Antibodies

11 pages, 3827 KiB

Open AccessEditor’s ChoiceArticle

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 - 2 Dec 2022

Cited by 5 | Viewed by 2656

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 AccessEditor’s ChoiceArticle

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 2741

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 19 | Viewed by 7704

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

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 8 | Viewed by 3233

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 - 8 Nov 2022

Cited by 5 | Viewed by 2319

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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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 8 | Viewed by 4610

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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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 6 | Viewed by 3807

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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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 14 | Viewed by 7215

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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15 pages, 5034 KiB

Open AccessEditor’s ChoiceArticle

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 2890

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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11 pages, 556 KiB

Open AccessEditor’s ChoiceArticle

Investigation of Possible Factors Influencing the Neutralizing Anti-SARS-CoV-2 Antibody Titer after Six Months from the Second Vaccination Dose in a Sample of Italian Nursing Home Personnel

by Alberto Modenese, Stefania Paduano, Rossana Bellucci, Simona Marchetti, Fulvio Bruno, Pietro Grazioli, Roberto Vivoli, Fabriziomaria Gobba and Annalisa Bargellini

Antibodies 2022, 11(3), 59; https://doi.org/10.3390/antib11030059 - 19 Sep 2022

Cited by 4 | Viewed by 2385

Abstract

The titer of the anti-SARS-CoV-2 antibodies produced after vaccination shows a relevant decay over time, as demonstrated in several studies. However, less is known on the possible factors affecting the entity of this decay. The aim of this study is to analyze a [...] Read more.

The titer of the anti-SARS-CoV-2 antibodies produced after vaccination shows a relevant decay over time, as demonstrated in several studies. However, less is known on the possible factors affecting the entity of this decay. The aim of this study is to analyze a group of individual factors which are possibly associated with anti-SARS-CoV-2 antibody titer decay six months after the second vaccine dose. We report here the results of a follow-up serological analysis and a questionnaire-based evaluation of a sample of workers from an Italian nursing home, vaccinated with two doses of BNT162b2 vaccine in early 2021. The baseline data were collected one month after the vaccine, while in the present analysis we report the data collected six months later. Our data show a relevant decay of the neutralizing antibody titer, even if for all the workers a largely positive response was detected. Moreover, our results demonstrate a possible association between younger age and the absence of previous COVID-19 infection, and a higher decay rate of the anti-SARS-CoV-2 antibodies titer. Full article

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

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13 pages, 2187 KiB

Open AccessEditor’s ChoiceArticle

In Vitro and In Vivo Characterization of a Broadly Neutralizing Anti-SARS-CoV-2 Antibody Isolated from a Semi-Immune Phage Display Library

by Edith González-González, Gregorio Carballo-Uicab, Juana Salinas-Trujano, María I. Cortés-Paniagua, Said Vázquez-Leyva, Luis Vallejo-Castillo, Ivette Mendoza-Salazar, Keyla Gómez-Castellano, Sonia M. Pérez-Tapia and Juan C. Almagro

Antibodies 2022, 11(3), 57; https://doi.org/10.3390/antib11030057 - 6 Sep 2022

Cited by 4 | Viewed by 3207

Abstract

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 [...] Read more.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 have shown reduced efficacy or have failed to neutralize the variants of concern (VOCs), particularly B.1.1.529 (Omicron). Previously, we reported the discovery and characterization of antibodies with high affinity for SARS-CoV-2 RBD Wuhan (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) strains. One of the antibodies, called IgG-A7, also blocked the interaction of human angiotensin-converting enzyme 2 (hACE2) with the RBDs of the three strains, suggesting it may be a broadly SARS-CoV-2 neutralizing antibody. Herein, we show that IgG-A7 efficiently neutralizes all the three SARS-CoV-2 strains in plaque reduction neutralization tests (PRNTs). In addition, we demonstrate that IgG-A7 fully protects K18-hACE2 transgenic mice infected with SARS-CoV-2 WT. Taken together, our findings indicate that IgG-A7 could be a suitable candidate for development of antibody-based drugs to treat and/or prevent SARS-CoV-2 VOCs infection. Full article

(This article belongs to the Topic Host Response against SARS-CoV-2 Infection: Implications for Diagnosis, Treatment and Vaccine Development)

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25 pages, 1041 KiB

Open AccessEditor’s ChoiceReview

Alternative Routes of Administration for Therapeutic Antibodies—State of the Art

by Aubin Pitiot, Nathalie Heuzé-Vourc’h and Thomas Sécher

Antibodies 2022, 11(3), 56; https://doi.org/10.3390/antib11030056 - 26 Aug 2022

Cited by 22 | Viewed by 8634

Abstract

Background: For the past two decades, there has been a huge expansion in the development of therapeutic antibodies, with 6 to 10 novel entities approved each year. Around 70% of these Abs are delivered through IV injection, a mode of administration allowing rapid [...] Read more.

Background: For the past two decades, there has been a huge expansion in the development of therapeutic antibodies, with 6 to 10 novel entities approved each year. Around 70% of these Abs are delivered through IV injection, a mode of administration allowing rapid and systemic delivery of the drug. However, according to the evidence presented in the literature, beyond the reduction of invasiveness, a better efficacy can be achieved with local delivery. Consequently, efforts have been made toward the development of innovative methods of administration, and in the formulation and engineering of novel Abs to improve their therapeutic index. Objective: This review presents an overview of the routes of administration used to deliver Abs, different from the IV route, whether approved or in the clinical evaluation stage. We provide a description of the physical and biological fundamentals for each route of administration, highlighting their relevance with examples of clinically-relevant Abs, and discussing their strengths and limitations. Methods: We reviewed and analyzed the current literature, published as of the 1 April 2022 using MEDLINE and EMBASE databases, as well as the FDA and EMA websites. Ongoing trials were identified using clinicaltrials.gov. Publications and data were identified using a list of general keywords. Conclusions: Apart from the most commonly used IV route, topical delivery of Abs has shown clinical successes, improving drug bioavailability and efficacy while reducing side-effects. However, additional research is necessary to understand the consequences of biological barriers associated with local delivery for Ab partitioning, in order to optimize delivery methods and devices, and to adapt Ab formulation to local delivery. Novel modes of administration for Abs might in fine allow a better support to patients, especially in the context of chronic diseases, as well as a reduction of the treatment cost. Full article

