified mammalian expressed hCDH17 extracellular domain protein (aa 23-787). Selective mAb binding was confirmed by flow cytometry and mAb internalization rate was assessed by immunofluorescence (IF). TORL-3-600 was generated from a fully humanized CDH17 mAb by MMAE conjugation with a cleavable linker. Cell membrane staining of CDH17 in patient tumor microarrays (TMAs), cell line (CDX) and patient derived xenografts (PDX) was evaluated by IHC assay. Selective binding of the TORL-3-600 to CDH17 was confirmed in human cancer cell lines and cells engineered to overexpress CDH17. Binding of TORL-3-600 to cell surface CDH17 induced Internalization and translocation to the lysosome of the protein-ADC complex for release of the MMAE payload. Treatment with TORL-3-600 induced significant regressions and tumor growth inhibition (TGI) in four CDH17-positive (CDH17+) human colorectal cancer (CRC) CDXs (103.6 - 140.4% TGI) and three CDH17+ CRC PDXs (63.3 - 102.2% TGI). Responses in these models were sustained for up to nine-weeks following cessation of treatment. Sustained inhibition of xenograft tumor progression was also observed in a cell line model of CDH17+ pancreatic cancer (85.8% TGI). In contrast, greatly reduced responses (37.3 - 58.2% TGI) to TORL-3-600 were observed in the CDH17- human colon cancer CDX and PDX models. Each of the doses tested in this study were well tolerated in mice with no dose-limiting toxicities observed. Analyses of large human patient TMAs by CDH17 IHC assay demonstrated detectable expression of CDH17 in 90.1% (173/193) of CRC, 51.8% (86/166) of gastric and 20.4% (69/331) of pancreatic cancers. These numbers suggest that an ADC directed against CDH17 could provide benefit to a significant number of patients diagnosed with these cancers. These data support the clinical development of TORL-3-600 for the treatment of CDH17+ cancers. TORL-3-600 has completed IND enabling toxicity studies with acceptable PK and toxicity profiles and is now in phase 1 clinical testing (NCT05948826). Citation Format: Neil A. O'Brien, Martina SJ McDermott, Jun Zhang, Ming Lu, Ke Wei Gong, Benjamin Hoffstrom, Wei Ping Jia, Tong Luo, Athena M. Madrid, Min Liang, John A. Glaspy, Dennis J. Slamon. TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer [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 1900.

Regulation of PD-L1 Expression by YY1 in Cancer: Therapeutic Efficacy of Targeting YY1.

During the last decade, we have witnessed several milestones in the treatment of various resistant cancers including immunotherapeutic strategies that have proven to be superior to conventional treatment options, such as chemotherapy and radiation. This approach utilizes the host's immune response, which is triggered by cancer cells expressing tumor-associated antigens or neoantigens. The responsive immune cytotoxic CD8+ T cells specifically target and kill tumor cells, leading to tumor regression and prolongation of survival in some cancers; however, some cancers may exhibit resistance due to the inactivation of anti-tumor CD8+ T cells. One mechanism by which the anti-tumor CD8+ T cells become dysfunctional is through the activation of the inhibitory receptor programmed death-1 (PD-1) by the corresponding tumor cells (or other cells in the tumor microenvironment (TME)) that express the programmed death ligand-1 (PD-L1). Hence, blocking the PD-1/PD-L1 interaction via specific monoclonal antibodies (mAbs) restores the CD8+ T cells' functions, leading to tumor regression. Accordingly, the Food and Drug Administration (FDA) has approved several checkpoint antibodies which act as immune checkpoint inhibitors. Their clinical use in various resistant cancers, such as metastatic melanoma and non-small-cell lung cancer (NSCLC), has shown significant clinical responses. We have investigated an alternative approach to prevent the expression of PD-L1 on tumor cells, through targeting the oncogenic transcription factor Yin Yang 1 (YY1), a known factor overexpressed in many cancers. We report the regulation of PD-L1 by YY1 at the transcriptional, post-transcriptional, and post-translational levels, resulting in the restoration of CD8+ T cells' anti-tumor functions. We have performed bioinformatic analyses to further explore the relationship between both YY1 and PD-L1 in cancer and to corroborate these findings. In addition to its regulation of PD-L1, YY1 has several other anti-cancer activities, such as the regulation of proliferation and cell viability, invasion, epithelial-mesenchymal transition (EMT), metastasis, and chemo-immuno-resistance. Thus, targeting YY1 will have a multitude of anti-tumor activities resulting in a significant obliteration of cancer oncogenic activities. Various strategies are proposed to selectively target YY1 in human cancers and present a promising novel therapeutic approach for treating unresponsive cancer phenotypes. These findings underscore the distinct regulatory roles of YY1 and PD-L1 (CD274) in cancer progression and therapeutic response.

