Antibodies

24 pages, 3262 KiB

Open AccessEditor’s ChoiceReview

Delivery of Drugs into Cancer Cells Using Antibody–Drug Conjugates Based on Receptor-Mediated Endocytosis and the Enhanced Permeability and Retention Effect

by Toshihiko Tashima

Antibodies 2022, 11(4), 78; https://doi.org/10.3390/antib11040078 - 19 Dec 2022

Cited by 7 | Viewed by 3776

Abstract

Innumerable people worldwide die of cancer every year, although pharmaceutical therapy has actualized many benefits in human health. For background, anti-cancer drug development is difficult due to the multifactorial pathogenesis and complicated pathology of cancers. Cancer cells excrete hydrophobic low-molecular anti-cancer drugs by [...] Read more.

Innumerable people worldwide die of cancer every year, although pharmaceutical therapy has actualized many benefits in human health. For background, anti-cancer drug development is difficult due to the multifactorial pathogenesis and complicated pathology of cancers. Cancer cells excrete hydrophobic low-molecular anti-cancer drugs by overexpressed efflux transporters such as multiple drug resistance 1 (MDR1) at the apical membrane. Mutation-driven drug resistance is also developed in cancer. Moreover, the poor distribution of drug to cancer cells is a serious problem, because patients suffer from off-target side effects. Thus, highly selective and effective drug delivery into solid cancer cells across the membrane should be established. It is known that substances (10–100 nm in diameter) such as monoclonal antibodies (mAbs) (approximately 14.2 nm in diameter) or nanoparticles spontaneously gather in solid tumor stroma or parenchyma through the capillary endothelial fenestration, ranging from 200–2000 nm, in neovasculatures due to the enhanced permeability and retention (EPR) effect. Furthermore, cancer antigens, such as HER2, Nectin-4, or TROP2, highly selectively expressed on the surface of cancer cells act as a receptor for receptor-mediated endocytosis (RME) using mAbs against such antigens. Thus, antibody–drug conjugates (ADCs) are promising anti-cancer pharmaceutical agents that fulfill accurate distribution due to the EPR effect and due to antibody–antigen binding and membrane permeability owing to RME. In this review, I introduce the implementation and possibility of highly selective anti-cancer drug delivery into solid cancer cells based on the EPR effect and RME using anti-cancer antigens ADCs with payloads through suitable linkers. Full article

(This article belongs to the Special Issue Targeting Tumor Cells with Antibodies Enhances Anti-Tumor Immunity)

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11 pages, 1851 KiB

Open AccessArticle

Heparin-Independent and Heparin-Dependent Anti-CXCL4 Antibodies Have a Reciprocal Expression in a Systemic Sclerosis Patients’ Cohort

by Raffaella Palazzo, Katia Stefanantoni, Marius Cadar, Alessia Butera, Valeria Riccieri, Roberto Lande and Loredana Frasca

Antibodies 2022, 11(4), 77; https://doi.org/10.3390/antib11040077 - 15 Dec 2022

Viewed by 1689

Abstract

Systemic sclerosis (SSc) is a chronic disease characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an early SSc biomarker that predicts worse disease outcome. We previously reported that CXCL4 is an autoantigen in SSc, and anti-CXCL4 [...] Read more.

Systemic sclerosis (SSc) is a chronic disease characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an early SSc biomarker that predicts worse disease outcome. We previously reported that CXCL4 is an autoantigen in SSc, and anti-CXCL4 antibodies correlated with IFN-I and were more abundant in patients with lung fibrosis. However, it is unclear whether antibodies to CXCL4 in SSc are only directed to CXCL4 or recognize complexes formed by CXCL4 and heparin. Here, by analyzing an SSc cohort, we addressed the occurrence of circulating heparin-dependent VS heparin-independent anti-CXCL4 antibodies and their relationship with a few disease parameters. We found that heparin-dependent, like the heparin-independent antibodies, are higher in SSc as compared to healthy donors; they are detectable in 24% and 30% of the SSc patients, respectively, and appear inversely correlated and mutually exclusive. Like the heparin-independent antibodies, heparin-dependent antibodies correlated with digital ulcers. However, in contrast to heparin-independent antibodies, heparin-dependent antibodies did not correlate with IFN-I, but were largely expressed in patients with pulmonary arterial hypertension. This pilot study indicates that heparin-dependent antibodies are worth studying in larger SSc cohorts to address whether they discriminate SSc sub-groups with different pathological characteristics and outcomes. Full article

