Generation Of mAbs Research Articles

Generation and Selection of Phage Display Antibody Libraries in Fab Format.

Monoclonal antibodies (mAbs) have exceptional utility as research reagents and pharmaceuticals. As a complement to both traditional and contemporary single-B-cell cloning technologies, the mining of antibody libraries via display technologies-which mimic and simplify B cells by physically linking phenotype (protein) to genotype (protein-encoding DNA or RNA)-has become an important method for mAb discovery. Among these display technologies, phage display has been particularly successful for the generation of mAbs that bind to a wide variety of antigens with exceptional specificities and affinities. Rather than multivalent whole antibodies, phage display typically uses monovalent antibody fragments, such as "fragment antigen binding" (Fab), as the format of choice. The ∼50-kDa Fab format consists of four immunoglobulin (Ig) domains on two polypeptide chains (light chain and shortened heavy chain), and exhibits its antigen binding site in a natural configuration found in bivalent IgG and other multivalent Ig molecules. The Fab fragment has a high melting temperature and a low tendency to aggregate, and can be readily converted to natural and nonnatural Ig formats without affecting antigen binding properties, which has made it a favored format for phage display for more than three decades. Here, I briefly summarize some of the approaches used for the generation and selection of phage display antibody libraries in Fab format, from human and nonhuman antibody repertoires.

A Monoclonal Antibody with a High Affinity for Ricin Isoforms D and E Provides Strong Protection against Ricin Poisoning.

Ricin is a highly potent toxin that has been used in various attempts at bioterrorism worldwide. Although a vaccine for preventing ricin poisoning (RiVax™) is in clinical development, there are currently no commercially available prophylaxis or treatments for ricin intoxication. Numerous studies have highlighted the potential of passive immunotherapy using anti-ricin monoclonal antibodies (mAbs) and have shown promising results in preclinical models. In this article, we describe the neutralizing and protective efficacy of a new generation of high-affinity anti-ricin mAbs, which bind and neutralize very efficiently both ricin isoforms D and E in vitro through cytotoxicity cell assays. In vivo, protection assay revealed that one of these mAbs (RicE5) conferred over 90% survival in a murine model challenged intranasally with a 5 LD50 of ricin and treated by intravenous administration of the mAbs 6 h post-intoxication. Notably, a 35% survival rate was observed even when treatment was administered 24 h post-exposure. Moreover, all surviving mice exhibited long-term immunity to high ricin doses. These findings offer promising results for the clinical development of a therapeutic candidate against ricin intoxication and may also pave the way for novel vaccination strategies against ricin or other toxins.

Generation of anti-SN38 antibody for loading efficacy and therapeutic monitoring of SN38-containing therapeutics

SN38, one of the most potent anti-tumor analogues of the camptothecins (CPTs), has limitations in its direct formulation as an anticancer agent due to its super toxicity and poor solubility in water and pharmaceutically approved solvents. However, it has garnered significant scientific interest as a payload in conjugated nanomedicine platforms (e.g., SN-38lip, NK012, SNB-101, and ADCs) to enhance their effectiveness and safety. The development of these platforms necessitates a convenient quantitative determination of SN38 in preclinical and clinical studies, a need that our study directly addresses, offering a practical solution to a pressing problem in cancer research and drug development. This study details the meticulous process of generating poly and monoclonal antibodies (pAb and mAb) against SN38 and their application to measure the SN38 in naked and conjugated forms of SN38-conjugated ADCs. For this purpose, two haptens of SN38 were synthesized by introducing the glycine or 4-amino-4-oxobutanyol(glycine) moiety as a conjugation functional group of the SN38. IR, NMR and mass spectrometric techniques confirmed the chemical modifications of the haptens. The haptens were then conjugated to each bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) protein. The SN38-KLH conjugates were meticulously examined for immunization and generation of pAb and mAb. The immunization efficiency, reactivity, binding affinity, specificity, and cross-reactivity of purified pAb and mAb against Irinotecan, a model for the emergence of an SN38 derivative in clinical settings, were evaluated using ELISA and western blotting (WB) techniques. Conjugation efficiency of the SN38 to the KLH was increased using 4-amino-4-oxobutanyol(glycine) moiety, as its immunization efficacy was more to generate pAb. Furthermore, only this hapten could immunized mice to generate mAb recognizing SN38 with nanomolar equilibrium affinity. Our recent findings strongly support the notion that the generated pAb employed in developing an ELISA effectively ascertains the presence of SN38 in SN38-conjugated ADC, with a test midpoint EC50 of 2.5 μg/mL.Our study's unique contribution to the field lies in the development of specific antibodies against SN38 for measuring it on ADC, a feat that has not been achieved before. These immunoassays can be readily applied to detect other SN38-conjugate therapeutic platforms, thereby enhancing their clinical knowledge translation. The affinity of both pAb and mAb also meets the acceptance criteria for quantifying SN38 in fluidic material, as well as in Therapeutic drug monitoring (TDM) studies, a crucial aspect of personalized medicine. The potential applications of the anti-SN38 antibodies extend to reducing SN38-induced systemic toxicity through an inverse targeting strategy, a novel approach that piques further interest in our findings.