Quantitative immunochromatographic assay for rapid and cost-effective on-site detection of benzo[a]pyrene in oilfield chemicals

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42–300 mg/kg, and limit of detection was 0.23–1.07 mg/kg. The recovery rate was 88–106% with a coefficient of variation of 1.46–6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection.

Impact of Methylthioxylose Substituents on the Biological Activities of Lipomannan and Lipoarabinomannan in Mycobacterium tuberculosis.

Two lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), play various, albeit incompletely defined, roles in the interactions of mycobacteria with the host. Growing evidence points to the modification of LM and LAM with discrete covalent substituents as a strategy used by these bacteria to modulate their biological activities. One such substituent, originally identified in Mycobacterium tuberculosis (Mtb), is a 5-methylthio-d-xylose (MTX) sugar, which accounts for the antioxidative properties of LAM. The widespread distribution of this motif across Mtb isolates from several epidemiologically important lineages have stimulated interest in MTX-modified LAM as a biomarker of tuberculosis infection. Yet, several lines of evidence indicate that MTX may not be restricted to Mtb and that this motif may substitute more acceptors than originally thought. Using a highly specific monoclonal antibody to the MTX capping motif of Mtb LAM, we here show that MTX motifs not only substitute the mannoside caps of LAM but also the mannan core of LM in Mtb. MTX substituents were also found on the LM and LAM of pathogenic, slow-growing nontuberculous mycobacteria. The presence of MTX substituents on the LM and LAM from Mtb enhances the pro-apoptotic properties of both lipoglycans on LPS-stimulated THP-1 macrophages. A comparison of the cytokines and chemokines produced by resting and LPS-activated THP-1 cells upon exposure to MTX-proficient versus MTX-deficient LM further indicates that MTX substituents confer anti-inflammatory properties upon LM. These findings add to our understanding of the glycan-based strategies employed by slow-growing pathogenic mycobacteria to alter the host immune response to infection.

Analytical Insights into Protein-Alum Interactions and Their Impact on Conformational Epitope.

Several alum-adjuvanted vaccines have been licensed in the past 40 years. Despite its extensive and continuous use, the immune mechanism of action of alum adjuvants is not yet completely understood. Many different variables during the formulation process have been assessed as critical for alum-adjuvanted vaccines, although most of them are still not yet fully understood. The absence of a clear understanding of all the possible variables regulating the mechanism of action and the behavior that alum adjuvant imposes on the protein antigen may also be related to analytical challenges. For this reason, there is an urgent need for a fast and simple tool that is possible without a preliminary sample manipulation and is able to control the amount and the degree of antigen adsorption levels and their consistency across different production processes. This work attempts to develop new analytical tools with the aim of directly quantifying and assessing both the content and/or the purity of formulated alum-adsorbed antigens, without any preliminary sample manipulation (e.g., antigen desorption) being reported. In addition, the different confirmation/behavior in terms of the response to specific monoclonal antibodies in the presence of different ratios of alum-OH adsorbent antigens have been investigated. As a proxy to develop new analytical tools, three recombinant protein adsorbed models were used as follows: Neisseria adhesin A (NadA), Neisserial Heparin Binding Antigen (NHBA), and factor H binding protein (fHbp) as antigens, as well as aluminum hydroxide (AH) as an adjuvant system. The selection of the adjuvanted system model was dictated due to the substantial quantity of the literature regarding the protein structure and immunological activities, meaning that they are well characterized, including their adhesion rate to alum. In conclusion, three different analytical tools were explored to quantify, detect, and study the behavior of antigens in the presence of the alum adjuvant.