(This article belongs to the Special Issue Women's Special Issue Series: Recent Development for Improved Therapeutic Antibodies)

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8 pages, 489 KiB

Open AccessEditor’s ChoiceReview

Mitochondrial Autoantibodies and the Role of Apoptosis in Pemphigus Vulgaris

by Dana M. Hutchison, Anna-Marie Hosking, Ellen M. Hong and Sergei A. Grando

Antibodies 2022, 11(3), 55; https://doi.org/10.3390/antib11030055 - 25 Aug 2022

Cited by 5 | Viewed by 4089

Abstract

Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocutaneous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of epidermal keratinocytes termed apoptolysis. Mitochondrial damage by [...] Read more.

Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocutaneous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of epidermal keratinocytes termed apoptolysis. Mitochondrial damage by antimitochondrial antibodies (AMA) has proven to be a critical link in this process. AMA act synergistically with other autoantibodies in the pathogenesis of PV. Importantly, absorption of AMA inhibits the ability of PVIgGs to induce blisters. Pharmacologic agents that protect mitochondrial function offer a new targeted approach to treating this severe immunoblistering disease. Full article

(This article belongs to the Special Issue Advances in the Use of Therapeutic Antibodies and Identification of Novel Autoantibodies in Dermatologic Disease)

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12 pages, 588 KiB

Open AccessEditor’s ChoiceReview

IgG Avidity Test as a Tool for Discrimination between Recent and Distant Toxoplasma gondii Infection—Current Status of Studies

by Lucyna Holec-Gąsior and Karolina Sołowińska

Antibodies 2022, 11(3), 52; https://doi.org/10.3390/antib11030052 - 15 Aug 2022

Cited by 10 | Viewed by 9416

Abstract

Toxoplasma gondii, an obligate intracellular protozoan parasite, is the causative agent of one of the most prevalent zoonoses worldwide. T. gondii infection is extremely important from a medical point of view, especially for pregnant women, newborns with congenital infections, and immunocompromised individuals. [...] Read more.

Toxoplasma gondii, an obligate intracellular protozoan parasite, is the causative agent of one of the most prevalent zoonoses worldwide. T. gondii infection is extremely important from a medical point of view, especially for pregnant women, newborns with congenital infections, and immunocompromised individuals. Thus, an accurate and proper diagnosis of this infection is essential. Among the available diagnostic tests, serology is commonly used. However, traditional serological techniques have certain limitations in evaluating the duration of T. gondii infection, which is problematic, especially for pregnant women. Avidity of T. gondii-specific IgG antibodies seems to be a significant tool for discrimination between recent and distant infections. This article describes the problem of diagnosis of T. gondii infection, with regard to IgG avidity tests. The IgG avidity test is a useful serological indicator of toxoplasmosis, which in many cases can confirm or exclude the active form of the disease. IgG antibodies produced in the recent primary T. gondii infection are of low avidity while IgG antibodies with high avidity are detected in the chronic phase of infection. Furthermore, this paper presents important topics of current research that concern the usage of parasite recombinant antigens that may improve the performance of IgG avidity tests. Full article

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

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19 pages, 2152 KiB

Open AccessEditor’s ChoiceArticle

On the Rapid Calculation of Binding Affinities for Antigen and Antibody Design and Affinity Maturation Simulations

by Simone Conti, Edmond Y. Lau and Victor Ovchinnikov

Antibodies 2022, 11(3), 51; https://doi.org/10.3390/antib11030051 - 3 Aug 2022

Cited by 6 | Viewed by 5456

Abstract

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are [...] Read more.

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process. Full article

(This article belongs to the Special Issue Computational Discovery of Antibodies)

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20 pages, 11965 KiB

Open AccessEditor’s ChoiceReview

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions

by Thomas Böldicke

Antibodies 2022, 11(3), 49; https://doi.org/10.3390/antib11030049 - 18 Jul 2022

Cited by 11 | Viewed by 5454

Abstract

Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies [...] Read more.

Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies with activated autologous T cells from cancer patients transduced with novel recombinant TCRs or chimeric antigen receptors have been successfully applied. Many TAAs and most neoantigens are expressed in the cytoplasm or nucleus of tumor cells. As alternative to adoptive T-cell therapy, the mRNA of intracellular tumor antigens can be depleted by RNAi, the corresponding genes or proteins deleted by CRISPR-Cas or inactivated by kinase inhibitors or by intrabodies, respectively. Intrabodies are suitable to knockdown TAAs and neoantigens without off-target effects. RNA sequencing and proteome analysis of single tumor cells combined with computational methods is bringing forward the identification of new neoantigens for the selection of anti-cancer intrabodies, which can be easily performed using phage display antibody repertoires. For specifically delivering intrabodies into tumor cells, the usage of new capsid-modified adeno-associated viruses and lipid nanoparticles coupled with specific ligands to cell surface receptors can be used and might bring cancer intrabodies into the clinic. Full article

(This article belongs to the Special Issue Antibody Drug and Target Discovery for Cancer Therapies)

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21 pages, 1407 KiB

Open AccessEditor’s ChoiceReview

Antibody-Based Approaches to Target Pancreatic Tumours

by Marie Sorbara, Pierre Cordelier and Nicolas Bery

Antibodies 2022, 11(3), 47; https://doi.org/10.3390/antib11030047 - 12 Jul 2022

Cited by 8 | Viewed by 5385

Abstract

Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are available, and they are confronted by mechanisms of resistance. Monoclonal [...] Read more.

Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are available, and they are confronted by mechanisms of resistance. Monoclonal antibody (mAb) molecules are highly specific biologics that can be directly used as a blocking agent or modified to deliver a drug payload depending on the desired outcome. They are widely used to target extracellular proteins, but they can also be employed to inhibit intracellular proteins, such as oncoproteins. While mAbs are a class of therapeutics that have been successfully employed to treat many cancers, they have shown only limited efficacy in pancreatic cancer as a monotherapy so far. In this review, we will discuss the challenges, opportunities and hopes to use mAbs for pancreatic cancer treatment, diagnostics and imagery. Full article

(This article belongs to the Special Issue Antibody Drug and Target Discovery for Cancer Therapies)

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16 pages, 4376 KiB

Open AccessEditor’s ChoiceReview

3D Models as a Tool to Assess the Anti-Tumor Efficacy of Therapeutic Antibodies: Advantages and Limitations

by Virginia Guzzeloni, Lorenzo Veschini, Federica Pedica, Elisabetta Ferrero and Marina Ferrarini

Antibodies 2022, 11(3), 46; https://doi.org/10.3390/antib11030046 - 8 Jul 2022

Cited by 5 | Viewed by 4188

Abstract

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely [...] Read more.

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology. Full article

(This article belongs to the Special Issue Women's Special Issue Series: Recent Development for Improved Therapeutic Antibodies)

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16 pages, 1638 KiB

Open AccessEditor’s ChoiceReview

FcγR-Mediated Trogocytosis 2.0: Revisiting History Gives Rise to a Unifying Hypothesis

by Margaret A. Lindorfer and Ronald P. Taylor

Antibodies 2022, 11(3), 45; https://doi.org/10.3390/antib11030045 - 5 Jul 2022

Cited by 10 | Viewed by 3855

Abstract

There is increasing interest in the clinical implications and immunology of trogocytosis, a process in which the receptors on acceptor cells remove and internalize cognate ligands from donor cells. We have reported that this phenomenon occurs in cancer immunotherapy, in which cells that [...] Read more.

There is increasing interest in the clinical implications and immunology of trogocytosis, a process in which the receptors on acceptor cells remove and internalize cognate ligands from donor cells. We have reported that this phenomenon occurs in cancer immunotherapy, in which cells that express FcγR remove and internalize CD20 and bound mAbs from malignant B cells. This process can be generalized to include other reactions including the immune adherence phenomenon and antibody-induced immunosuppression. We discuss in detail FcγR-mediated trogocytosis and the evidence supporting a proposed predominant role for liver sinusoidal endothelial cells via the action of the inhibitory receptor FcγRIIb2. We describe experiments to test the validity of this hypothesis. The elucidation of the details of FcγR-mediated trogocytosis has the potential to allow for the development of novel therapies that can potentially block or enhance this reaction, depending upon whether the process leads to unfavorable or positive biological effects. Full article

(This article belongs to the Special Issue Women's Special Issue Series: Recent Development for Improved Therapeutic Antibodies)

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11 pages, 561 KiB

Open AccessEditor’s ChoiceReview

Deciphering the Contribution of BP230 Autoantibodies in Bullous Pemphigoid

by Connor Cole, Luca Borradori and Kyle T. Amber

Antibodies 2022, 11(3), 44; https://doi.org/10.3390/antib11030044 - 28 Jun 2022

Cited by 6 | Viewed by 5425

Abstract

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease predominantly affecting elderly patients and carries significant morbidity and mortality. Patients typically suffer from severe itch with eczematous lesions, urticarial plaques, and/or tense blisters. BP is characterized by the presence of circulating autoantibodies against [...] Read more.

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease predominantly affecting elderly patients and carries significant morbidity and mortality. Patients typically suffer from severe itch with eczematous lesions, urticarial plaques, and/or tense blisters. BP is characterized by the presence of circulating autoantibodies against two components of the hemidesmosome, BP180 and BP230. The transmembrane BP180, also known as type XVII collagen or BPAG2, represents the primary pathogenic autoantigen in BP, whereas the intracellular BP230 autoantigen is thought to play a minor role in disease pathogenesis. Although experimental data exist suggesting that anti-BP230 antibodies are secondarily formed following initial tissue damage mediated by antibodies targeting extracellular antigenic regions of BP180, there is emerging evidence that anti-BP230 IgG autoantibodies alone directly contribute to tissue damage. It has been further claimed that a subset of patients has a milder variant of BP driven solely by anti-BP230 autoantibodies. Furthermore, the presence of anti-BP230 autoantibodies might correlate with distinct clinical features. This review summarizes the current understanding of the role of BP230 and anti-BP230 antibodies in BP pathogenesis. Full article

(This article belongs to the Special Issue Advances in the Use of Therapeutic Antibodies and Identification of Novel Autoantibodies in Dermatologic Disease)

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13 pages, 2815 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Development of a Novel Anti-EpCAM Monoclonal Antibody for Various Applications

by Guanjie Li, Hiroyuki Suzuki, Teizo Asano, Tomohiro Tanaka, Hiroyoshi Suzuki, Mika K. Kaneko and Yukinari Kato

Antibodies 2022, 11(2), 41; https://doi.org/10.3390/antib11020041 - 8 Jun 2022

Cited by 16 | Viewed by 4538

Abstract

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis [...] Read more.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG₁, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The K_D of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10⁻⁸ M and 3.2 × 10⁻⁸ M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications. Full article

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

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10 pages, 1008 KiB

Open AccessEditor’s ChoiceArticle

Kinetics of the Neutralizing and Spike SARS-CoV-2 Antibodies following the Sinovac Inactivated Virus Vaccine Compared to the Pfizer mRNA Vaccine in Singapore

by Chin Shern Lau, 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(2), 38; https://doi.org/10.3390/antib11020038 - 27 May 2022

Cited by 10 | Viewed by 3373

Abstract

Introduction: We compared the early total spike antibody (S-Ab) and neutralizing antibody (N-Ab) responses to two vaccines. Methods: We studied 96 Pfizer and 34 Sinovac vaccinees over a 14-month period from January 2021 to February 2022. All vaccinees received three doses of one [...] Read more.

