Mouse Monoclonal Antibody Research Articles

Use of the immunohistochemical marker HBME-1 to optimize the diagnosis of follicular carcinomas

Background. In clinical practice, there are often patients with a diagnosis of benign follicular adenoma (FA) and follicular tumor with an uncertain malignant potential, whose diagnoses must be changed to malignant processes due to relapse or metastasis observed over time. The purpose of the study was to optimize the differential diagnosis of FA and follicular thyroid carcinoma (FTC) using the immunohistochemical (IHC) marker HBME-1 on the histological material of patients who were operated for nodular goiter with a cytological conclusion corresponding to the Bethesda categories III–V. Materials and methods. One hundred and twenty-four patients underwent surgery for follicular nodular neoplasms with a cytological conclusion according to Bethesda Gray zone and had a histological diagnosis of FTC in 23 ca­ses (18.55 %) and FA in 101 cases (81.45 %). IHC analysis was performed using mouse monoclonal antibodies against human HBME-1 (Bio SB, USA). Evaluation criteria corresponded to the strength of staining (from 0 to 3). Results. According to the intensity of IHC staining, there was an increase in the diagnosis of FTC (p < 0.05): 0 — 0 %, 1 — 12.7 %, 2 — 25 %, 3 — 36.4 %. Histopathological findings were reexamined for FA that had the strongest staining reaction: in 3 (2.97 %) cases, the diagnosis was changed to FTC. Accor­dingly, the total number of detected FTC increased to 26 (20.96 %). These cases showed a good positive result regarding IHC study with HMBE-1 on FA preparations for a differential diagnosis between FA and FTC. Conclusions. HBME-1 can serve as an IHC marker for the differential diagnosis of FA and FTC in cases considered as FA, as after the study, the diagnosis was changed to FTC in 2.97 % of patients.

Exploring the Biological Activity of a Humanized Anti-CD99 ScFv and Antibody for Targeting T Cell Malignancies

CD99, a type I transmembrane protein, emerges as a promising therapeutic target due to its heightened expression in T cell acute lymphoblastic leukemia (T-ALL). This characteristic renders it a potential marker for minimal residual disease detection and an appealing target for antibody-based treatments. Previous studies have revealed that a mouse monoclonal antibody, mAb MT99/3, selectively binds to CD99, triggering apoptosis in T-ALL/T-LBL cells while preserving the integrity of healthy cells. By targeting CD99, mAb MT99/3 suppresses antigen presentation and disrupts T cell functions, offering promise for addressing hyperresponsive T cell conditions. To facilitate clinical translation, we developed a humanized ScFv variant of mAb MT99/3, termed HuScFvMT99/3 in “ScFvkh” design. Structural analysis confirms its resemblance to the original antibody, and the immunoreactivity of HuScFvMT99/3 against CD99 is preserved. The fully humanized version of antibody HuMT99/3 was further engineered, exhibiting similar binding affinity at the 10−10 M level and specificity to the CD99 epitope without antigenic shift. HuMT99/3 demonstrates remarkable selectivity, recognizing both malignant and normal T cells but inducing apoptosis only in T-ALL/T-LBL cells, highlighting its potential for safe and targeted therapy.

Immunohistochemical Localization of Fibroblast Activation Protein in Coronary Arteries with Different Forms of Atherosclerosis