(This article belongs to the Special Issue New, Old, and Shared Antibody Specificities in Autoimmune Diseases)

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10 pages, 926 KiB

Open AccessArticle

SARS-CoV-2 Virus-like Particles (VLPs) Specifically Detect Humoral Immune Reactions in an ELISA-Based Platform

by Stefan Hirschberg, Hannes Bauer, Julian Kamhieh-Milz, Frauke Ringel, Christoph Harms, Omar Kamal Eddin, Axel Pruß, Katja Hanack and Kai Schulze-Forster

Antibodies 2022, 11(4), 76; https://doi.org/10.3390/antib11040076 - 12 Dec 2022

Cited by 2 | Viewed by 2816

Abstract

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, [...] Read more.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen. Full article

(This article belongs to the Special Issue The Role of Antibodies in SARS-CoV-2 Infection)

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11 pages, 3827 KiB

Open AccessArticle

Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry

by Nami Tateyama, Teizo Asano, Hiroyuki Suzuki, Guanjie Li, Takeo Yoshikawa, Tomohiro Tanaka, Mika K. Kaneko and Yukinari Kato

Antibodies 2022, 11(4), 75; https://doi.org/10.3390/antib11040075 - 02 Dec 2022

Cited by 1 | Viewed by 2063

Abstract

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved [...] Read more.

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C₃Mab-3 (rat IgG_2a, kappa), and C₃Mab-4 (rat IgG_2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C₃Mab-3, C₃Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C₃Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C₃Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C₃Mab-3, C₃Mab-4, and J073E5. Full article

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14 pages, 4130 KiB

Open AccessArticle

A Defucosylated Anti-EpCAM Monoclonal Antibody (EpMab-37-mG_2a-f) Exerts Antitumor Activity in Xenograft Model

by Teizo Asano, Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Tomokazu Ohishi, Manabu Kawada, Takeo Yoshikawa, Mika K. Kaneko and Yukinari Kato

Antibodies 2022, 11(4), 74; https://doi.org/10.3390/antib11040074 - 24 Nov 2022

Cited by 8 | Viewed by 2137

Abstract

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor [...] Read more.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG₁, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG_2a-type of EpMab-37 (EpMab-37-mG_2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG_2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (K_D): 2.9 × 10⁻⁸ M and 1.8 × 10⁻⁸ M, respectively] by flow cytometry. EpMab-37-mG_2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG_2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG_2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers. Full article

(This article belongs to the Special Issue Monoclonal Antibody-Directed Therapy Series II)

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25 pages, 480 KiB

Open AccessEditor’s ChoiceReview

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

by Alain Beck, Christine Nowak, Deborah Meshulam, Kristina Reynolds, David Chen, Dennis B. Pacardo, Samantha B. Nicholls, Gregory J. Carven, Zhenyu Gu, Jing Fang, Dongdong Wang, Amit Katiyar, Tao Xiang and Hongcheng Liu

Antibodies 2022, 11(4), 73; https://doi.org/10.3390/antib11040073 - 20 Nov 2022

Cited by 11 | Viewed by 5623

Abstract

Since the first approval of the anti-CD3 recombinant monoclonal antibody (mAb), muromonab-CD3, a mouse antibody for the prevention of transplant rejection, by the US Food and Drug Administration (FDA) in 1986, mAb therapeutics have become increasingly important to medical care. A wealth of [...] Read more.

