International Journal of Molecular Sciences

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21 pages, 3328 KiB

Open AccessArticle

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

by Zora Novakova, Nikola Belousova, Catherine A. Foss, Barbora Havlinova, Marketa Gresova, Gargi Das, Ala Lisok, Adam Prada, Marketa Barinkova, Martin Hubalek, Martin G. Pomper and Cyril Barinka

Int. J. Mol. Sci. 2020, 21(18), 6672; https://doi.org/10.3390/ijms21186672 - 12 Sep 2020

Cited by 3 | Viewed by 3577

Abstract

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv [...] Read more.

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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17 pages, 3382 KiB

Open AccessArticle

Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries

by Kyungjae Kang, Kicheon Kim, Se-Ra Lee, Yoonji Kim, Joo Eon Lee, Yong Sun Lee, Ju-Hyeon Lim, Chung-Su Lim, Yu Jung Kim, Seung Il Baek, Du Hyun Song, Jin Tae Hong and Dae Young Kim

Int. J. Mol. Sci. 2020, 21(17), 6354; https://doi.org/10.3390/ijms21176354 - 01 Sep 2020

Cited by 9 | Viewed by 3512

Abstract

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, [...] Read more.

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (K_D = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, K_D = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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14 pages, 2484 KiB

Open AccessArticle

An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor

by Sebastian W. Meister, Linnea C. Hjelm, Melanie Dannemeyer, Hanna Tegel, Hanna Lindberg, Stefan Ståhl and John Löfblom

Int. J. Mol. Sci. 2020, 21(8), 2999; https://doi.org/10.3390/ijms21082999 - 23 Apr 2020

Cited by 12 | Viewed by 4710

Abstract

The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (Z [...] Read more.

The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (Z_SYM73) via binding to the transferrin receptor (TfR). Z_SYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule Z_SYM73. Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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17 pages, 5633 KiB

Open AccessArticle

Identification of a Novel Linear B-Cell Epitope on the Nucleocapsid Protein of Porcine Deltacoronavirus

by Jiayu Fu, Rui Chen, Jingfei Hu, Huan Qu, Yujia Zhao, Sanjie Cao, Xintian Wen, Yiping Wen, Rui Wu, Qin Zhao, Xiaoping Ma and Xiaobo Huang

Int. J. Mol. Sci. 2020, 21(2), 648; https://doi.org/10.3390/ijms21020648 - 19 Jan 2020

Cited by 18 | Viewed by 4542

Abstract

Porcine deltacoronavirus (PDCoV), first identified in 2012, is a swine enteropathogen now found in many countries. The nucleocapsid (N) protein, a core component of PDCoV, is essential for virus replication and is a significant candidate in the development of diagnostics for PDCoV. In [...] Read more.

Porcine deltacoronavirus (PDCoV), first identified in 2012, is a swine enteropathogen now found in many countries. The nucleocapsid (N) protein, a core component of PDCoV, is essential for virus replication and is a significant candidate in the development of diagnostics for PDCoV. In this study, monoclonal antibodies (mAbs) were generated and tested for reactivity with three truncations of the full protein (N1, N2, N3) that contained partial overlaps; of the five monoclonals chosen tested, each reacted with only the N3 truncation. The antibody designated 4E88 had highest binding affinity with the N protein and was chosen for in-depth examination. The 4E88 epitope was located to amino acids 308-AKPKQQKKPKK-318 by testing the 4E88 monoclonal for reactivity with a series of N3 truncations, then the minimal epitope, 309-KPKQQKKPK-317 (designated EP-4E88), was pinpointed by testing the 4E88 monoclonal for reactivity with a series of synthetic peptides of this region. Homology analysis showed that the EP-4E88 sequence is highly conserved among PDCoV strains, and also shares high similarity with sparrow coronavirus (HKU17), Asian leopard cat coronavirus (ALCCoV), quail coronavirus (UAE-HKU30), and sparrow deltacoronavirus (SpDCoV). Of note, the PDCoV EP-4E88 sequence shared very low similarity (<22.2%) with other porcine coronaviruses (PEDV, TGEV, PRCV, SADS-CoV, PHEV), demonstrating that it is an epitope that can be used for distinguishing PDCoV and other porcine coronavirus. 3D structural analysis revealed that amino acids of EP-4E88 were in close proximity and may be exposed on the surface of the N protein. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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Review