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblasts in areas of active tissue remodeling. It plays a potentially important role in cardiac remodeling, atherosclerotic plaque formation, and plaque rupture. Given the distinct pathophysiology and morphology of different forms of atherosclerosis, we analyzed FAP expression in human coronary vessels with no coronary artery disease, atherosclerotic plaques at different levels of progression, and other distinct forms of coronary disease in post bypass vein grafting and cardiac allograft vasculopathy after a heart transplant. Methods: Immunohistochemical staining with monoclonal F19 mouse anti-human FAP antibody was performed to identify FAP in human atherosclerotic plaques, coronary bypass atherosclerosis, and post-transplant arteriosclerosis. The presence and distribution of FAP in different types and stages of human atherosclerosis were compared. Results: There was no FAP staining in patients with no significant coronary disease. All different types of human atherosclerotic lesioning lesions showed the presence of FAP expression, with different staining patterns in advanced atherosclerotic plaque, vein graft atherosclerosis lesions, and arteriosclerosis after a heart transplant. Conclusions: These data suggest that FAP may be a potential diagnostic marker and target for interventions, not only in coronary atherosclerotic plaque, but also in other forms of coronary disease, which have distinct pathophysiologies and currently limited treatment options.

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

Preparation and Construction of Chimeric Humanized Broadly Reactive Antibody 10H10 to Protein E of Tick-Borne Encephalitis Virus.

A full-length humanized chimeric antibody 10H10ch that specifically interacts with the surface glycoprotein E of flaviviruses was obtained. To construct it, we used variable fragments of the heavy and light chains of the monoclonal antibody 10H10 that form the active center of the antibody and a fragment of the constant part of the heavy chain of the human IgG1 antibody. The resulting full-length chimeric humanized antibody 10H10ch specifically interacted with the E protein of flaviviruses pathogenic to humans, such as tick-borne encephalitis, Zika, West Nile, and dengue viruses. An immunochemical assessment of the interaction constants of the 10H10ch antibody with a panel of native and recombinant flavivirus antigens by ELISA and biolayer interferometry showed that the dissociation constant (Kd) of the chimeric antibody is in the nanomolar region and is comparable to that of the high-affinity mouse monoclonal antibody 10H10. The possibility of using the resulting chimeric humanized antibody 10H10ch for the diagnosis, prevention, and treatment of various flavivirus infections is discussed.

Hypothyroidism Induced by a TSH Receptor Peptide-Implications for Thyroid Autoimmunity.

Background: The "neutral" thyrotropin receptor autoantibodies (N-TSHR-Ab) directed at the TSHR ectodomain's hinge region have been shown to induce thyroid cell damage in vitro. During these earlier studies, we developed a mouse monoclonal antibody (MC1) specific for a peptide (amino acid 322-340) in the region (MC1-Mab) which was able to induce thyroid cell stress and apoptosis when administered invivo. Methods: In order to examine the effect of invivo generated N-TSHR-Abs, rather than an acutely administered monoclonal antibody, we immunized Balb/c mice with the hinge region peptide over 18 weeks. Serum TSHR antibodies, specific TSHR hinge region antibodies, serum thyroglobulin (TG) and anti-TG as well as thyroxine and thyrotropin (TSH) levels were examined to evaluate the response to the immunization. Histological examination of the thyroid glands and flow cytometry of spleen T cells, B cells and macrophages were also performed to explore the underlying mechanisms. Results: We found that TSHR-peptide immunized mice developed N-TSHR-Abs against the peptide which resulted in thyroid damage shown by thyroid follicular destruction with follicular cell apoptosis, M1 macrophage infiltration, thyroglobulin release, and induction of thyroglobulin antibodies. This resulted in hypothyroidism with increased TSH levels. Conclusion: This study demonstrated that endogenous neutral antibodies to the TSHR could induce thyroid cell damage from apoptosis and M1 macrophage infiltration and resulted in hypothyroidism.

The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1

Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.