Since the first approval of the anti-CD3 recombinant monoclonal antibody (mAb), muromonab-CD3, a mouse antibody for the prevention of transplant rejection, by the US Food and Drug Administration (FDA) in 1986, mAb therapeutics have become increasingly important to medical care. A wealth of information about mAbs regarding their structure, stability, post-translation modifications, and the relationship between modification and function has been reported. Yet, substantial resources are still required throughout development and commercialization to have appropriate control strategies to maintain consistent product quality, safety, and efficacy. A typical feature of mAbs is charge heterogeneity, which stems from a variety of modifications, including modifications that are common to many mAbs or unique to a specific molecule or process. Charge heterogeneity is highly sensitive to process changes and thus a good indicator of a robust process. It is a high-risk quality attribute that could potentially fail the specification and comparability required for batch disposition. Failure to meet product specifications or comparability can substantially affect clinical development timelines. To mitigate these risks, the general rule is to maintain a comparable charge profile when process changes are inevitably introduced during development and even after commercialization. Otherwise, new peaks or varied levels of acidic and basic species must be justified based on scientific knowledge and clinical experience for a specific molecule. Here, we summarize the current understanding of mAb charge variants and outline risk-based control strategies to support process development and ultimately commercialization. Full article

8 pages, 242 KiB

Open AccessCase Report

Cross-Reactive Antibodies in Tick-Borne Encephalitis: Case Report and Literature Review

by Tatjana Vilibic-Cavlek, Thomas Ferenc, Mateja Vujica Ferenc, Maja Bogdanic, Tanja Potocnik-Hunjadi, Dario Sabadi, Vladimir Savic, Ljubo Barbic, Vladimir Stevanovic, Federica Monaco, Eddy Listes and Giovanni Savini

Antibodies 2022, 11(4), 72; https://doi.org/10.3390/antib11040072 - 20 Nov 2022

Cited by 5 | Viewed by 1962

Abstract

Flaviviruses are a heterogeneous group of viruses that may induce broad antigenic cross-reactivity. We present a patient who was admitted to the infectious disease department with symptoms suggestive of aseptic meningitis. During the clinical workup, the patient reported a tick bite two weeks [...] Read more.

Flaviviruses are a heterogeneous group of viruses that may induce broad antigenic cross-reactivity. We present a patient who was admitted to the infectious disease department with symptoms suggestive of aseptic meningitis. During the clinical workup, the patient reported a tick bite two weeks before the disease onset. High titers of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) were found in both serum and cerebrospinal fluid (CSF) samples, indicating acute TBEV infection. West Nile virus (WNV) and Usutu virus (USUV) IgM and/or IgG antibodies were also detected, and a virus neutralization test (VNT) was performed. A high titer of TBEV neutralizing (NT) antibodies (640) was detected, which confirmed acute TBE. However, NT antibodies to WNV and USUV were also detected (titer 80 for both viruses). After TBEV and WNV IgG avidity evaluation, previous flavivirus infection was highly suspected (avidity index 82% and 89%, respectively). Blood, CSF, and urine samples were negative for respective viruses’ RNA. The presented case highlights the challenges in flavivirus serodiagnosis. In the published literature, different degrees of cross-reactivity or cross-neutralization between TBEV and dengue, louping ill, Omsk hemorrhagic fever, Langat, and Powassan virus were also observed. Therefore, the serology results should be interpreted with caution, including the possibility of cross-reactivity. In areas where several flaviviruses co-circulate VNT is recommended for disease confirmation. Full article

16 pages, 2251 KiB

Open AccessEditor’s ChoiceReview

Structural Investigation of Therapeutic Antibodies Using Hydroxyl Radical Protein Footprinting Methods

by Corie Y. Ralston and Joshua S. Sharp

Antibodies 2022, 11(4), 71; https://doi.org/10.3390/antib11040071 - 14 Nov 2022

Cited by 7 | Viewed by 2470

Abstract

Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to [...] Read more.

Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to refine antibody design. However, many standard structural techniques require specialized sample preparation that may perturb antibody structure or require high concentrations or other conditions that are far from the conditions conducive to the accurate determination of antigen binding or kinetics. We describe here in this minireview the relatively new method of hydroxyl radical protein footprinting, a solution-state method that can provide structural and kinetic information on antibodies or antibody–antigen interactions useful for therapeutic antibody design. We provide a brief history of hydroxyl radical footprinting, examples of current implementations, and recent advances in throughput and accessibility. Full article

(This article belongs to the Special Issue Higher Order Structure Characterization of Therapeutic Antibodies-Second Volume)