Introduction: We compared the early total spike antibody (S-Ab) and neutralizing antibody (N-Ab) responses to two vaccines. Methods: We studied 96 Pfizer and 34 Sinovac vaccinees over a 14-month period from January 2021 to February 2022. All vaccinees received three doses of one type of vaccine. Antibody levels (Roche Elecsys total S-Ab and the Snibe N-Ab) were tested 10 days after the first dose, 20 days after the second dose, and 20 days after the booster dose. Results: At all time points, the mRNA vaccine generated higher S-Ab and N-Ab responses than the inactivated virus vaccine (S-Ab: first dose 2.48 vs. 0.4 BAU/mL, second dose 2174 vs. 98 BAU/mL, third dose 15,004 vs. 525 BAU/mL; N-Ab: first dose 0.05 vs. 0.02 µg/mL, second dose 3.48 vs. 0.38 µg/mL, third dose 19.8 vs. 0.89 µg/mL). mRNA vaccine recipients had a 6.2/22.2/28.6-fold higher S-Ab and 2.5/9.2/22.2-fold higher N-Ab response than inactivated virus vaccine recipients after the first/second/third inoculations, respectively. Mann–Whitney U analysis confirmed the significant difference in S-Ab and N-Ab titers between vaccination groups at each time point. Conclusions: The mRNA vaccines generated a more robust S-Ab and N-Ab response than the inactivated virus vaccine at all time points after the first, second, and third vaccinations. Full article

(This article belongs to the Special Issue Antibodies, B Cell Responses and Immune Responses to SARS-CoV-2 Infections)

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17 pages, 8390 KiB

Open AccessFeature PaperEditor’s ChoiceReview

A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting

by Teruo Akuta, Toshiaki Maruyama, Chiaki Sakuma, Masataka Nakagawa, Yui Tomioka, Kevin Entzminger, Jonathan K. Fleming, Ryo Sato, Takashi Shibata, Yasunori Kurosawa, C. J. Okumura and Tsutomu Arakawa

Antibodies 2022, 11(2), 36; https://doi.org/10.3390/antib11020036 - 12 May 2022

Cited by 8 | Viewed by 9145

Abstract

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. [...] Read more.

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein. Full article

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

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19 pages, 3042 KiB

Open AccessEditor’s ChoiceArticle

Mammalian Display Platform for the Maturation of Bispecific TCR-Based Molecules

by Janine Dilchert, Martin Hofmann, Felix Unverdorben, Roland Kontermann and Sebastian Bunk

Antibodies 2022, 11(2), 34; https://doi.org/10.3390/antib11020034 - 10 May 2022

Cited by 6 | Viewed by 5671

Abstract

Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, [...] Read more.

Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, but also requires a complex maturation process to increase the naturally low affinity and stability of TCRs. Even though TCR domains can be matured via phage and yeast display, these techniques share the disadvantages of non-human glycosylation patterns and the need for a later reformatting into the final bispecific format. Here, we describe the development and application of a Chinese Hamster Ovary (CHO) display for affinity engineering of TCRs in the context of the final bispecific TCR format. The recombinase-mediated cassette exchange (RCME)-based system allows for stable, single-copy integration of bispecific TCR molecules with high efficiency into a defined genetic locus of CHO cells. We used the system to isolate affinity-increased variants of bispecific T cell engaging receptor (TCER) molecules from a library encoding different CDR variants of a model TCR targeting preferentially expressed antigen in melanoma (PRAME). When expressed as a soluble protein, the selected TCER molecules exhibited strong reactivity against PRAME-positive tumor cells associated with a pronounced cytokine release from activated T cells. The obtained data support the usage of the CHO display-based maturation system for TCR affinity maturation in the context of the final bispecific TCER format. Full article

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10 pages, 536 KiB

Open AccessEditor’s ChoiceReview

Advances in Chimeric Antigen Receptor (CAR) T-Cell Therapies for the Treatment of Primary Brain Tumors

by Christopher W. Mount and Luis Nicolas Gonzalez Castro

Antibodies 2022, 11(2), 31; https://doi.org/10.3390/antib11020031 - 27 Apr 2022

Cited by 5 | Viewed by 4436

Abstract

Immunotherapy has revolutionized the care of cancer patients. A diverse set of strategies to overcome cancer immunosuppression and enhance the tumor-directed immune response are in clinical use, but have not achieved transformative benefits for brain tumor patients. Adoptive cell therapies, which employ a [...] Read more.

Immunotherapy has revolutionized the care of cancer patients. A diverse set of strategies to overcome cancer immunosuppression and enhance the tumor-directed immune response are in clinical use, but have not achieved transformative benefits for brain tumor patients. Adoptive cell therapies, which employ a patient’s own immune cells to generate directed anti-tumor activity, are emerging technologies that hold promise to improve the treatment of primary brain tumors in children and adults. Here, we review recent advances in chimeric antigen receptor (CAR) T-cell therapies for the treatment of aggressive primary brain tumors, including glioblastoma and diffuse midline glioma, H3 K27M-mutant. We highlight current approaches, discuss encouraging investigational data, and describe key challenges in the development and implementation of these types of therapies in the neuro-oncology setting. Full article

(This article belongs to the Special Issue CAR-T Cell Metabolism)

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13 pages, 1244 KiB

Open AccessEditor’s ChoiceReview

Precision-Cut Tumor Slices (PCTS) as an Ex Vivo Model in Immunotherapy Research

by Paraskevi Dimou, Sumita Trivedi, Maria Liousia, Reena R. D'Souza and Astero Klampatsa

Antibodies 2022, 11(2), 26; https://doi.org/10.3390/antib11020026 - 6 Apr 2022

Cited by 11 | Viewed by 7384

Abstract

Precision-cut tumor slices (PCTS) have recently emerged as important ex vivo human tumor models, offering the opportunity to study individual patient responses to targeted immunotherapies, including CAR-T cell therapies. In this review, an outline of different human tumor models available in laboratory settings [...] Read more.