Anti-inflammatory effect of gallic acid-conjugated chitooligosaccharides in lipopolysaccharide-stimulated RAW 264.7 macrophages

The aim of the present study is to investigate the anti-inflammatory effect of gallic acid grafted onto COS chains (GA-COS), focusing on the reduction of nitric oxide production, downregulation of inflammatory signals such as inducible nitric oxide synthase (iNOS), gene expression of cytokines such as TNF-α, IL-1β, IL-6, and nuclear factor kappa B (NF-κB) signalling, including the p50 and p65 subunits. The anti-inflammatory effect is mediated through the reduction of nitric oxide production and downregulation of inflammatory proteins such as inducible nitric oxide synthase (iNOS), gene expression of cytokines like TNF-α, IL-1β, IL-6, and nuclear factor kappa B (NF-κB) signalling, including the p50 and p65 subunits. Target proteins were identified by western blot analysis with specific monoclonal antibodies. The levels of gene expression were determined by the RT-PCR method. The results demonstrate that GA-COS effectively reduces nitric oxide generation and downregulates iNOS protein and cytokine expression and NF-κB signalling in lipopolysaccharide (LPS) -induced RAW 264.7 cells. GA-COS exhibits significantly enhanced anti-inflammatory activity compared to the free chitooligosaccharide chain. This study lays the groundwork for future research to demonstrate that GA-COS holds significant potential as a novel compound for the prevention of inflammatory diseases.

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay (ic-ELISA) for Copper ion (Cu2+) in Raw Meat Products

Adding an appropriate amount of copper to feed can promote the growth and development of livestock; however, a large amount of heavy metal copper can accumulate in livestock through the enrichment effect, which poses a serious threat to human health. Traditional Cu2+ detection relies heavily on complex and expensive instruments, such as inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS); thus, convenient and simple rapid detection technologies are urgently needed. In this paper, synthesized copper antigens were used to immunize mice and highly specific anticopper monoclonal antibodies were obtained, which were verified to exhibit high affinity and specificity. Based on the above antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid detection of copper content in pork. The standard inhibition curve of the method was obtained by antigen–antibody working concentration screening, in which the half inhibitory concentration (IC50) was 11.888 ng/mL, the limit of detection (LOD) was 0.841 ng/mL and the correlation coefficient R2 of the curve was 0.998. In the additive recovery experiment, the recovery rate ranged from 90% to 110%, and the coefficient of variation (CV) was less than 10%, indicating that the method achieved high accuracy and precision. Finally, the results of ic-ELISA combined with Bland-Altman analysis showed a high correlation with ICP-MS, and the correlation coefficient (R2) reached 0.990 when the copper concentration was less than 200 ng/mL. Thus, the ic-ELISA method exhibits high reliability.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

A comprehensive Exdia TRF-LFIA for simultaneous quantification of GFAP and NT-proBNP in distinguishing ischemic and hemorrhagic stroke

The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available.We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used.To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS.Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

Terahertz Lattice enhanced Quasi-Anapole Immunosensor assisted by protein antibody and AuNPs

Terahertz (THz) metasurface immunosensor has the characteristics of label-free detection, good biocompatibility and high specificity because the antibody-modified of biomarkers has the ability to specifically recognize corresponding biomarkers and increase the detection sensitivity to the concentration of the recognized antigen. The limit of detection (LoD) of THz metasurface immunosensor is highly dependent on resonant field enhancement of metasurface. The anapole mode shows a radiationless state with nontrivial oscillating current configuration. To observe such mode in farfield, in this work, the concept of quasi-anapole (QA) mode is presented which is induced when the electric and toroidal dipoles with identical intensity radiate in antiphase on the metasurface with the unit cell of two split ring resonators (SRRs) on both sides of the central cut-wire. In addition, by coupling the first-order lattice mode to the QA mode, lattice-enhanced QA mode (LQA) was investigated and observed to further enhance field confinement and decrease radiative loss. As a practical application, immunosensor was designed and fabricated by functionalized gold nanoparticles conjugated with the specific monoclonal antibody onto the LQA metasurface to detect the cell concentration of SARS-CoV-2 spike protein. The experimental results indicate that the response of LQA immunosensor depends heavily on the concentration of the Anti-S modified on its surface. When the concentration of Anti-S is 50 pg/ml, the LoD reaches to 3 pg/ml (39 fmol). Such THz LQA immunosensor also shows good specific identification and validates its effectiveness in biomedical sensing.