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8 pages, 516 KiB

Open AccessEditor’s ChoiceArticle

Neutralizing and Total/IgG Spike Antibody Responses Following Homologous CoronaVac vs. BNT162b2 Vaccination Up to 90 Days Post-Booster

by Chin Shern Lau, John Thundyil, May Lin Helen Oh, Soon Kieng Phua, Ya Li Liang, Yanfeng Li, Jianxin Huo, Yuhan Huang, Biyan Zhang, Shengli Xu and Tar Choon Aw

Antibodies 2022, 11(4), 70; https://doi.org/10.3390/antib11040070 - 08 Nov 2022

Cited by 4 | Viewed by 1787

Abstract

Introduction: We documented the total spike antibody (S-Ab), IgG S-Ab and neutralizing antibody (N-Ab) responses of BNT162b2/CoronaVac vaccinees up to 90 days post-booster dose. Methods: We included 32 homologous regimen CoronaVac vaccinees and 136 BNT162b2 mRNA vaccinees. We tested their total S-Ab (Roche), [...] Read more.

Introduction: We documented the total spike antibody (S-Ab), IgG S-Ab and neutralizing antibody (N-Ab) responses of BNT162b2/CoronaVac vaccinees up to 90 days post-booster dose. Methods: We included 32 homologous regimen CoronaVac vaccinees and 136 BNT162b2 mRNA vaccinees. We tested their total S-Ab (Roche), IgG (Abbott) and N-Ab (Snibe) levels at set time points from January 2021 to April 2022. All subjects were deemed to be COVID-19-naïve either via clinical history (CoronaVac vaccinees) or nucleocapsid antibody testing (BNT162b2 vaccinees). Results: All antibodies peaked 20–30 days post-inoculation. In BNT162b2 vaccinees, all post-booster antibodies were significantly higher than second-dose peaks. In CoronaVac vaccinees, IgG showed no significant differences between peak third-/second-dose titers (difference of 56.0 BAU/mL, 95% CI of −17.1 to 129, p = 0.0894). The post-vaccination titers of all antibodies in BNT162b2 vaccinees were significantly higher than those in CoronaVac vaccinees at all time points. Post-booster, all antibodies declined in 90 days; the final total/IgG/N-Ab titers were 7536 BAU/mL, 1276 BAU/mL and 12.5 μg/mL in BNT162b2 vaccinees and 646 BAU/mL, 62.4 BAU/mL and 0.44 μg/mL in CoronaVac vaccinees. Conclusion: The mRNA vaccine generated more robust total S-Ab, IgG and N-Ab responses after the second and third vaccinations. Full article

(This article belongs to the Special Issue The Role of Antibodies in SARS-CoV-2 Infection)

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13 pages, 711 KiB

Open AccessArticle

Seroprevalence of Anti-SARS-CoV-2 Antibodies in Chattogram Metropolitan Area, Bangladesh

by Jahan Ara, Md. Sirazul Islam, Md. Tarek Ul Quader, Anan Das, F. M. Yasir Hasib, Mohammad Saiful Islam, Tazrina Rahman, Seemanta Das, M. A. Hassan Chowdhury, Goutam Buddha Das and Sharmin Chowdhury

Antibodies 2022, 11(4), 69; https://doi.org/10.3390/antib11040069 - 07 Nov 2022

Cited by 3 | Viewed by 2622

Abstract

Seroprevalence studies of COVID-19 are used to assess the degree of undetected transmission in the community and different groups such as health care workers (HCWs) are deemed vulnerable due to their workplace hazards. The present study estimated the seroprevalence and quantified the titer [...] Read more.

Seroprevalence studies of COVID-19 are used to assess the degree of undetected transmission in the community and different groups such as health care workers (HCWs) are deemed vulnerable due to their workplace hazards. The present study estimated the seroprevalence and quantified the titer of anti-SARS-CoV-2 antibody (IgG) and its association with different factors. This cross-sectional study observed HCWs, in indoor and outdoor patients (non-COVID-19) and garment workers in the Chattogram metropolitan area (CMA, N = 748) from six hospitals and two garment factories. Qualitative and quantitative ELISA were used to identify and quantify antibodies (IgG) in the serum samples. Descriptive, univariable, and multivariable statistical analysis were performed. Overall seroprevalence and among HCWs, in indoor and outdoor patients, and garment workers were 66.99% (95% CI: 63.40–70.40%), 68.99% (95% CI: 63.8–73.7%), 81.37% (95% CI: 74.7–86.7%), and 50.56% (95% CI: 43.5–57.5%), respectively. Seroprevalence and mean titer was 44.47% (95% CI: 38.6–50.4%) and 53.71 DU/mL in the non-vaccinated population, respectively, while it was higher in the population who received a first dose (61.66%, 95% CI: 54.8–68.0%, 159.08 DU/mL) and both doses (100%, 95% CI: 98.4–100%, 255.46 DU/mL). This study emphasizes the role of vaccine in antibody production; the second dose of vaccine significantly increased the seroprevalence and titer and both were low in natural infection. Full article