Membrane-bound receptor for advanced glycation end products (RAGE) is a stable biomarker of low-quality sperm

Abstract STUDY QUESTION Does receptor for advanced glycation end products (RAGE) on the surface membrane of the sperm cell function as a biomarker of low-quality sperm? SUMMARY ANSWER Membrane-bound RAGE at a cellular level directly correlates with low sperm motility, high cell permeability, decreased mitochondrial function, DNA fragmentation, and higher levels of apoptosis. WHAT IS KNOWN ALREADY RAGE has previously been measured by ELISA in low-quality sperm in diabetic men and has been shown to correlate with DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay). STUDY DESIGN, SIZE, DURATION Semen samples were recovered from 60 non-obese, non-diabetic and non-smoking subjects, washed with fresh media, and analysed directly or purified further by differential gradient centrifugation (DGC) or fractionated by direct swim-up before being analysed for sperm motility and molecular health parameters, including cell membrane permeability, cell death, mitochondrial membrane potential, DNA fragmentation, and RAGE protein expression. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm motility assessments were carried out by computer-assisted sperm analysis (CASA) on 1000 spermatozoa for washed samples and 300 spermatozoa for purified samples. Molecular sperm health parameters were evaluated using flow cytometry with the use of the following markers: DAPI for cell membrane permeability, Annexin V/DAPI for cell death (apoptosis and necrosis), MitoTracker® Red CMXRos for mitochondrial membrane potential, TUNEL assay for DNA fragmentation and 8-hydroxy-2-deoxyguanosine for identification of oxidative damage to sperm DNA, and contrasted to membrane-bound RAGE expression levels, which were evaluated using an anti-RAGE monoclonal mouse antibody. MAIN RESULTS AND THE ROLE OF CHANCE RAGE protein was shown to be present on the acrosomal and equatorial regions of sperm, with the levels of membrane bound receptor strongly correlating with poor sperm health across all parameters tested; motility (R2 = 0.5441, P < 0.0001) and mitochondrial membrane potential (R2 = 0.6181, P < 0.0001) being of particular note. The analysis was performed at a single cell level thereby removing confounding complications from soluble forms of the RAGE protein that can be found in seminal plasma. The expression of the RAGE protein was shown to be stable over time and its levels are therefore not subject to variation in sample handling or preparation time. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Inclusion criteria for this study were non-diabetic, non-obese and non-smoking participants to assess the distribution of RAGE expression in the general population, thereby excluding disease conditions that may increase RAGE expression in sperm or contribute to low sperm quality. The study does not address how RAGE expression may be affected in other patient subpopulations or disease states associated with male infertility. Sperm analysis by flow cytometry is not amenable to the study of males with a low sperm count. WIDER IMPLICATIONS OF THE FINDINGS Results of this study suggest that RAGE expression is a molecular maker of sperm cell health, which may be used for improvements in assisted reproduction through the removal of RAGE expressing sperm and facilitate in the diagnoses of unexplained infertility through its use as a biomarker of male infertility. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Irish Research Council under the Government of Ireland Programme (GOIPG/2015/3729) and the Enterprise Ireland Innovation Partnership Programme (IP-2020-0952). All authors declare no competing interests.

Gastric Precancerous Lesions: From Progenitor Cell and Microsatellite Instability to Clinical Interpretation of Gastric Cancer Risk

Aim: to evaluate the possibility of the MMR-system status, microsatellite instability (MSI) usage in the differential diagnosis of gastric mucosa dysplasia, determination of the gastric adenocarcinoma development risk. Material and methods. The study included gastric mucosa specimens of 75 patients: 25 with high-grade dysplasia, 25 with low-grade dysplasia, 25 were indefinite for dysplasia. Gastrobiopsy specimens were examined histologically, immunohistochemically using mouse monoclonal antibodies (Diagnostic BioSystems, USA) to the MMR system proteins: MLH-1 (clone G168-15, dilution 1:50), MSH2 (clone DBM15.82, dilution 1:100), MSH6 (clone 44, dilution 1:50), PMS2 (clone A16-4, ready to use). MSI was studied with multiplex PCR evaluation of DNA microsatellites (NR-21, NR-24, NR-27, BAT-25, BAT-26) from paraffin sections, their analysis with capillary electrophoresis. The obtained data were processed with the Statistica 10.0 (StatSoft, USA), presented using descriptive, analytical statistics. VOSviewer (1.6.20) was used to visualize the bibliometric analysis. Results. MMR-deficient cases were found in low (2.8 %) and high-grade (2.8 %) dysplasia with the immunohistochemical evaluation of MMR-system proteins in gastric mucosa specimens. In all indefinite for dysplasia cases MMR-system proteins remained unaffected. Three MSI-positive cases (6.5 %) were detected by PCR with two low-grade dysplasia, one high-grade dysplasia cases. All identified cases were also immunohistochemically MSI-positive. Conclusion. Determination of MSI can be used as an auxiliary study within a panel of biomarkers aimed to support the decision-making of a pathologist in the alternative of “indefinite for dysplasia” or “definite dysplasia — obligate precancer”.