Analysis of retreatment with monoclonal antibodies in chronic/episodic migraine: Real world data

ObjectiveTo analyze the response to retreatment in patients with chronic/episodic migraine who discontinued therapy with erenumab/fremanezumab after 1 year of treatment. MethodsObservational, retrospective, single-center, multidisciplinary study in patients with chronic/episodic migraine who received therapy with erenumab/fremanezumab for at least 1 year and discontinued it after achieving an adequate response (optimization). The evaluation of the response after retreatment included the following variables: DMM, MIDAS, and HIT-6 scales at the beginning of retreatment and 3 months later. The response was evaluated in different subgroups (episodic/chronic, erenumab/fremanezumab, and time until retreatment). Results48 patients were included. 70.8% (n=34) required retreatment with mAb, with a median of 3.9 (2.9–6.4) months until reintroduction. Clinical response after retreatment was achieved in 67.6% (n=23) of patients. No statistically significant differences were found in the analyzed subgroups. ConclusionInterruption of treatment with erenumab/fremanezumab for chronic/episodic migraine produces a clinical worsening of the disease requiring retreatment in most cases, approximately after 4 months. Two out of three patients respond positively after restarting monoclonal therapy. This response does not appear to be related to the type of migraine, the specific monoclonal antibody prescribed, or the time to retreatment.

PCSK9 inhibition protects mice from food allergy

The Proprotein Convertase Subtilisin Kexin of type 9 (PCSK9) has been identified in 2003 as the third gene involved in familial hypercholesterolemia. PCSK9 binds to the membrane low-density lipoprotein receptor (LDLR) and promotes its cellular internalization and lysosomal degradation. Beyond this canonical role, PCSK9 was recently described to be involved in several immune responses. However, to date, the contribution of PCSK9 in food allergy remains unknown. Here, we showed that Pcsk9 deficiency or pharmacological inhibition of circulating PCSK9 with a specific monoclonal antibody (m-Ab) protected mice against symptoms of gliadin-induced-food allergy, such as increased intestinal transit time and ear oedema. Furthermore, specific PCSK9 inhibition during the elicitation steps of allergic process was sufficient to ensure anti-allergic effects in mice. Interestingly, the protective effect of PCSK9 inhibition against food allergy symptoms was independent of the LDLR as PCSK9 inhibitors remained effective in Ldlr deficient mice. In vitro, we showed that recombinant gain of function PCSK9 (PCSK9 D374Y) increased the percentage of mature bone marrow derived dendritic cells (BMDCs), promoted naïve T cell proliferation and potentiated the gliadin induced basophils degranulation. Altogether, our data demonstrate that PCSK9 inhibition is protective against gliadin induced food allergy in a LDLR-independent manner.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. HuAR9.6 bound a discontinuous, SEA domain epitope with an overall affinity of 88 nmol/L. Binding affinity depended on the specific SEA domain(s) present, and glycosylation modestly enhanced affinity driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDO). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs, and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

Development of an ostrich-derived single-chain variable fragment (scFv) against PTPRN extracellular domain

In type 1 diabetes, the immune system destroys pancreatic beta cells in an autoimmune condition. To overcome this disease, a specific monoclonal antibody that binds to pancreatic beta cells could be used for targeted immunotherapy. Protein tyrosine phosphatase receptor N (PTPRN) is one of the important surface antigen candidates. Due to its high sequence homology among mammals, so far, no single-chain monoclonal antibody has been produced against this receptor. In this study, we developed a novel single-chain variable fragment (scFv) against the PTPRN extracellular domain. To this aim, ostrich species was used as a host is far phylogenetically birds from mammals to construct a phage display library for the first time. An ostrich-derived scfv phage display library was prepared and biopanning steps were done to enrich and screen for isolating the best anti-PTPRN binders. An scFv with appropriate affinity and specificity to the PTPRN extracellular domain was selected and characterized by ELISA, western blotting, and flow cytometry. The anti-PTPRN scFv developed in this study could be introduced as an effective tool that can pave the way for the creation of antibody-based targeting systems in cooperation with the detection and therapy of type I diabetes.

Development of a colloidal gold immunochromatographic strip for rapid detection of ribavirin in pig urine

ABSTRACT A specific and sensitive monoclonal antibody (mAb) for ribavirin (RBV) was generated by immunizing BALB/c mice with RBV – bovine serum albumin. Using the mAb, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic (CGI) test strip were developed for detecting RBV in pig urine. Under optimum assay conditions, IC50 was 0.08 ng/mL in 0.05 M phosphate-buffered saline and the recovery rate in real-world samples ranged between 77.3% and 99%. The cut-off limit of the CGI test strip for RBV was 5 ng/mL in pig urine. The cross-reactivity rate of the test strip to the tested antiviral drugs including acyclovir and moroxydine hydrochloride and the metabolites 1,2,4-triazole-3-carboxamide and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxylic acid was below 0.1%. The results indicate that ic-ELISA and the CGI test strip have high sensitivity and specificity and that these methods are suitable for the rapid detection of RBV in pig urine.