(This article belongs to the Special Issue Coronavirus-Targeting Antibodies)

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39 pages, 3622 KiB

Open AccessArticle

Shared 6mer Peptides of Human and Omicron (21K and 21L) at SARS-CoV-2 Mutation Sites

by Yekbun Adiguzel and Yehuda Shoenfeld

Antibodies 2022, 11(4), 68; https://doi.org/10.3390/antib11040068 - 25 Oct 2022

Cited by 2 | Viewed by 1809

Abstract

We investigated the short sequences involving Omicron 21K and Omicron 21L variants to reveal any possible molecular mimicry-associated autoimmunity risks and changes in those. We first identified common 6mers of the viral and human protein sequences present for both the mutant (Omicron) and [...] Read more.

We investigated the short sequences involving Omicron 21K and Omicron 21L variants to reveal any possible molecular mimicry-associated autoimmunity risks and changes in those. We first identified common 6mers of the viral and human protein sequences present for both the mutant (Omicron) and nonmutant (SARS-CoV-2) versions of the same viral sequence and then predicted the binding affinities of those sequences to the HLA supertype representatives. We evaluated change in the potential autoimmunity risk, through comparative assessment of the nonmutant and mutant viral sequences and their similar human peptides with common 6mers and affinities to the same HLA allele. This change is the lost and the new, or de novo, autoimmunity risk, associated with the mutations in the Omicron 21K and Omicron 21L variants. Accordingly, e.g., the affinity of virus-similar sequences of the Ig heavy chain junction regions shifted from the HLA-B*15:01 to the HLA-A*01:01 allele at the mutant sequences. Additionally, peptides of different human proteins sharing 6mers with SARS-CoV-2 proteins at the mutation sites of interest and with affinities to the HLA-B*07:02 allele, such as the respective SARS-CoV-2 sequences, were lost. Among all, any possible molecular mimicry-associated novel risk appeared to be prominent in HLA-A*24:02 and HLA-B*27:05 serotypes upon infection with Omicron 21L. Associated disease, pathway, and tissue expression data supported possible new risks for the HLA-B*27:05 and HLA-A*01:01 serotypes, while the risks for the HLA-B*07:02 serotypes could have been lost or diminished, and those for the HLA-A*03:01 serotypes could have been retained, for the individuals infected with Omicron variants under study. These are likely to affect the complications related to cross-reactions influencing the relevant HLA serotypes upon infection with Omicron 21K and Omicron 21L. Full article

(This article belongs to the Special Issue The Role of Antibodies in SARS-CoV-2 Infection)

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Graphical abstract

14 pages, 2653 KiB

Open AccessEditor’s ChoiceArticle

Rational Design and In Vivo Characterization of mRNA-Encoded Broadly Neutralizing Antibody Combinations against HIV-1

by Elisabeth Narayanan, Samantha Falcone, Sayda M. Elbashir, Husain Attarwala, Kimberly Hassett, Michael S. Seaman, Andrea Carfi and Sunny Himansu

Antibodies 2022, 11(4), 67; https://doi.org/10.3390/antib11040067 - 24 Oct 2022

Cited by 5 | Viewed by 3405

Abstract

Monoclonal antibodies have been used successfully as recombinant protein therapy; however, for HIV, multiple broadly neutralizing antibodies may be necessary. We used the mRNA-LNP platform for in vivo co-expression of 3 broadly neutralizing antibodies, PGDM1400, PGT121, and N6, directed against the HIV-1 envelope [...] Read more.