Precision-cut tumor slices (PCTS) have recently emerged as important ex vivo human tumor models, offering the opportunity to study individual patient responses to targeted immunotherapies, including CAR-T cell therapies. In this review, an outline of different human tumor models available in laboratory settings is provided, with a focus on the unique characteristics of PCTS. Standard PCTS generation and maintenance procedures are outlined, followed by an in-depth overview of PCTS utilization in preclinical research aiming to better understand the unique functional characteristics of cytotoxic T cells within human tumors. Furthermore, recent studies using PCTS as an ex vivo model for predicting patient responses to immunotherapies and other targeted therapies against solid tumors are thoroughly presented. Finally, the advantages and limitations of the PCTS models are discussed. PCTS are expected to gain momentum and be fully utilized as a significant tool towards better patient stratification and personalized medicine. Full article

(This article belongs to the Special Issue CAR-T Cell Metabolism)

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17 pages, 1427 KiB

Open AccessEditor’s ChoiceReview

Cellular, Antibody and Cytokine Pathways in Children with Acute SARS-CoV-2 Infection and MIS-C—Can We Match the Puzzle?

by Snezhina Lazova, Yulia Dimitrova, Diana Hristova, Iren Tzotcheva and Tsvetelina Velikova

Antibodies 2022, 11(2), 25; https://doi.org/10.3390/antib11020025 - 1 Apr 2022

Cited by 13 | Viewed by 4412

Abstract

The newly identified strain of the Coronaviridae family called severe acute respiratory syndrome (SARS-CoV-2) recently became the most significant health threat for adults and children. Some main predictors of severe clinical course in patients with SARS-CoV-2 infection are age and concomitant health conditions. [...] Read more.

The newly identified strain of the Coronaviridae family called severe acute respiratory syndrome (SARS-CoV-2) recently became the most significant health threat for adults and children. Some main predictors of severe clinical course in patients with SARS-CoV-2 infection are age and concomitant health conditions. Therefore, the proper evaluation of SARS-CoV-2-specific immunity is urgently required to understand and predict the spectrum of possible clinical phenotypes and recommend vaccination options and regimens in children. Furthermore, it is critical to characterize the nature of SARS-CoV-2-specific immune responses in children following asymptomatic infection and COVID-19 and other related conditions such as multisystem inflammatory syndrome (MIS-C), para-infectious and late postinfectious consequences. Recent studies involving children revealed a variety of cytokines, T cells and antibody responses in the pathogenesis of the disease. Moreover, different clinical scenarios in children were observed-asymptomatic seroprevalence, acute SARS-CoV-2 infection, and rarely severe COVID-19 with typical cytokine storm, MIS-C, long COVID-19, etc. Therefore, to gain a better clinical view, adequate diagnostic criteria and treatment algorithms, it is essential to create a realistic picture of the immunological puzzle of SARS-CoV-2 infection in different age groups. Finally, it was demonstrated that children may exert a potent and prolonged adaptive anti-SARS-CoV-2 immune response, with significant cross-reactions against other human Corona Viruses, that might contribute to disease sparing effect in this age range. However, the immunopathology of the virus has to be elucidated first. Full article

(This article belongs to the Special Issue Antibodies, B Cell Responses and Immune Responses to SARS-CoV-2 Infections)

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19 pages, 3287 KiB

Open AccessEditor’s ChoiceArticle

Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations

by Amy Y. Xu, Nicholas J. Clark, Joseph Pollastrini, Maribel Espinoza, Hyo-Jin Kim, Sekhar Kanapuram, Bruce Kerwin, Michael J. Treuheit, Susan Krueger, Arnold McAuley and Joseph E. Curtis

Antibodies 2022, 11(2), 24; https://doi.org/10.3390/antib11020024 - 31 Mar 2022

Cited by 9 | Viewed by 4801

Abstract

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (C_NaCl) and monoclonal antibody (mAb) concentration (C [...] Read more.

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (C_NaCl) and monoclonal antibody (mAb) concentration (C_mAb) were assessed using the diffusion interaction parameter k_D and second virial coefficient B₂₂ measured from solutions with low to moderate C_mAb. The effective structure factor S(q)_eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with C_NaCl, but also with increasing C_mAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including k_D and S(q)_eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low C_mAb. Full article

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

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14 pages, 2186 KiB

Open AccessEditor’s ChoiceArticle

Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope

by Adva Mechaly, Eran Diamant, Ron Alcalay, Alon Ben David, Eyal Dor, Amram Torgeman, Ada Barnea, Meni Girshengorn, Lilach Levin, Eyal Epstein, Ariel Tennenhouse, Sarel J. Fleishman, Ran Zichel and Ohad Mazor

Antibodies 2022, 11(1), 21; https://doi.org/10.3390/antib11010021 - 16 Mar 2022

Cited by 5 | Viewed by 4241

Abstract

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays [...] Read more.

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes. Full article

(This article belongs to the Special Issue Phage and Ribosome Display for Antibody Discovery and Optimization)

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15 pages, 1215 KiB

Open AccessEditor’s ChoiceArticle

Reactivity of Rheumatoid Arthritis-Associated Citrulline-Dependent Antibodies to Epstein-Barr Virus Nuclear Antigen1-3

by Ilaria Fanelli, Paolo Rovero, Paul Robert Hansen, Jette Lautrup Frederiksen, Gunnar Houen and Nicole Hartwig Trier

Antibodies 2022, 11(1), 20; https://doi.org/10.3390/antib11010020 - 11 Mar 2022

Cited by 8 | Viewed by 3557

Abstract

Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence [...] Read more.

Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence points to a link between the Epstein-Barr virus and RA. In this study, we analyzed ACPA reactivity to citrullinated peptides originating from Epstein-Barr nuclear antigens (EBNA1, EBNA2, and EBNA3) in order to elaborate the diagnostic potential of citrullinated EBNA peptides. Moreover, ACPA cross-reactivity to citrullinated peptides from myelin basic protein (MBP) was analyzed, as citrullinated MBP recently was described to be associated with multiple sclerosis, and some degree of sequence homology between MBP and citrullinated EBNA exists. A peptide from EBNA2, (EBNA2-A, GQGRGRWRG-Cit-GSKGRGRMH) reacted with approximately 70% of all RA sera, whereas only limited reactivity was detected to EBNA1 and EBNA3 peptides. Moreover, screening of ACPA reactivity to hybrid peptides of EBNA3-A (EPDSRDQQS-Cit-GQRRGDENRG) and EBNA2-A and peptides containing citrulline close to the N-terminal confirmed that ACPA sera contain different populations of ACPAs. No notable ACPA reactivity to MBP peptides was found, confirming that ACPAs are specific for RA, and that other factors than the presence of a central Cit-Gly motif are crucial for antibody binding. Collectively, these findings illustrate that citrullinated EBNA2 is an optimal candidate for ACPA detection, supporting current evidence that EBV is linked to RA onset. Full article

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10 pages, 2477 KiB

Open AccessEditor’s ChoiceArticle

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21

by Cesare Di Nitto, Dario Neri, Tobias Weiss, Michael Weller and Roberto De Luca

Antibodies 2022, 11(1), 19; https://doi.org/10.3390/antib11010019 - 4 Mar 2022

Cited by 6 | Viewed by 5801

Abstract

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory [...] Read more.

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product. Full article

(This article belongs to the Special Issue Cytokine-Targeting Antibodies and Immuno-Cytokines)

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43 pages, 4424 KiB

Open AccessEditor’s ChoiceArticle

Ramifications of the HLA-I Allelic Reactivity of Anti-HLA-E*01:01 and Anti-HLA-E*01:03 Heavy Chain Monoclonal Antibodies in Comparison with Anti-HLA-I IgG Reactivity in Non-Alloimmunized Males, Melanoma-Vaccine Recipients, and End-Stage Renal Disease Patients

by Mepur H. Ravindranath, Narendranath M. Ravindranath, Fatiha El Hilali, Senthamil R. Selvan and Edward J. Filippone

Antibodies 2022, 11(1), 18; https://doi.org/10.3390/antib11010018 - 2 Mar 2022

Cited by 5 | Viewed by 3568

Abstract

Serum anti-HLA-I IgG are present in non-alloimmunized males, cancer patients, and transplant recipients. Anti-HLA-I antibodies are also present in intravenous immunoglobulin (IVIg), prepared from the plasma of thousands of healthy donors. However, the HLA-Ia reactivity of IVIg diminishes markedly after passing through HLA-E [...] Read more.

Serum anti-HLA-I IgG are present in non-alloimmunized males, cancer patients, and transplant recipients. Anti-HLA-I antibodies are also present in intravenous immunoglobulin (IVIg), prepared from the plasma of thousands of healthy donors. However, the HLA-Ia reactivity of IVIg diminishes markedly after passing through HLA-E HC-affinity columns, suggesting that the HLA-I reactivity is due to antibodies formed against HLA-E. Hence, we examined whether anti-HLA-E antibodies can react to HLA-I alleles. Monoclonal IgG antibodies (mAbs) against HCs of two HLA-E alleles were generated in Balb/C mice. The antibodies were analyzed using multiplex bead assays on a Luminex platform for HLA-I reactivity. Beads coated with an array of HLA heterodimers admixed with HCs (LABScreen) were used to examine the binding of IgG to different HLA-Ia (31-HLA-A, 50-HLA-B, and 16-HLA-C) and Ib (2-HLA-E, one each of HLA-F and HLA-G) alleles. A striking diversity in the HLA-Ia and/or HLA-Ib reactivity of mAbs was observed. The number of the mAbs reactive to (1) only HLA-E (n = 25); (2) all HLA-Ib isomers (n = 8); (3) HLA-E and HLA-B (n = 5); (4) HLA-E, HLA-B, and HLA-C (n = 30); (5) HLA-E, HLA-A*1101, HLA-B, and HLA-C (n = 83); (6) HLA-E, HLA-A, HLA-B, and HLA-C (n = 54); and (7) HLA-Ib and HLA-Ia (n = 8), in addition to four other minor groups. Monospecificity and polyreactivity were corroborated by HLA-E monospecific and HLA-I shared sequences. The diverse HLA-I reactivity of the mAbs are compared with the pattern of HLA-I reactivity of serum-IgG in non-alloimmunized males, cancer patients, and ESKD patients. The findings unravel the diagnostic potential of the HLA-E monospecific-mAbs and immunomodulatory potentials of IVIg highly mimicking HLA-I polyreactive-mAbs. Full article

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

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41 pages, 1578 KiB

Open AccessEditor’s ChoiceReview

Immune- and Non-Immune-Mediated Adverse Effects of Monoclonal Antibody Therapy: A Survey of 110 Approved Antibodies

by Brian A. Baldo

Antibodies 2022, 11(1), 17; https://doi.org/10.3390/antib11010017 - 25 Feb 2022

Cited by 28 | Viewed by 13264

Abstract

Identification of new disease-associated biomarkers; specific targeting of such markers by monoclonal antibodies (mAbs); and application of advances in recombinant technology, including the production of humanized and fully human antibodies, has enabled many improved treatment outcomes and successful new biological treatments of some [...] Read more.

Identification of new disease-associated biomarkers; specific targeting of such markers by monoclonal antibodies (mAbs); and application of advances in recombinant technology, including the production of humanized and fully human antibodies, has enabled many improved treatment outcomes and successful new biological treatments of some diseases previously neglected or with poor prognoses. Of the 110 mAbs preparations currently approved by the FDA and/or EMA, 46 (including 13 antibody–drug conjugates) recognizing 29 different targets are indicated for the treatment of cancers, and 66, recognizing 48 different targets, are indicated for non-cancer disorders. Despite their specific targeting with the expected accompanying reduced collateral damage for normal healthy non-involved cells, mAbs, may cause types I (anaphylaxis, urticaria), II (e.g., hemolytic anemia, possibly early-onset neutropenia), III (serum sickness, pneumonitis), and IV (Stevens–Johnson syndrome, toxic epidermal necrolysis) hypersensitivities as well as other cutaneous, pulmonary, cardiac, and liver adverse events. MAbs can provoke severe infusion reactions that resemble anaphylaxis and induce a number of systemic, potentially life-threatening syndromes with low frequency. A common feature of most of these syndromes is the release of a cascade of cytokines associated with inflammatory and immunological processes. Epidermal growth factor receptor-targeted antibodies may provoke papulopustular and mucocutaneous eruptions that are not immune-mediated. Full article