B-245 Novel monoclonal antibodies validated specifically for immunohistochemistry by performance assessment on tissue microarrays

Abstract Background Immunohistochemistry (IHC) is a laboratory immunoassay that is a pillar of diagnostic pathology, particularly in the identification and characterization of neoplasms and other diseases/disorders. Though performed routinely, rigorous SOP protocols must be determined empirically for each molecular target, beginning with antibody selection. Both traditional polyclonal and hybridoma-based monoclonal antibodies have been employed in the past, though monoclonal, and particularly recombinant monoclonal, antibodies are preferred. However, any reagent used for diagnostic purposes must be meticulously validated to minimize, if not eliminate, erroneous interpretations of IHC assays. Thus, the extent of validation is the pivotal aspect for establishing an antibody as a reliable diagnostic tool for IHC applications. Methods GeneTex has assembled an extensive catalog of rabbit and mouse monoclonal antibodies validated specifically for IHC. These antibodies are tested on both human normal tissue and cancer tissue microarrays, with upwards of 76 normal and numerous tumor tissues included. Images of the staining are available for all the tested tissues. This broad spectrum of tissues allows a researcher to assess the performance of the antibody on known positive and negative samples, thereby minimizing the risk of false positive or false negative readouts (Gown, 2016). In addition, the data images are of sufficient resolution to determine proper cellular localization of the signal. This special catalog of IHC-optimized reagents are listed under the “HistoMAX” designation. New antibodies will be added continuously, many of them being fully recombinant rabbit monoclonals manufactured using a multi-parameter FACS-based methodology to select antigen-specific IgG+ memory B cells from an immunized rabbit with subsequent cloning of the matched IgG heavy and light chains (Starkie et al., 2016). These recombinant antibodies will be validated following the same approach described above. Results Presently more than 130 mouse or rabbit monoclonal antibodies are available in the HistoMAX line of IHC-validated products and are directed against a spectrum of important targets. This includes antibodies for the commonly analyzed cytokeratins 5, 6, 7, 18, 19, and 20, among many others. Antibodies for CTLA4 and PD-L1 headline products for immunology and immunotherapy research, while other reagents are directed against various cadherins. Hormone receptor antibodies are also represented, with antibodies against estrogen receptor alpha, progesterone receptor, Her2/ErbB2, and the androgen receptor. There are also antibodies against various key “Cluster of Differentiation” (CD) proteins as well as other membrane proteins such as ACE2. Many antibodies are relevant to the study of human malignancies, such as CD66e/CEACAM5/CEA, MSH6, p63, MUC1, Collagen IV, EpCAM, PAX6, PAX8, GP2, TRIM29, S100, and GAD65. All antibodies were evaluated as described above. Conclusions Antibodies are application-specific reagents, and the best antibodies for IHC are validated through systematic staining of a multitude of normal and abnormal tissues. This is the only way that an antibody’s performance for IHC can be critically assessed. The HistoMAX reagents consist only of those monoclonal antibodies that have generated expected signals, or the lack of a signal, in a wide range of tissues in a manner consistent with the literature.