Monoclonal antibodies have been used successfully as recombinant protein therapy; however, for HIV, multiple broadly neutralizing antibodies may be necessary. We used the mRNA-LNP platform for in vivo co-expression of 3 broadly neutralizing antibodies, PGDM1400, PGT121, and N6, directed against the HIV-1 envelope protein. mRNA-encoded HIV-1 antibodies were engineered as single-chain Fc (scFv-Fc) to overcome heavy- and light-chain mismatch. In vitro neutralization breadth and potency of the constructs were compared to their parental IgG form. We assessed the ability of these scFv-Fcs to be expressed individually and in combination in vivo, and neutralization and pharmacokinetics were compared to the corresponding full-length IgGs. Single-chain PGDM1400 and PGT121 exhibited neutralization potency comparable to parental IgG, achieving peak systemic concentrations ≥ 30.81 μg/mL in mice; full-length N6 IgG achieved a peak concentration of 974 μg/mL, but did not tolerate single-chain conversion. The mRNA combination encoding full-length N6 IgG and single-chain PGDM1400 and PGT121 was efficiently expressed in mice, achieving high systemic concentration and desired neutralization potency. Analysis of mice sera demonstrated each antibody contributed towards neutralization of multiple HIV-1 pseudoviruses. Together, these data show that the mRNA-LNP platform provides a promising approach for antibody-based HIV treatment and is well-suited for development of combination therapeutics. Full article

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

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12 pages, 918 KiB

Open AccessArticle

A Simple Method for the Prediction of Therapeutic Proteins (Monoclonal and Polyclonal Antibodies and Non-Antibody Proteins) for First-in-Pediatric Dose Selection: Application of Salisbury Rule

by Iftekhar Mahmood

Antibodies 2022, 11(4), 66; https://doi.org/10.3390/antib11040066 - 21 Oct 2022

Cited by 2 | Viewed by 1926

Abstract

In order to conduct a pediatric clinical trial, it is important to optimize pediatric dose as accurately as possible. In this study, a simple weight-based method known as ‘Salisbury Rule’ was used to predict pediatric dose for therapeutic proteins and was then compared [...] Read more.

In order to conduct a pediatric clinical trial, it is important to optimize pediatric dose as accurately as possible. In this study, a simple weight-based method known as ‘Salisbury Rule’ was used to predict pediatric dose for therapeutic proteins and was then compared with the observed pediatric dose. The observed dose was obtained mainly from the FDA package insert and if dosing information was not available from the FDA package insert then the observed dose was based on the dose given to an age group in a particular study. It was noted that the recommended doses of most of the therapeutic proteins were extrapolated to pediatrics from adult dose based on per kilogram (kg) body weight basis. Since it is widely believed that pediatric dose should be selected based on the pediatric clearance (CL), a CL based pediatric dose was projected from the following equation: Dose in children = Adult dose × (Observed CL in children/Observed adult CL). In this study, this dose was also considered observed pediatric dose for comparison. A ±30% prediction error (predicted vs. observed) was considered acceptable. There were 21 monoclonal antibodies, 5 polyclonal antibodies in children ≥ 2 years of age, 4 polyclonal antibodies in preterm and term neonates, and 11 therapeutic proteins (non-antibodies) in the study. In children < 30 kg body weight, the predicted doses were within 0.5–1.5-fold prediction error for 87% (monoclonal antibody), 100% (polyclonal antibody), and 92% (non-antibodies) observations. In children > 30 kg body weight, the predicted doses were within 0.5–1.5-fold prediction error for 96% (monoclonal antibody), 100% (polyclonal antibody), and 100% (non-antibodies) observations. The Salisbury Rule mimics more to CL-based dose rather than per kg body weight-based extrapolated dose from adults. The Salisbury Rule for the pediatric dose prediction can be used to select first-in-children dose in pediatric clinical trials and may be in clinical settings. Full article

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45 pages, 4126 KiB

Open AccessEditor’s ChoiceArticle

IMGT^® Nomenclature of Engineered IGHG Variants Involved in Antibody Effector Properties and Formats

by Marie-Paule Lefranc and Gérard Lefranc

Antibodies 2022, 11(4), 65; https://doi.org/10.3390/antib11040065 - 18 Oct 2022

Cited by 3 | Viewed by 2874

Abstract

The constant region of the immunoglobulin (IG) or antibody heavy gamma chain is frequently engineered to modify the effector properties of the therapeutic monoclonal antibodies. These variants are classified in regards to their effects on effector functions, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), [...] Read more.