Generation and Characterization of Human IgE mAbs to PR-10 Allergens

Generation and Characterization of Human IgE mAbs to PR-10 Allergens

Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

A First-in-Human Randomized Study to Assess the Safety, Tolerability, Pharmacokinetics, and Neutralization Profile of Two Investigational Long-Acting Anti-SARS-CoV-2 Monoclonal Antibodies.

COVID-19 remains a significant risk for the immunocompromised given their lower responsiveness to vaccination or infection. Therefore, passive immunity through long-acting monoclonal antibodies (mAbs) offers a needed approach for pre-exposure prophylaxis (PrEP). Our study evaluated safety, anti-SARS-CoV-2 neutralizing activity, nasal penetration, and pharmacokinetics (PK) of two half-life-extended investigational mAbs, AER001 and AER002, providing the first demonstration of upper airway penetration of mAbs with the LS-modification. This randomized, double-blind, placebo-controlled phaseI study enrolled healthy adults (n = 80) who received two long-acting COVID mAbs (AER001 and AER002), AER002 alone, or placebo. The dose ranged from 100mg (mg) to 1200mg per mAb component. The primary objective was to describe the safety and tolerability following intravenous (IV) administration. Secondary objectives were to describe PK, anti-drug antibodies (ADA), neutralization activity levels, and safety evaluation through 6months of follow-up. The majority (97.6%) of the reported adverse events (AE) post administration were of grade1 severity. There were no serious adverse events (SAE) or ADAs. AER001 and AER002 successfully achieved an extended half-life of 105days and 97.5days, respectively. Participants receiving AER001 and AER002 (300mg each) or AER002 (300mg) alone showed 15- and 26-fold higher neutralization levels against D614G and omicron BA.1 than the placebo group24h post-administration. Single 300 or 1200mg IV dose of AER001 and AER002 resulted in nasal mucosa transudation of approximately 2.5% and 2.7%, respectively. AER001 and AER002 showed an acceptable safety profile and extended half-life. High serum neutralization activity was observed against D614G and Omicron BA.1 compared to the placebo group. These data support that LS-modified mAbs can achieve durability, safety, potency, and upper airway tissue penetration and will guide the development of the next generation of mAbs for COVID-19 prevention and treatment. EudraCT Number 2022-001709-35 (COV-2022-001).