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18 pages, 4903 KiB

Open AccessReview

Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers

by André L. B. Bitencourt, Raquel M. Campos, Erika N. Cline, William L. Klein and Adriano Sebollela

Int. J. Mol. Sci. 2020, 21(23), 8920; https://doi.org/10.3390/ijms21238920 - 24 Nov 2020

Cited by 8 | Viewed by 3492

Abstract

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by [...] Read more.

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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41 pages, 3532 KiB

Open AccessReview

Toward Drug-Like Multispecific Antibodies by Design

by Manali S. Sawant, Craig N. Streu, Lina Wu and Peter M. Tessier

Int. J. Mol. Sci. 2020, 21(20), 7496; https://doi.org/10.3390/ijms21207496 - 12 Oct 2020

Cited by 35 | Viewed by 9171

Abstract

The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal [...] Read more.

The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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14 pages, 1285 KiB

Open AccessReview

Modular Chimeric Antigen Receptor Systems for Universal CAR T Cell Retargeting

by Ashley R. Sutherland, Madeline N. Owens and C. Ronald Geyer

Int. J. Mol. Sci. 2020, 21(19), 7222; https://doi.org/10.3390/ijms21197222 - 30 Sep 2020

Cited by 29 | Viewed by 6489

Abstract

The engineering of T cells through expression of chimeric antigen receptors (CARs) against tumor-associated antigens (TAAs) has shown significant potential for use as an anti-cancer therapeutic. The development of strategies for flexible and modular CAR T systems is accelerating, allowing for multiple antigen [...] Read more.

The engineering of T cells through expression of chimeric antigen receptors (CARs) against tumor-associated antigens (TAAs) has shown significant potential for use as an anti-cancer therapeutic. The development of strategies for flexible and modular CAR T systems is accelerating, allowing for multiple antigen targeting, precise programming, and adaptable solutions in the field of cellular immunotherapy. Moving beyond the fixed antigen specificity of traditional CAR T systems, the modular CAR T technology splits the T cell signaling domains and the targeting elements through use of a switch molecule. The activity of CAR T cells depends on the presence of the switch, offering dose-titratable response and precise control over CAR T cells. In this review, we summarize developments in universal or modular CAR T strategies that expand on current CAR T systems and open the door for more customizable T cell activity. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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42 pages, 3576 KiB

Open AccessReview

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

by Annamaria Sandomenico, Jwala P. Sivaccumar and Menotti Ruvo

Int. J. Mol. Sci. 2020, 21(17), 6324; https://doi.org/10.3390/ijms21176324 - 31 Aug 2020

Cited by 56 | Viewed by 9588

Abstract

Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. [...] Read more.

Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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28 pages, 3708 KiB

Open AccessReview

Antibody-Drug Conjugates: The New Frontier of Chemotherapy

by Sara Ponziani, Giulia Di Vittorio, Giuseppina Pitari, Anna Maria Cimini, Matteo Ardini, Roberta Gentile, Stefano Iacobelli, Gianluca Sala, Emily Capone, David J. Flavell, Rodolfo Ippoliti and Francesco Giansanti

Int. J. Mol. Sci. 2020, 21(15), 5510; https://doi.org/10.3390/ijms21155510 - 31 Jul 2020

Cited by 83 | Viewed by 9965

Abstract

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels [...] Read more.

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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24 pages, 1188 KiB

Open AccessReview

B-Cell Maturation Antigen (BCMA) as a Target for New Drug Development in Relapsed and/or Refractory Multiple Myeloma

by Hanley N. Abramson

Int. J. Mol. Sci. 2020, 21(15), 5192; https://doi.org/10.3390/ijms21155192 - 22 Jul 2020

Cited by 24 | Viewed by 8579

Abstract

During the past two decades there has been a major shift in the choice of agents to treat multiple myeloma, whether newly diagnosed or in the relapsed/refractory stage. The introduction of new drug classes, such as proteasome inhibitors, immunomodulators, and anti-CD38 and anti-SLAMF7 [...] Read more.

