Epitope Analysis of Murine and Chimeric Monoclonal Antibodies Recognizing the Cancer Testis Antigen PRAME

Misyurin, V. A.; Finashutina, Yu. P.; Turba, A. A.; Larina, M. V.; Солопова, О. Н.; Лыжко, Н А; Кесаева, Л. А.; Kasatkina, N. N.; Алиев, Т. К.; Misyurin, A V; Kirpichnikov, Mikhaǐl P.

doi:10.1134/s1607672920030072

Cited by 7 publications

(4 citation statements)

References 6 publications

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“…Recently, an epitope analysis of a panel of mAbs targeting PRAME protein has been reported [20]. Here, we explored the used of BLI biosensors to capture a fragment of the PRAME protein recognized by a newly generated monoclonal antibody.…”

Section: Discussionmentioning

confidence: 99%

“…However, they may display a relatively low affinity for the full-length parent proteins where the structure of the epitope may differ considerably from that of the original immunogen. In the case of PRAME, the epitope analysis of a panel of mAbs [20] and the development of a polyclonal antibody against the predicted extracellular PRAME 310-331 peptide [16] have been recently described. Capture experiments of protein fragments with antibody-immobilized sensor chips can also be performed in this instance using, for example, label-free devices that provide real-time evidence of the capture and of the release of the bound epitope [21,22].…”

Section: Introductionmentioning

confidence: 99%

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Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Sivaccumar

Leonardi

Iaccarino

et al. 2021

IJMS

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Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Sivaccumar

Leonardi

Iaccarino

et al. 2021

IJMS

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“…Ethyl N-(11-azido-3,6,9-trioxaundecyloxy) acetimidate 1 was prepared as described [42]; NHS derivatives of 6-carboxyfluorescein, 6-carboxy-Alexa488, and BDP-FL were obtained from Lumiprobe. The 6H8 monoclonal antibody was obtained as described in a previous study [58]. All NMR data for the synthesized compounds were obtained in DMSO-d 6 solution at 30 • C. The 1D 1 H, 13 C, and 15 N NMR spectra were acquired using a Bruker AVANCE-III-600 spectrometer equipped with a room-temperature probe.…”

Section: General Methodsmentioning

confidence: 99%

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Sapozhnikova

Misyurin²,

Ryazantsev

et al. 2021

IJMS

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Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

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“…For the present study, we choose a PRAME-specific monoclonal IgG1 [ 32 , 33 ] as the tumor-targeting antibody. Preferentially expressed antigen of melanoma (PRAME) is a small human protein which is seldom expressed in the cytosol of normal cells (it is found in small amounts in the cytosol of cells of the male reproductive system) but is present in the membrane of melanoma and acute leukemia tumor cells.…”

Section: Introductionmentioning

confidence: 99%

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody–Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control

Sapozhnikova

Gulyak

Brylev

et al. 2023

IJMS

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A universal approach to the construction of antibody–drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug–antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

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Epitope Analysis of Murine and Chimeric Monoclonal Antibodies Recognizing the Cancer Testis Antigen PRAME

Cited by 7 publications

References 6 publications

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody–Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control

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