Recent Advances in the Development of Monoclonal Antibodies and Next-Generation Antibodies.

mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B lymphocytes with myeloma cells, which resulted in generation of single mAbs against a specific Ag. Along with hybridoma technology, several novel and alternative methods have been developed to improve mAb generation, ranging from electrofusion to the discovery of completely novel technologies such as B cell immortalization; phage, yeast, bacterial, ribosome, and mammalian display systems; DNA/RNA encoded Abs; single B cell technology; transgenic animals; and artificial intelligence/machine learning. This commentary outlines the evolution, methodology, advantages, and limitations of various mAb production techniques. Furthermore, with the advent of next-generation Ab technologies such as single-chain variable fragments, nanobodies, bispecific Abs, Fc-engineered Abs, Ab biosimilars, Ab mimetics, and Ab-drug conjugates, the healthcare and pharmaceutical sectors have become resourceful to develop highly specific mAb treatments against various diseases such as cancer and autoimmune and infectious diseases.

Generation of recombinant mAbs to vaccinia virus displaying high affinity and potent neutralization.

Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.

Generation and Study of Antibodies against Two Triangular Trimers Derived from Aβ.

Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aβ) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aβ assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aβ. These antibodies are the first mAbs generated against Aβ oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aβ42, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aβ enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.

Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology.

Efficient induction of target-specific antibodies can be elicited upon immunization with highly immunogenic virus-like particles (VLPs) decorated with desired membrane-anchored target antigens (Ags). However, for example, for diagnostic purposes, monoclonal antibodies (mAbs) are required to enable the histological examination of formaldehyde-fixed paraffin-embedded (FFPE) biopsy tissue samples. Aiming at the generation of FFPE-antigen-specific mAbs and as a proof of concept (POC), we first established a simplified protocol using only formaldehyde and 90 °C heat fixation (FF90) of cells expressing the target Ag nerve growth factor receptor (NGFR). The FF90 procedure was validated using flow cytometric analysis and two mAbs recognizing either the native and FFPE-Ag or exclusively the native Ag. C-terminally truncated NGFR (trNGFR)-displaying native and FF90-treated VLPs derived from HIV-1 did not reveal distinctive changes in particle morphology using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Mice were subsequently repetitively immunized with trNGFR-decorated FF90-VLPs and hybridoma technology was used to establish mAb-producing cell clones. In multiple screening rounds, nine cell clones were identified producing mAbs distinctively recognizing epitopes in FF90- and FFPE-NGFR. This POC of a new methodology should foster the future generation of mAbs selectively targeting FFPE-fixed cell-surface Ags.

Novel anti-PTEN C2 domain monoclonal antibodies to analyse the expression and function of PTEN isoform variants.

PTEN is a major tumor suppressor gene frequently mutated in human tumors, and germline PTEN gene mutations are the molecular diagnostic of PTEN Hamartoma Tumor Syndrome (PHTS), a heterogeneous disorder that manifests with multiple hamartomas, cancer predisposition, and neurodevelopmental alterations. A diversity of translational and splicing PTEN isoforms exist, as well as PTEN C-terminal truncated variants generated by disease-associated nonsense mutations. However, most of the available anti-PTEN monoclonal antibodies (mAb) recognize epitopes at the PTEN C-terminal tail, which may introduce a bias in the analysis of the expression of PTEN isoforms and variants. We here describe the generation and precise characterization of anti-PTEN mAb recognizing the PTEN C2-domain, and their use to monitor the expression and function of PTEN isoforms and PTEN missense and nonsense mutations associated to disease. These anti-PTEN C2 domain mAb are suitable to study the pathogenicity of PTEN C-terminal truncations that retain stability and function but have lost the PTEN C-terminal epitopes. The use of well-defined anti-PTEN mAb recognizing distinct PTEN regions, as the ones here described, will help to understand the deleterious effects of specific PTEN mutations in human disease.

New high-throughput screening method for Chinese hamster ovary cell lines expressing low reduced monoclonal antibody levels: application of a system controlling the gas phase over cell lysates in miniature bioreactors and facilitating multiple sample setup.