Systemic administration of a potent mouse monoclonal antibody provides multisite protection against HPV16 infection

Although preventive vaccines for Human Papillomaviruses (HPV) are available, a definitive cure for the viral infection itself is currently lacking. There is a sizable population that remains inaccessible to HPV vaccination due to reasons such as high costs or lack of availability of the vaccines. Therefore, there remains a significant population susceptible to HPV infection. Persistent multisite infections with high-risk HPV types can cause cancer at several different anatomic sites.Especially HPV16 is a key etiologic factor for cervical, other ano-genital and oropharyngeal cancers. Therefore, it is imperative to develop pharmaceutical interventions for the treatment of viral infections. In this study, a panel of 9 neutralizing antibodies was screened using the hybridoma technique, with 20F6 being identified as the representative antibody. The purified 20F6 exhibited an IC50 of 0.0011 μg/ml against HPV16, demonstrating potent viral inhibitory activity. Moreover, it displayed cross-neutralizing efficacy towards other Alphapapillom 9 subtypes including HPV31, HPV33, HPV52, and HPV58 with respective IC50 values of 2.0 μg/ml, 7.3 μg/ml, 1.7 μg/ml, and 3.0 μg/ml. 20F6 recognizes the linear epitope MSLW, the first four amino-acids located at the very N-terminus of the HPV16 L1 protein. Administration of 20F6, 24 h prior to and following HPV16 pseudo-virion (PSV) challenge, conferred protection against infection in mice at doses as low as 1 mg/kg. Following intraperitoneal administration of 20F6, neutralizing antibodies were consistently detected at both oral and vaginal sites, indicating that prophylactic systemic administration of 20F6 may confer efficient protection against multiple susceptible mucosal sites.

Immunohistochemical Expression of Vitamin D receptor in Breast Cancer in Erbil city

Background & objectives: Vitamin D receptor is a nuclear receptor that modulates the activity of genes. The purpose of this study was to evaluate the frequency of vitamin D receptor expression in breast carcinoma by immunohistochemistry and to investigate the association between vitamin D receptor expression with some clinicopathologic factors, like tumor grading, staging, histologic type (ductal and non-ductal) and the hormonal status. Methods: One hundred formalin-fixed paraffin embedded tissue blocks of mastectomy and core biopsy specimens diagnosed as carcinoma, were obtained from a private laboratory in Erbil city/Iraq from October 2020-October 2022. In this study, vitamin D antibody (a mouse monoclonal antibody) was applied to a breast cancer tissue samples and its expression was assessed. Results: Nuclear vitamin D receptor was expressed in 32% of the cases, and a strong relation was noted between vitamin D receptor expression and low-grade tumor (p = 0.034), estrogen receptor (p-value =0.007), and progesterone receptor (p = 0.045) positivity, while no significant association was noted between vitamin D receptor expression and other clinicopathologic parameters like age, lymph node status, tumor stage, the type of the cancer and human epidermal growth factor receptor 2 status. Conclusion: increased Vitamin D receptor expression in breast tumors appears to be associated with a better prognosis. Vitamin D receptor expressed in one third of cases in which there was significant association with tumor grade and hormonal receptors.

Immunohistochemical expression of B-cell lymphoma 2 gene -Bcl-2 in endometrial carcinoma