During the past two decades there has been a major shift in the choice of agents to treat multiple myeloma, whether newly diagnosed or in the relapsed/refractory stage. The introduction of new drug classes, such as proteasome inhibitors, immunomodulators, and anti-CD38 and anti-SLAMF7 monoclonal antibodies, coupled with autologous stem cell transplantation, has approximately doubled the disease’s five-year survival rate. However, this positive news is tempered by the realization that these measures are not curative and patients eventually relapse and/or become resistant to the drug’s effects. Thus, there is a need to discover newer myeloma-driving molecular markers and develop innovative drugs designed to precisely regulate the actions of such putative targets. B cell maturation antigen (BCMA), which is found almost exclusively on the surfaces of malignant plasma cells to the exclusion of other cell types, including their normal counterparts, has emerged as a specific target of interest in this regard. Immunotherapeutic agents have been at the forefront of research designed to block BCMA activity. These agents encompass monoclonal antibodies, such as the drug conjugate belantamab mafodotin; bispecific T-cell engager strategies exemplified by AMG 420; and chimeric antigen receptor (CAR) T-cell therapeutics that include idecabtagene vicleucel (bb2121) and JNJ-68284528. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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11 pages, 486 KiB

Open AccessReview

CD38 and Anti-CD38 Monoclonal Antibodies in AL Amyloidosis: Targeting Plasma Cells and beyond

by Dario Roccatello, Roberta Fenoglio, Savino Sciascia, Carla Naretto, Daniela Rossi, Michela Ferro, Antonella Barreca, Fabio Malavasi and Simone Baldovino

Int. J. Mol. Sci. 2020, 21(11), 4129; https://doi.org/10.3390/ijms21114129 - 10 Jun 2020

Cited by 19 | Viewed by 4807

Abstract

Immunoglobulin light chain amyloidosis (AL amyloidosis) is a rare systemic disease characterized by monoclonal light chains (LCs) depositing in tissue as insoluble fibrils, causing irreversible tissue damage. The mechanisms involved in aggregation and deposition of LCs are not fully understood, but CD138/38 plasma [...] Read more.

Immunoglobulin light chain amyloidosis (AL amyloidosis) is a rare systemic disease characterized by monoclonal light chains (LCs) depositing in tissue as insoluble fibrils, causing irreversible tissue damage. The mechanisms involved in aggregation and deposition of LCs are not fully understood, but CD138/38 plasma cells (PCs) are undoubtedly involved in monoclonal LC production.CD38 is a pleiotropic molecule detectable on the surface of PCs and maintained during the neoplastic transformation in multiple myeloma (MM). CD38 is expressed on T, B and NK cell populations as well, though at a lower cell surface density. CD38 is an ideal target in the management of PC dyscrasia, including AL amyloidosis, and indeed anti-CD38 monoclonal antibodies (MoAbs) have promising therapeutic potential. Anti-CD38 MoAbs act both as PC-depleting agents and as modulators of the balance of the immune cells. These aspects, together with their interaction with Fc receptors (FcRs) and neonatal FcRs, are specifically addressed in this paper. Moreover, the initiallyavailable experiences with the anti-CD38 MoAb DARA in AL amyloidosis are reviewed. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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16 pages, 1914 KiB

Open AccessTechnical Note

A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins

by Julie Pelletier, Hervé Agonsanou, Fabiana Manica, Elise G. Lavoie, Mabrouka Salem, Patrick Luyindula, Romuald Brice Babou Kammoe and Jean Sévigny

Int. J. Mol. Sci. 2020, 21(19), 7074; https://doi.org/10.3390/ijms21197074 - 25 Sep 2020

Viewed by 2726

Abstract

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique [...] Read more.

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins. Full article

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

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