The constant region of the immunoglobulin (IG) or antibody heavy gamma chain is frequently engineered to modify the effector properties of the therapeutic monoclonal antibodies. These variants are classified in regards to their effects on effector functions, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) enhancement or reduction, B cell inhibition by the coengagement of antigen and FcγR on the same cell, on half-life increase, and/or on structure such as prevention of IgG4 half-IG exchange, hexamerisation, knobs-into-holes and the heteropairing H-H of bispecific antibodies, absence of disulfide bridge inter H-L, absence of glycosylation site, and site-specific drug attachment engineered cysteine. The IMGT engineered variant identifier is comprised of the species and gene name (and eventually allele), the letter ‘v’ followed by a number (assigned chronologically), and for each concerned domain (e.g, CH1, h, CH2 and CH3), the novel AA (single letter abbreviation) and IMGT position according to the IMGT unique numbering for the C-domain and between parentheses, the Eu numbering. IMGT engineered variants are described with detailed amino acid changes, visualized in motifs based on the IMGT numbering bridging genes, sequences, and structures for higher order description. Full article

(This article belongs to the Special Issue Higher Order Structure Characterization of Therapeutic Antibodies-Second Volume)

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10 pages, 1067 KiB

Open AccessArticle

Integrating Single Domain Antibodies into Field-Deployable Rapid Assays

by George P. Anderson, Lisa C. Shriver-Lake, Jinny L. Liu and Ellen R. Goldman

Antibodies 2022, 11(4), 64; https://doi.org/10.3390/antib11040064 - 17 Oct 2022

Cited by 1 | Viewed by 2019

Abstract

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to [...] Read more.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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5 pages, 198 KiB

Open AccessCase Report

Alloimmune Neutropenia in a Neonate: Case Report and Review of Literature

by Arun Prasath, Alanna Grafius, Mona Bonanno, Steven Ambrusko and Jayasree Nair

Antibodies 2022, 11(4), 63; https://doi.org/10.3390/antib11040063 - 03 Oct 2022

Cited by 1 | Viewed by 2042

Abstract

Neonatal alloimmune neutropenia, variably referred to in the literature as NAIN, FNAIN or NIN, is a disorder of neutrophil destruction in newborns similar to better-known conditions such as hemolytic disease of the newborn and neonatal alloimmune thrombocytopenia (FNAIT). Infants affected by this self-limiting [...] Read more.

Neonatal alloimmune neutropenia, variably referred to in the literature as NAIN, FNAIN or NIN, is a disorder of neutrophil destruction in newborns similar to better-known conditions such as hemolytic disease of the newborn and neonatal alloimmune thrombocytopenia (FNAIT). Infants affected by this self-limiting condition can present asymptomatically or have a wide range of symptoms, from skin manifestations and mucositis to severe infections such as sepsis and pneumonia. In our case, we report an otherwise asymptomatic term infant born with severe neutropenia to a mother affected by COVID-19 in the 3rd trimester. However, it is unclear if COVID-19 contributed to our patients’ neutropenia. Diagnostic testing eventually revealed the presence of anti-neutrophil antibodies, confirming the diagnosis of alloimmune neutropenia. The infant was conservatively managed with early discharge prior to resolution of neutropenia and close post-discharge follow up. Full article

9 pages, 869 KiB

Open AccessEditor’s ChoiceReview

IgY Antibodies as Biotherapeutics in Biomedicine

by Diana León-Núñez, María Fernanda Vizcaíno-López, Magdalena Escorcia, Dolores Correa, Elizabeth Pérez-Hernández and Fernando Gómez-Chávez

Antibodies 2022, 11(4), 62; https://doi.org/10.3390/antib11040062 - 29 Sep 2022

Cited by 8 | Viewed by 4773

Abstract

Since the discovery of antibodies by Emil Von Behring and Shibasaburo Kitasato during the 19th century, their potential for use as biotechnological reagents has been exploited in different fields, such as basic and applied research, diagnosis, and the treatment of multiple diseases. Antibodies [...] Read more.