Characterization of the monoclonal antibody and the immunodominant B-cell epitope of African swine fever virus pA104R by using mouse model.

The African swine fever virus (ASFV) structural protein pA104R is the only histone-like protein encoded by eukaryotic viruses. pA104R is an essential DNA-binding protein required for DNA replication and genome packaging of ASFV, which are vital for pathogen survival and proliferation. pA104R is an important target molecule for diagnosing, treating, and immune prevention of ASFV. This study characterized monoclonal antibodies (mAbs) against pA104R and found them to recognize natural pA104R in ASFV strains with different genotypes, showing high conservation. Confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Furthermore, the pA104R protein functions through the polar interactions between the binding amino acid sites; however, these interactions may be blocked by the recognition of generated mAbs. Characterizing the immunodominant B-cell epitope of the ASFV critical proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and vaccine candidate targets.IMPORTANCEAfrican swine fever (ASF) is a highly pathogenic, lethal, and contagious viral disease affecting domestic pigs and wild boars. As no effective vaccine or other treatments have been developed, the control of African swine fever virus (ASFV) relies heavily on virus detection and diagnosis. A potential serological target is the structural protein pA104R. However, the molecular basis of pA104R antigenicity remains unclear, and a specific monoclonal antibody (mAb) against this protein is still unavailable. In this study, mAbs against pA104R were characterized and found to recognize natural pA104R in ASFV strains with different genotypes. In addition, confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Characteristics of the immunodominant B-cell epitope of ASFV proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and identifying vaccine candidate targets.

Highly specific and super-sensitive Dot-ELISA and colloidal gold immunochromatographic strip for detecting Xanthomonas oryzae pv. oryzicola of rice bacterial leaf streak

Rice bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a worldwide destructive rice bacterial disease, and seriously affects the global rice industry. Effective integrated management measures for BLS depend on the timely and proper detection of Xoc. Here, two highly specific and super-sensitive monoclonal antibodies (MAbs) against Xoc were first prepared using the cultured Xoc RS105 as an immunogen. Then, two serological assays, Dot enzyme-linked immunosorbent assay (Dot-ELISA) and Colloidal gold immunochromatographic strip (CGICS), were developed for the super-sensitive and broad-spectrum detection of Xoc in homogenates prepared from field-collected rice plants. The newly created Dot-ELISA and CGICS assays can detect ten different Xoc strains from various provinces in China and other countries without cross-reactivity with the other ten tested plant bacteria. Furthermore, Dot-ELISA and CGICS can detect Xoc in bacterial suspensions diluted up to 9.78 × 103 CFU/mL and 4.88 × 103 CFU/mL, respectively, or in rice plant tissue homogenates diluted up to 1:51,200 (w/v, g/mL). Surprisingly, both Dot-ELISA and CGICS serological assays were more sensitive than the conventional PCR. Additionally, analysis results using field-collected rice samples showed that the newly created Dot-ELISA and CGICS were reliable in detecting Xoc in rice tissues. Thus, the two serological assays were highly valuable and effective for the diagnosis of rice bacterial leaf streak in rice-grown areas and for inspection and quarantine of Xoc.

Neutralizing monoclonal antibodies against the Gc fusion loop region of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus, prevalent in more than 30 countries worldwide. Human infection by this virus leads to severe illness, with an average case fatality of 40%. There is currently no approved vaccine or drug to treat the disease. Neutralizing antibodies are a promising approach to treat virus infectious diseases. This study generated 37 mouse-derived specific monoclonal antibodies against CCHFV Gc subunit. Neutralization assays using pseudotyped virus and authentic CCHFV identified Gc8, Gc13, and Gc35 as neutralizing antibodies. Among them, Gc13 had the highest neutralizing activity and binding affinity with CCHFV Gc. Consistently, Gc13, but not Gc8 or Gc35, showed in vivo protective efficacy (62.5% survival rate) against CCHFV infection in a lethal mouse infection model. Further characterization studies suggested that Gc8 and Gc13 may recognize a similar, linear epitope in domain II of CCHFV Gc, while Gc35 may recognize a different epitope in Gc. Cryo-electron microscopy of Gc-Fab complexes indicated that both Gc8 and Gc13 bind to the conserved fusion loop region and Gc13 had stronger interactions with sGc-trimers. This was supported by the ability of Gc13 to block CCHFV GP-mediated membrane fusion. Overall, this study provides new therapeutic strategies to treat CCHF and new insights into the interaction between antibodies with CCHFV Gc proteins.