Interchain disulfide bonds in monoclonal antibodies may be reduced during large-scale mAb production using Chinese hamster ovary (CHO) cells. This reaction lowers the mAb product yield and purity; however, it may be prevented by screening cell lines that are unsusceptible to reduction and using them in mAb production. Antibody reduction susceptibility may be cell line-dependent. To the best of our knowledge, however, an efficient method of screening reduction-unsusceptible CHO cell lines has not been previously reported. Here, we report a novel screening method that can simultaneously detect and identify mAb reduction susceptibility in lysates containing ≤ 48 CHO cell lines. This evaluation system was equally effective and generated similar results at all culture scales, including 250 mL, 3L, and 1000L. Furthermore, we discovered that reduction-susceptible cell lines contained higher total intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ concentrations than reduction-unsusceptible cell lines, regardless of whether they expressed immunoglobulin (Ig)G4 or IgG1. NADPH or NADP+ supplementation in the lysate of reduction-unsusceptible cells resulted in mAb reduction. Application of the innovative CHO cell line screening approach could mitigate or prevent reductions in large-scale mAb generation from CHO cells.

Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses.

Marek's disease (MD) caused by pathogenic Marek's disease virus type 1 (MDV-1) is one of the most important neoplastic diseases of poultry. MDV-1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV-1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV-1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.

Molecular disease mechanisms of human antineuronal monoclonal autoantibodies.

Autoantibodies targeting brain antigens can mediate a wide range of neurological symptoms ranging from epileptic seizures to psychosis to dementia. Although earlier experimental work indicated that autoantibodies can be directly pathogenic, detailed studies on disease mechanisms, biophysical autoantibody properties, and target interactions were hampered by the availability of human material and the paucity of monospecific disease-related autoantibodies. The emerging generation of patient-derived monoclonal autoantibodies (mAbs) provides a novel platform for the detailed characterization of immunobiology and autoantibody pathogenicity in vitro and in animal models. This Feature Review focuses on recent advances in mAb generation and discusses their potential as powerful scientific tools for high-resolution imaging, antigenic target identification, atomic-level structural analyses, and the development of antibody-selective immunotherapies.

Use of monoclonal antibodies in cancer immunotherapy: types and mechanisms of action.

Immunotherapy is one of the most innovative treatments in the current field of oncology and consists of stimulating the immune system to eliminate tumoral cells. Monoclonal antibodies (mAbs) are glycoproteins secreted by B-cells capable of recognizing and neutralizing foreign organisms or antigens. Structurally, they are composed of two heavy and two light chains. The generation of therapeutic mAbs is one of the most developed and fastest-growing areas of the biotechnological and pharmaceutical industries and is an important adjunct to cancer therapy. Several antibodies have been approved for human administration and can be mouse-derived, chimeric, humanized, or fully human. mAbs main mechanism of action includes the lysis of the tumoral cells through inducing apoptosis, phagocytosis, complement activation, or signaling inhibition.

Abstract 4259: PT199, a next generation anti-CD73 mAb that inhibits both membrane-bound and soluble CD73 activity to completion without “hook effect”