Background and objectives: Apoptosis, a process of cell death, is inhibited by the B-cell lymphoma 2 protein, leading to the prolonged survival of cells. Participation of Bcl-2 in starting and advancement of endometrial carcinoma remains inconclusive, with varying results observed. The aims of this study were to assess the frequency of B-cell lymphoma 2 expression in endometrial carcinoma, and to investigate the connection between B-cell lymphoma 2 expression with some clinicopathological parameters. Methods: A retrospective study was carried out for seventy-eight formalin fixed paraffin embedded blocks of total abdominal hysterectomy specimens diagnosed as endometrial carcinoma, which were randomly obtained from a private laboratory and Rizgary teaching hospital laboratory in Erbil city during June 2021-June 2022. In this study, endometrial carcinoma cases were subjected to the utilization of B-cell lymphoma 2 gene, which is a Mouse monoclonal antibody, and its expression was evaluated. Results: The expression of Bcl-2 was observed in 61.5% of the studied cases. The majority (93.6%) of EC cases were of low grade, although Bcl-2 expression was observed at a greater frequency (64.4%) in low-grade endometrial carcinoma, but there was no significant association. Additionally, no statistically significant correlation was found between B-cell lymphoma 2 gene expression and other clinicopathological parameters like, age, myometrial invasion, lympho-vascular invasion, lymph node status and tumor stage. Conclusion: Bcl-2 was expressed in 61.5% of the studied cases with no significant association with any of the variables, additional research is necessary to assess the expression of B-cell lymphoma 2 gene in relation to endometrial carcinoma invasion and metastasis.

TLE1 Expression in Different Sarcoma Types and its Relation to Clinicopathological Parameters

Background and objectives: Split genes that contain Transducin-Like Enhancer encode transcriptional corepressors that are essential for hematopoiesis and embryogenesis in humans. Synovial sarcomas express Transducin-Like Enhancer-1, a distinct factor which hardly has been shown in other soft tissues malignancies. Transducin-Like Enhancer-1 expression was recently found to be sensitive but not entirely specific for synovial sarcoma. Methods: This retrospective study included eighty paraffin embedded formalin-fixed blocks of different sarcomas which were gathered and picked out of the files of Rizgary teaching hospital lab and from certain private labs in Hawler, during June 2015-December 2022. In this study, Transducin-Like Enhancer-1, a mouse monoclonal antibody was applied on sarcoma cases and its expression was assessed. Results: around one third (30%) of the patients had synovial sarcoma, (16.3%) had undifferentiated pleomorphic sarcoma, and the rest had other types. More than half (55%) of the sarcomas were of high grade, and the Transducin-Like Enhancer-1 score was strongly reactive in 33.8% of the patients. It was also strong in all (100%) the patients with synovial sarcoma, compared with 23.1% of patients with undifferentiated pleomorphic sarcoma, and 0% of the other types of sarcoma (p < 0.001). Conclusion: this study demonstrates that, when synovial sarcoma was kept in the differential diagnosis of spindle cell soft tissue sarcomas, Transducin-Like Enhancer-1 was a highly sensitive and relatively a specific marker.

Anti-DEFA5 Monoclonal Antibody Clones 1A8 and 4F5 Immunoreactive Bioassay for Diagnosing Inflammatory Bowel Disease.

Robust evidence suggests that the aberrant expression of α defensin 5 protein (DEFA5) in colon inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn's colitis, can be exploited as a reliable diagnostic biomarker to differential diagnosis of Crohn's colitis (CC) from Ulcerative colitis (UC) in otherwise indeterminate colitis (IC). We evaluated the specificity of the commercially available anti-DEFA5 antibodies and showed further validation of their appropriateness for a given application is required. We established two mouse monoclonal DEFA5 antibody clones 1A8 and 4F5 by immunizing the mice with purified recombinant protein and validated the specificity, selectivity and cross reactivity in recognizing the endogenous and recombinant DEFA5 protein, especially for Immunohistochemistry, Western blot, Immunoprecipitation, or enzyme-linked immunosorbent assay. Clones 1A8 and 4F5 recognized effectively the endogenous DEFA5 in active human diverticulitis (DV), UC, CC or IC disease samples, including transiently transfected HEK293T cells expressing DEFA5 with high degree of specificity and minimal non-confounding cross reactivity. 1A8 and 4F5 clones are worth studying in larger IBD cohorts to fully address whether DEFA5 expression may be used as a diagnostic biomarker to discrimination of the diagnosis of UC from CC or IC into authentic CC or UC or a colitis with different pathological characteristics.

