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Antibodies, Volume 13, Issue 3 (September 2024) – 6 articles

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25 pages, 1969 KiB

Open AccessArticle

NK Cytotoxicity Mediated by NK-92 Cell Lines Expressing Combinations of Two Allelic Variants for FCGR3

by Marta Freitas Monteiro, Maria Papaserafeim, Matteo Andreani, Aline Réal, Athanasios Kouklas, Daniela Reis Galvão, Jörg D. Seebach and Gisella L. Puga Yung

Antibodies 2024, 13(3), 55; https://doi.org/10.3390/antib13030055 - 12 Jul 2024

Viewed by 66

Abstract

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by ligands of activating/inhibitory NK receptors, respectively. Allelic variants of the FCGR3A gene include the high-affinity single-nucleotide polymorphism (SNP) rs396991 (V176F), which is associated with the efficacy of monoclonal antibody (mAb) therapies, and the SNP rs10127939 (L66H/R). The contribution of FCGR3A SNPs to NK cell effector functions remains controversial; therefore, we generated a panel of eight NK-92 cell lines expressing specific combinations of these SNPs and tested their cytotoxicities. NK-92 cells were stably transfected with plasmids containing different combinations of FCGR3A SNPs. Messenger RNA and FcγRIIIa/CD16 cell surface expressions were detected using new generation sequencing (NGS) and flow cytometry, respectively. All FcγRIIIa/CD16-transfected NK-92 cell lines exhibited robust ADCC against three different target cell lines with minor differences. In addition, enhanced direct NK cytotoxicity against K562 target cells was observed, suggesting a mechanistic role of FcγRIIIa/CD16 in direct NK cytotoxicity. In conclusion, we generated eight FcγRIIIa/CD16-transfected NK-92 cell lines carrying different combinations of two of the most studied FCGR3A SNPs, representing the major genotypes described in the European population. The functional characterization of these cell lines revealed differences in ADCC and direct NK cytotoxicity that may have implications for the design of adoptive cancer immunotherapies using NK cells and tumor antigen-directed mAbs. Full article

(This article belongs to the Special Issue Emerging Antibody Engineering Strategies and Applications for Immunotherapy of Cancer)

25 pages, 7363 KiB

Open AccessArticle

Inter-Antibody Variability in the Clinical Pharmacokinetics of Monoclonal Antibodies Characterized Using Population Physiologically Based Pharmacokinetic Modeling

by Mokshada Kumar, Sravani Lanke, Alka Yadav, Mfonabasi Ette, Donald E. Mager and Dhaval K. Shah

Antibodies 2024, 13(3), 54; https://doi.org/10.3390/antib13030054 - 9 Jul 2024

Viewed by 1014

Abstract

The objective of this work was to develop a population physiologically based pharmacokinetic (popPBPK) model to characterize the variability in the clinical PK of monoclonal antibodies (mAbs) following intravenous (IV) and subcutaneous (SC) administration. An extensive literature search was conducted and clinical PK data for FDA-approved as well as non-approved mAbs were collected. Training and validation datasets of 44 and 9 mAbs exhibiting linear pharmacokinetics were used for model development. The variability in antibody PK was captured by accounting for different rate constants of pinocytosis (CL_up) and intracellular degradation (k_deg) for different mAbs. Typical values for CL_up and k_deg and their respective inter-antibody variabilities (

ω_{C l u p}

ω_{K d e g})

were estimated to be 0.32 L/h/L and 26.1

h^{- 1}

(73% and 46%). Varied absorption profiles following SC dosing were characterized by incorporating inter-antibody variability in local degradation (k_SC) and rate of lymphatic uptake (S_Lu) of mAbs. Estimates for typical k_SC and S_Lu values, and

ω_{K s c}, ω_{S_L u},

were found to be 0.0015

h^{- 1}

and 0.54 (193%, and 49%). FDA-approved mAbs showed less local degradation (0.0014

h^{- 1}

vs. 0.0038

h^{- 1}

) compared with other clinically tested mAbs, whereas no substantial differences in physiological processes involved in disposition were observed. To evaluate the generalizability of estimated PK parameters and model validation, the final popPBPK model was used to simulate the range of expected PK for mAbs following SC administration of nine different mAbs that were not used for model-building purposes. The predicted PK of all nine mAbs was within the expected range specified a priori. Thus, the popPBPK model presented here may serve as a tool to predict the clinical PK of mAbs with linear disposition before administering them to humans. The model may also support preclinical-to-clinical translation and ‘first-in-human’ dose determination for mAbs. Full article

(This article belongs to the Section Antibody-Based Therapeutics)