Since the discovery of antibodies by Emil Von Behring and Shibasaburo Kitasato during the 19th century, their potential for use as biotechnological reagents has been exploited in different fields, such as basic and applied research, diagnosis, and the treatment of multiple diseases. Antibodies are relatively easy to obtain from any species with an adaptive immune system, but birds are animals characterized by relatively easy care and maintenance. In addition, the antibodies they produce can be purified from the egg yolk, allowing a system for obtaining them without performing invasive practices, which favors the three “rs” of animal care in experimentation, i.e., replacing, reducing, and refining. In this work, we carry out a brief descriptive review of the most outstanding characteristics of so-called “IgY technology” and the use of IgY antibodies from birds for basic experimentation, diagnosis, and treatment of human beings and animals. Full article

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

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17 pages, 5595 KiB

Open AccessArticle

Yeast Surface Display Platform for Rapid Selection of an Antibody Library via Sequential Counter Antigen Flow Cytometry

by Bhupal Ban, Robert C. Blake II and Diane A. Blake

Antibodies 2022, 11(4), 61; https://doi.org/10.3390/antib11040061 - 27 Sep 2022

Cited by 2 | Viewed by 4359

Abstract

Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules [...] Read more.

Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules (haptens). In this approach, we coupled 4 to 15 sequential cycles of error-prone PCR to introduce heterogeneity into the sequence of an 12F6 scFv antibody that binds to chelated uranium; the resulting full-length constructs were combined to create a yeast-displayed scFv-library with high diversity. We also developed a stringent selection technique utilizing fluorescence-activated cell sorting; this was based on sequentially dropping the target antigen concentration, while concomitantly increasing the concentration of potential cross-reactive haptens in subsequent selection cycles. As a proof of the efficacy this approach, we confirmed that the antibodies identified via this approach retained binding to the target antigen (UO₂²⁺ complexed to a chelator), while binding with lesser affinity than the parental scFv to a structurally related haptens (the same chelator complexed to other metal ions). As will be described in this report, these scFv variants perform more efficiently in sensor-based assay than the parental 12F6 antibody. Combining the generation of scFv libraries via error-prone PCR with selection of yeast-displayed antibodies by fluorescence activated cell sorting will provide an efficient new method for the isolation of scFvs and other binding proteins with high affinity and specificity. Full article

(This article belongs to the Section Antibody Discovery and Engineering)

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15 pages, 5034 KiB

Open AccessArticle

High-Resolution Epitope Mapping and Affinity Binding Analysis Comparing a New Anti-Human LAG3 Rabbit Antibody Clone to the Commonly Used Mouse 17B4 Clone

by P. Daniel Warren, Mark S. Dodson, Margaret H. Smith, Terry H. Landowski, John Douglas Palting and Penny Towne

Antibodies 2022, 11(4), 60; https://doi.org/10.3390/antib11040060 - 21 Sep 2022

Cited by 3 | Viewed by 2291

Abstract

Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applications. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare [...] Read more.

Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applications. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare the binding characteristics of a new anti-LAG3 rabbit antibody clone, SP464, with the thirty-year old and extensively used anti-LAG3 mouse 17B4 clone. The rabbit SP464 clone exhibited between 20× to 30× greater binding to LAG3 than did the mouse 17B4 clone. Using these tools, we precisely mapped the relative locations of the epitopes of these two antibodies. The SP464 and 17B4 minimal epitopes were localized to separate, but overlapping, sub-fragments within the amino-terminal fifteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Application of this approach for quantifying the effects of alanine substitutions along the minimal SP464 epitope identified two amino acids essential for binding and four amino acids that likely contribute towards binding. Together, ACE, SPR, and IHC constitute a powerful orthologous approach for comparing antibody-binding characteristics and for fine mapping of linear epitopes within short immunogens. Our results indicate that the rabbit clone SP464 may be useful for assessing LAG3 expression. Full article

(This article belongs to the Special Issue Monoclonal Antibody-Directed Therapy Series II)

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Antibodies, Volume 11, Issue 4 (December 2022) – 19 articles

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