A New Chimeric Antibody against the HIV-1 Fusion Inhibitory Peptide MT-C34 with a High Affinity and Fc-Mediated Cellular Cytotoxicity.

Peptides from heptad repeat (HR1 and HR2) regions of gp41 are effective inhibitors of HIV-1 entry that block the fusion of viral and cellular membranes, but the generation of antibodies highly specific for these peptides is challenging. We have previously described a mouse hybridoma that recognizes MT-C34-related peptides derived from HR2. It was used for the selection of HIV-1-resistant CD4 lymphocytes engineered to express the MT-C34 peptide via a CRISPR/Cas9-mediated knock-in into the CXCR4 locus. In this study, we cloned variable domains of this antibody and generated a recombinant chimeric antibody (chAb) by combining it with the constant regions of the humanized antibody Trastuzumab. The new chAb displayed a high specificity and two-fold higher level of affinity than the parental mouse monoclonal antibody. In addition, chAb mediated up to 27-43% of the antibody-dependent cellular cytotoxicity towards cells expressing MT-C34 on their surface. The anti-MT-C34 chAb can be easily generated using plasmids available for the research community and can serve as a valuable tool for the detection, purification, and even subsequent elimination of HIV-1-resistant CD4 cells or CAR cells engineered to fight HIV-1 infection.

IL1RAP-specific T cell engager depletes acute myeloid leukemia stem cells

BackgroundThe interleukin-1 receptor accessory protein (IL1RAP) is highly expressed on acute myeloid leukemia (AML) bulk blasts and leukemic stem cells (LSCs), but not on normal hematopoietic stem cells (HSCs), providing an opportunity to target and eliminate the disease, while sparing normal hematopoiesis. Herein, we report the activity of BIF002, a novel anti-IL1RAP/CD3 T cell engager (TCE) in AML.MethodsAntibodies to IL1RAP were isolated from CD138+ B cells collected from the immunized mice by optoelectric positioning and single cell sequencing. Individual mouse monoclonal antibodies (mAbs) were produced and characterized, from which we generated BIF002, an anti-human IL1RAP/CD3 TCE using Fab arm exchange. Mutations in human IgG1 Fc were introduced to reduce FcγR binding. The antileukemic activity of BIF002 was characterized in vitro and in vivo using multiple cell lines and patient derived AML samples.ResultsIL1RAP was found to be highly expressed on most human AML cell lines and primary blasts, including CD34+ LSC-enriched subpopulation from patients with both de novo and relapsed/refractory (R/R) leukemia, but not on normal HSCs. In co-culture of T cells from healthy donors and IL1RAPhigh AML cell lines and primary blasts, BIF002 induced dose- and effector-to-target (E:T) ratio-dependent T cell activation and leukemic cell lysis at subnanomolar concentrations. BIF002 administered intravenously along with human T cells led to depletion of leukemic cells, and significantly prolonged survival of IL1RAPhigh MOLM13 or AML patient-derived xenografts with no off-target side effects, compared to controls. Of note, BiF002 effectively redirects T cells to eliminate LSCs, as evidenced by the absence of disease initiation in secondary recipients of bone marrow (BM) from BIF002+T cells-treated donors (median survival not reached; all survived > 200 days) compared with recipients of BM from vehicle- (median survival: 26 days; p = 0.0004) or isotype control antibody+T cells-treated donors (26 days; p = 0.0002).ConclusionsThe novel anti-IL1RAP/CD3 TCE, BIF002, eradicates LSCs and significantly prolongs survival of AML xenografts, representing a promising, novel treatment for AML.