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8 pages, 769 KiB

Open AccessCommunication

A Bead-Based Nonradioactive Immunoassay for Autoantibody Testing in a Mouse Model of Myasthenia Gravis

by Afrin Bahauddin, Kyra Curtis, Jutatip Guptarak and Ruksana Huda

Antibodies 2024, 13(3), 53; https://doi.org/10.3390/antib13030053 - 1 Jul 2024

Viewed by 378

Abstract

Serological testing for anti-acetylcholine receptor (AChR) autoantibodies is not only crucial for the diagnosing, disease monitoring, and treatment management of patients with myasthenia gravis (MG) but also for preclinical studies utilizing MG disease models. However, there are no specific guidelines on which methods to use in clinical diagnostic or research laboratories to detect or quantify any MG-specific autoantibodies. Conventional autoantibody assays, particularly those for anti-AChR antibodies, are varied and mostly laboratory-specific. Here, we report our new nonradioactive immunoprecipitation–immunoblotting method for assessing autoantibodies (anti-AChR antibodies) in a mouse model of MG. This simple, efficient, reproducible, and cost-effective assay appears superior to the enzyme-linked immunosorbent assay but comparable to the radioimmunoprecipitation or cell-based assay in specificity and sensitivity. Thus, the newly developed assay can serve as a valuable alternative to classical assays and is suitable for routine testing of AChR-specific autoantibodies in preclinical studies. The further optimization of our assay may facilitate its application in the diagnosis and therapeutic management of patients with MG. Full article

(This article belongs to the Section Antibody-Based Diagnostics)

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21 pages, 3608 KiB

Open AccessArticle

Characterization of the Charge Heterogeneity of a Monoclonal Antibody That Binds to Both Cation Exchange and Anion Exchange Columns under the Same Binding Conditions

by Ming-Ching Hsieh, Jingming Zhang, Liangjie Tang, Cheng-Yen Huang, Yang Shen, Alice Matathia, Jun Qian and Babita Saxena Parekh

Antibodies 2024, 13(3), 52; https://doi.org/10.3390/antib13030052 - 30 Jun 2024

Viewed by 412

Abstract

Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody’s surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography. Full article

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15 pages, 8868 KiB

Open AccessArticle

¹⁷⁷Lu Anti-Angiogenic Radioimmunotherapy Targeting ATP Synthase in Gastric Cancer Model

by Bok-Nam Park, Young-Sil An, Su-Min Kim, Su-Jin Lee, Yong-Jin Park and Joon-Kee Yoon

Antibodies 2024, 13(3), 51; https://doi.org/10.3390/antib13030051 - 27 Jun 2024

Viewed by 278

Abstract

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate synthase (ATPS) monoclonal antibody (mAb) with the radioisotope ¹⁷⁷Lu using DOTA as a chelating agent. ¹⁷⁷Lu-DOTA-ATPS mAb demonstrated high labeling efficiency (99.0%) and stability in serum. MKN-45 cancer cells exhibited the highest cellular uptake, which could be specifically blocked by unlabeled ATPS mAb. In mice, ¹⁷⁷Lu-DOTA-ATPS mAb accumulated significantly in tumors, with a tumor uptake of 16.0 ± 1.5%ID/g on day 7. ¹⁷⁷Lu-DOTA-ATPS mAb treatment significantly reduced the viability of MKN-45 cells in a dose-dependent manner. In a xenograft tumor model, this radioimmunotherapy strategy led to substantial tumor growth inhibition (82.8%). Furthermore, combining ¹⁷⁷Lu-DOTA-ATPS mAb with sunitinib, an anti-angiogenic drug, enhanced the therapeutic efficacy of sunitinib in the mouse model. Our study successfully developed ¹⁷⁷Lu-DOTA-ATPS mAb, a radioimmunotherapy agent targeting tumor blood vessels. This approach demonstrates significant promise for inhibiting tumor growth, both as a single therapy and in combination with other anti-cancer drugs. Full article

(This article belongs to the Special Issue Emerging Antibody Engineering Strategies and Applications for Immunotherapy of Cancer)

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20 pages, 2248 KiB

Open AccessArticle

Development, Optimization and Evaluation of a Sensitive Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detection of Chicken-Based IgY Polyclonal Antibodies against Toxins of D. polylepis Venom

by Stephen Wilson Kpordze, Gideon Mutie Kikuvi, James Hungo Kimotho and Victor Atunga Mobegi

Antibodies 2024, 13(3), 50; https://doi.org/10.3390/antib13030050 - 21 Jun 2024

Cited by 1 | Viewed by 450

Abstract

Life-threatening medical issues can result from snakebite, and hence this is a public health concern. In many tropical and subtropical nations such as Kenya, where a wide variety of poisonous snakes are prevalent, diagnosis of snakebite in health facilities is imperative. Different antivenoms are needed to treat the venom of different snake species. Nonetheless, it might be difficult for medical professionals to identify the exact snake species that envenomated a patient due to the similarities of several snake envenomations’ clinical symptoms. Therefore, the necessity for an assay or technique for identifying venomous species is critical. The current study sought to develop a sensitive ELISA prototype for the detection of D. polylepis venom in Kenya using generated chicken-based IgY polyclonal antibodies. Serum samples containing specific chicken-based IgY antibodies previously raised against D. polylepis venom toxins were used in the assay development. ELISA parameters were optimized, and the developed assay was assessed for applicability. The limit of detection (LoD) of the ELISA for neurotoxic venoms was determined to be 0.01 µg/mL. Successful discrimination between neurotoxic and cytotoxic venoms was achieved by the ensuing inhibition ELISA assay. The developed assay showed the capability of identifying venoms in blood samples (from spiked and venom-challenged blood samples) of BALB/c mice, providing compelling evidence of the strategy’s usefulness. This assay could help physicians diagnose and manage victims of snakebites through the evaluation of clinical samples. Full article

(This article belongs to the Section Antibody-Based Diagnostics)

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