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14 pages, 2088 KiB

Open AccessEditor’s ChoiceReview

The Role of Bispecific Antibodies in Non-Hodgkin’s Lymphoma: From Structure to Prospective Clinical Use

by Rita Tavarozzi and Enrica Manzato

Antibodies 2022, 11(1), 16; https://doi.org/10.3390/antib11010016 - 21 Feb 2022

Cited by 8 | Viewed by 7748

Abstract

Bispecific antibodies (bsAbs) are molecules that simultaneously bind two different antigens (Ags). bsAbs represent a very active field in tumor immunotherapy with more than one hundred molecules currently being tested. More specifically, they have elicited a great interest in the setting of non-Hodgkin’s [...] Read more.

Bispecific antibodies (bsAbs) are molecules that simultaneously bind two different antigens (Ags). bsAbs represent a very active field in tumor immunotherapy with more than one hundred molecules currently being tested. More specifically, they have elicited a great interest in the setting of non-Hodgkin’s lymphoma (NHLs), where they could represent a viable option for more fragile patients or those resistant to other conventional therapies. This review aims to give a brief overview of the different available bsAb formats and their mechanisms of action, pinpointing the differences between IgG-like and non-IgG-like classes and will then focus on those in advanced clinical development for NHLs. Full article

(This article belongs to the Special Issue Reviews on Antibodies and Antigens)

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13 pages, 1493 KiB

Open AccessEditor’s ChoiceArticle

Anti-SARS-CoV-2 Omicron Antibodies Isolated from a SARS-CoV-2 Delta Semi-Immune Phage Display Library

by Ivette Mendoza-Salazar, Keyla M. Gómez-Castellano, Edith González-González, Ramsés Gamboa-Suasnavart, Stefany D. Rodríguez-Luna, Giovanni Santiago-Casas, María I. Cortés-Paniagua, Sonia M. Pérez-Tapia and Juan C. Almagro

Antibodies 2022, 11(1), 13; https://doi.org/10.3390/antib11010013 - 10 Feb 2022

Cited by 7 | Viewed by 5185

Abstract

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient [...] Read more.

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile. Full article

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22 pages, 3270 KiB

Open AccessEditor’s ChoiceArticle

Cryopreservation of Natural Killer Cells Pre-Complexed with Innate Cell Engagers

by Uwe Reusch, Kristina Ellwanger, Ivica Fucek, Thomas Müller, Ute Schniegler-Mattox, Joachim Koch and Michael Tesar

Antibodies 2022, 11(1), 12; https://doi.org/10.3390/antib11010012 - 9 Feb 2022

Cited by 7 | Viewed by 8588

Abstract

Innate cell engager (ICE^®) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the destruction of tumor cells. Pre-complexing of ICE^® constructs with adoptive NK cells is a novel [...] Read more.

Innate cell engager (ICE^®) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the destruction of tumor cells. Pre-complexing of ICE^® constructs with adoptive NK cells is a novel approach to enhance NK cell activity. The suitability of such complexes for cryopreservation, whilst retaining the biological activity and specificity, may enable the development of off-the-shelf NK cell products. This study investigates the binding affinity of ICE^® constructs targeting EpCAM and NK cell receptors CD16A, NKG2D, or NKp46 to the corresponding antigens, the ICE^® antitumor activity, and feasibility of cryopreservation. Cell surface retention assays using primary NK cells confirmed a substantially slower ICE^® construct dissociation kinetics compared with control molecules, suggesting the formation of durable complexes independently of the CD16A polymorphism. The high-affinity NK cell and EpCAM/CD16A ICE^® complexes were superior to those engaging NKG2D or NKp46 receptors when tested for the NK-cell-mediated elimination of EpCAM-expressing tumor cells. Moreover, the potency and efficacy of these complexes were unaffected after a single freeze–thaw cycle. CD16A-selective ICE^® drug candidates complexed with NK cells hold promise as novel cryopreserved off-the-shelf NK cell products with chimeric antigen receptor-like NK cell properties, capable of effective depletion of tumor cells. Full article

(This article belongs to the Special Issue Antibodies for Effector Cell Redirection)

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18 pages, 2608 KiB

Open AccessEditor’s ChoiceArticle

Functional Changes of Therapeutic Antibodies upon Exposure to Pro-Oxidative Agents

by Maxime Lecerf, Robin Lacombe, Alexia Kanyavuz and Jordan D. Dimitrov

Antibodies 2022, 11(1), 11; https://doi.org/10.3390/antib11010011 - 2 Feb 2022

Cited by 5 | Viewed by 3564

Abstract

Therapeutic monoclonal antibodies have exerted a transformative impact on clinical practice in last two decades. However, development of a therapeutic antibody remains a complex process. Various physiochemical and functional liabilities can compromise the production or the therapeutic efficacy of antibodies. One of these [...] Read more.

Therapeutic monoclonal antibodies have exerted a transformative impact on clinical practice in last two decades. However, development of a therapeutic antibody remains a complex process. Various physiochemical and functional liabilities can compromise the production or the therapeutic efficacy of antibodies. One of these liabilities is the susceptibility to oxidation. In the present study, we portrayed an oxidation-dependent vulnerability of immunoglobulins that can be of concern for therapeutic antibodies. By using a library of 119 monoclonal IgG1 molecules, containing variable domain matching clinical-stage antibodies, we demonstrated that a substantial number of these molecules acquired antigen-binding polyreactivity upon exposure to ferrous ions. Statistical analyses revealed that the potential for induction of polyreactivity by the redox-active metal ions correlated with a higher number of somatic mutations in V genes encoding variable domains of heavy and light immunoglobulin chains. Moreover, the sensitive antibodies used with biased frequencies particular V gene families encoding variable domains of their light chains. Besides the exposure to ferrous ions the induction of polyreactivity of therapeutic antibodies occurred after contact with an unrelated pro-oxidative substance—hypochlorite ions. Our data also revealed that induction of polyreactivity by pro-oxidative agents did not impact the binding of antibodies to their cognate antigens. The results from this study may contribute for better selection of antibody therapeutics with suitable developability profiles. Full article