Abstract CD73, also known as ecto-5’-nucleotidase, is implicated in promoting immunosuppressive tumor microenvironment (TME) through production of adenosine. The first generation of anti-CD73 therapeutic antibodies currently in clinical studies have largely exhibited subdued clinical activities mainly due to several unfavorable characteristics such as incomplete enzyme inhibition and/or “hook effect”. These weaknesses are caused by the unfavorable target binding sites and enzyme inhibition mechanisms, which explains why these molecules do not inhibit soluble CD73 enzyme activity, or why the enzyme inhibition depends on certain stochiometric ratios of CD73 and the antibody and exhibits “hook effect” in dose-response curves. Here we report a next generation humanized monoclonal antibody against human CD73 (hCD73), PT199, which binds to a unique site on human CD73 that confers a different mechanism of enzyme inhibition and consequently does not have the flaws associated with the first generation of anti-CD73 mAbs currently in clinic. By directly binding to an epitope that is in or near the CD73 catalytic domain, PT199 potently inhibits both soluble shed CD73 and membrane-bound CD73 activity to completion without “hook effect”. Enzyme kinetic studies demonstrate that PT199 inhibits CD73 enzyme activity in a noncompetitive manner, allowing complete CD73 inhibition even at high AMP concentrations reported in TME. In a cell-based functional assay, PT199 inhibition of CD73 alleviates the immunosuppression effect of AMP on T cell proliferation. Compared with the first-generation molecules, PT199 completely inhibits CD73 activity in cancer patient serum. Furthermore, when administrated to hCD73 knock-in mice, PT199 greatly elevates endogenous AMP level, demonstrating a robust pharmacodynamic effect in blocking the conversion of AMP to adenosine. Comparative mechanistic studies indicate that PT199 binds to both the “open conformation” (inactive enzyme state) and the “closed conformation” (active enzyme state) with high affinity but with shifted binding curves, suggesting that PT199 may inhibit the CD73 enzyme activity by preventing the transition of the enzyme from the “open conformation” to the “closed conformation”. PT199 did not cause B cell reduction in either acute or repeated dosing studies in NHP. In NHP toxicology studies, PT199 exhibits no clinical observations at up to 300 mg/kg weekly repeated dosing and has an excellent PK profile typical of mAbs. Taken together, our data confirms PT199 as a potential best-in-class anti-hCD73 mAb. Phase 1 clinical trial is planned to start in the first quarter of 2022. Citation Format: Haiqun (John) Jia, Jack Li, Fen Pei, Lorraine Greenwood, Lauren Pejza, Yan Long, Kelan Chen, Jessica Perer, Ming Wang, Hui Zou. PT199, a next generation anti-CD73 mAb that inhibits both membrane-bound and soluble CD73 activity to completion without “hook effect” [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4259.

An aggregation-induced emission immunoassay for broad detection of polychlorinated biphenyls in chicken and crab.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) with multiple variants, which may be harmful to human health by absorption and bioaccumulation. To ensure food safety, it is necessary to develop multi-residue immunoassays for broad recognition of PCBs. In this study, by mimicking the generic core structure of PCBs, three haptens have been designed and synthesized for monoclonal antibody (mAb) generation. A carboxylic acid derivative of PCB80 was a hapten that induced a mAb with broad recognition of PCBs. The results of ELISA further identified that the mAb could recognize 11 different kinds of PCBs; half-maximal inhibition concentrations (IC50) ranged from 33.12 to 476.42ng/mL. Subsequently, using aggregation-induced emission luminogen (AIEgen) nanobeads as the tracer for the output signal, the IC50 value of the various PCBs was improved to 6.38-252.1ng/mL. The limit of detection (LOD) varied from 0.32 to 42.15ng/mL. Recoveries of 76.90-95.74% and intra-assay coefficients of variation of 8.5-14.4% were obtained with spiked chicken and crab meat samples. Matrix interference was eliminated by dilution, and no false-positive and false-negative results were observed. The developed assay provides a simple, broad-spectrum, and sensitive tool for detecting PCBs, with high-throughput possibilities for large-scale screening of PCBs in food.

Development of swine immune reagents for analysis of immune correlates for vaccines, infection, and in biomedical research

Abstract The goal of the USDA-NIFA Swine Immune Toolkit Initiative has been to generate priority immune reagents based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. So far in our efforts we express soluble proteins in yeast system and then produce panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. So far we generated several new panels of mAbs reactive to porcine IL-6, IL-13, IFN-γ, IL-17A, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. Reactivity tests of labeled anti-IL-6 and anti-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assay are in progress. Our results have confirmed the reactivity of porcine IL-17A, IFN-γ and CXCL10 mAbs. A sensitive sandwich ELISA is now available for IL-17A, IL-13 and CXCL10; other targets are being screened for best mAb pairs for ELISA. Planning has been initiated for the generation of IL-5 and IL-21 mAbs; and use of SLA-I & -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease, vaccine and biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy

Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identified six amino acid differences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived single-domain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.