Selection of antibody-binding covalent aptamers

Aptamers are oligonucleotides with antibody-like binding function, selected from large combinatorial libraries. In this study, we modified a DNA aptamer library with N-hydroxysuccinimide esters, enabling covalent conjugation with cognate proteins. We selected for the ability to bind to mouse monoclonal antibodies, resulting in the isolation of two distinct covalent binding motifs. The covalent aptamers are specific for the Fc region of mouse monoclonal IgG1 and are cross-reactive with mouse IgG2a and other IgGs. Investigation into the covalent conjugation of the aptamers revealed a dependence on micromolar concentrations of Cu2+ ions which can be explained by residual catalyst remaining after modification of the aptamer library. The aptamers were successfully used as adapters in the formation of antibody-oligonucleotide conjugates (AOCs) for use in detection of HIV protein p24 and super-resolution imaging of actin. This work introduces a new method for the site-specific modification of native monoclonal antibodies and may be useful in applications requiring AOCs or other antibody conjugates.

Hazelnut proteins detection in cookies using an immersible label-free photonic chip sensor

Rapid and sensitive methods to detect allergenic proteins in foods at the point-of-need could help to protect allergic consumers from life-threatening accidental exposure. In this context, the development of a label-free optical immunosensor for detecting hazelnut proteins in cookies is presented. The sensor is based on silicon photonic chips containing two integrated Mach-Zehnder interferometers (MZI) with light input and output on one side of the chip, and sensing window openings on the other side. Coupling with a white-light LED and a spectrometer for signal recording is achieved via a bifurcated fiber. The sensing window of one of the MZIs is modified with a mouse monoclonal antibody against hazelnut proteins, while the other sensing window is modified with bovine serum albumin to serve as blank. The assay follows a two-step sandwich immunoassay format, involving a 10-min reaction with the hazelnut proteins calibrator or cookie sample, followed by a 2-min reaction with a mixture of monoclonal antibodies. All reactions were performed by immersing the chip side where the MZIs windows are located into the solutions. The recorded spectrum is processed in real-time to transform the spectrum shifts due to immunoreactions taking place on the sensing windows of the two MZIs into phase shifts. The developed assay detects hazelnut proteins at concentrations as low as 25 ng/mL in calibrators prepared in buffer or in aqueous extract of hazelnut-free cookies. Additionally, cookies containing hazelnuts were analyzed demonstrating the sensor ability for fast and sensitive detection of the hazelnut proteins in commercial products.

Immuno-targeting the ectopic phosphorylation sites of PDGFRA generated by MAN2A1-FER fusion in HCC.

HCC is one of the most lethal cancers for humans. Mannosidase alpha class 2A member 1 (MAN2A1)-FER is one of the most frequent oncogenic fusion genes in HCC. In this report, we showed that MAN2A1-FER ectopically phosphorylated the extracellular domains of PDGFRA, MET, AXL, and N-cadherin. The ectopic phosphorylation of these transmembrane proteins led to the activation of their kinase activities and initiated the activation cascades of their downstream signaling molecules. A panel of mouse monoclonal antibodies was developed to recognize the ectopic phosphorylation sites of PDGFRA. The analyses showed that these antibodies bound to the specific phosphotyrosine epitopes in the extracellular domain of PDGFRA with high affinity and specificity. The treatment of MAN2A1-FER-positive cancer HUH7 with one of the antibodies called 2-3B-G8 led to the deactivation of cell growth signaling pathways and cell growth arrest while having minimal impact on HUH7ko cells where MAN2A1-FER expression was disrupted. The treatment of 2-3B-G8 antibody also led to a large number of cell deaths of MAN2A1-FER-positive cancer cells such as HUH7, HEPG2, SNU449, etc., while the same treatment had no impact on HUH7ko cells. When severe combined immunodeficiency mice xenografted with HEPG2 or HUH7 were treated with monomethyl auristatin E-conjugated 2-3B-G8 antibody, it slowed the progression of tumor growth, eliminated the metastasis, and reduced the mortality, in comparison with the controls. Targeting the cancer-specific ectopic phosphorylation sites of PDGFRA induced by MAN2A1-FER may hold promise as an effective treatment for liver cancer.