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18 pages, 2882 KiB

Open AccessEditor’s ChoiceArticle

Construction of a Humanized Artificial VHH Library Reproducing Structural Features of Camelid VHHs for Therapeutics

by Taihei Murakami, Shigefumi Kumachi, Yasuhiro Matsunaga, Miwa Sato, Kanako Wakabayashi-Nakao, Hidekazu Masaki, Ryo Yonehara, Maiko Motohashi, Naoto Nemoto and Masayuki Tsuchiya

Antibodies 2022, 11(1), 10; https://doi.org/10.3390/antib11010010 - 30 Jan 2022

Cited by 17 | Viewed by 10438

Abstract

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the [...] Read more.

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening. Full article

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14 pages, 912 KiB

Open AccessEditor’s ChoiceReview

Applications of Antibody-Based Antigen Delivery Targeted to Dendritic Cells In Vivo

by Jessica Bourque and Daniel Hawiger

Antibodies 2022, 11(1), 8; https://doi.org/10.3390/antib11010008 - 25 Jan 2022

Cited by 9 | Viewed by 7368

Abstract

Recombinant immunoglobulins, derived from monoclonal antibodies recognizing the defined surface epitopes expressed on dendritic cells, have been employed for the past two decades to deliver antigens to dendritic cells in vivo, serving as critical tools for the investigation of the corresponding T cell [...] Read more.

Recombinant immunoglobulins, derived from monoclonal antibodies recognizing the defined surface epitopes expressed on dendritic cells, have been employed for the past two decades to deliver antigens to dendritic cells in vivo, serving as critical tools for the investigation of the corresponding T cell responses. These approaches originated with the development of the recombinant chimeric antibody against a multilectin receptor, DEC-205, which is present on subsets of murine and human conventional dendritic cells. Following the widespread application of antigen targeting through DEC-205, similar approaches then utilized other epitopes as entry points for antigens delivered by specific antibodies to multiple types of dendritic cells. Overall, these antigen-delivery methodologies helped to reveal the mechanisms underlying tolerogenic and immunogenic T cell responses orchestrated by dendritic cells. Here, we discuss the relevant experimental strategies as well as their future perspectives, including their translational relevance. Full article

(This article belongs to the Special Issue Reviews on Antibodies and Antigens)

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13 pages, 1390 KiB

Open AccessEditor’s ChoiceReview

Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT/HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT/TTS

by Emmanuel J. Favaloro, Leonardo Pasalic and Giuseppe Lippi

Antibodies 2022, 11(1), 7; https://doi.org/10.3390/antib11010007 - 21 Jan 2022

Cited by 19 | Viewed by 5015

Abstract

Antibodies against platelet factor 4 (PF4), a protein released from alpha-granules of activated platelets, may cause a number of pathophysiological conditions. The most commonly known is heparin-induced thrombocytopenia (HIT), which develops in a small proportion of people treated with the anticoagulant drug heparin. [...] Read more.

Antibodies against platelet factor 4 (PF4), a protein released from alpha-granules of activated platelets, may cause a number of pathophysiological conditions. The most commonly known is heparin-induced thrombocytopenia (HIT), which develops in a small proportion of people treated with the anticoagulant drug heparin. Notably, PF4 binds with high affinity to heparin, and in HIT, complexes of PF4/H may, in a small proportion of susceptible patients, trigger the development of anti-PF4 antibodies and subsequent platelet activation and aggregation, ultimately leading to the development of pathological thrombosis at sites of vessel occlusion. Of more modern interest, antibodies against PF4 may also arise in patients with COVID-19 (Coronavirus Disease 2019) or in patients who have been vaccinated against COVID-19, especially in recipients of adenovirus-based vaccines. For this latter group of patients, the terms VITT (vaccine-induced [immune] thrombotic thrombocytopenia) and TTS (thrombotic thrombocytopenia syndrome) have been coined. Another category associated with this pathophysiology comprises those in whom a precipitating event is not clear; this category is referred to as ‘spontaneous HIT-like syndrome’. Despite its name, it arises as an HIT-mimicking disorder but without antecedent heparin exposure. In this narrative review, we describe the development of antibodies against PF4, and associated pathophysiology, in such conditions. Full article

(This article belongs to the Special Issue Reviews on Antibodies and Antigens)

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14 pages, 2288 KiB

Open AccessEditor’s ChoiceArticle

Engineering an Enhanced EGFR Engager: Humanization of Cetuximab for Improved Developability

by Dennis R. Goulet, Soumili Chatterjee, Wai-Ping Lee, Andrew B. Waight, Yi Zhu and Amanda Nga-Sze Mak

Antibodies 2022, 11(1), 6; https://doi.org/10.3390/antib11010006 - 13 Jan 2022

Cited by 9 | Viewed by 5681

Abstract

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers [...] Read more.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering. Full article

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

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13 pages, 1008 KiB

Open AccessEditor’s ChoiceReview

New Opportunities in Glycan Engineering for Therapeutic Proteins

by Xiaotian Zhong, Aaron M. D’Antona, John J. Scarcelli and Jason C. Rouse

Antibodies 2022, 11(1), 5; https://doi.org/10.3390/antib11010005 - 10 Jan 2022

Cited by 8 | Viewed by 9401

Abstract

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports [...] Read more.

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins. Full article

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16 pages, 4766 KiB

Open AccessEditor’s ChoiceArticle

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro

by Ashleigh J. Nicaise, Amye McDonald, Erin Rushing Sears, Trell Sturgis and Barbara L. F. Kaplan

Antibodies 2022, 11(1), 4; https://doi.org/10.3390/antib11010004 - 9 Jan 2022

Cited by 5 | Viewed by 3554

Abstract

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune [...] Read more.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD’s effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD’s EAE disease attenuation. Full article

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