diagnostics Review Point Mutation Specific Antibodies in B-Cell and T-Cell Lymphomas and Leukemias: Targeting IDH2, KRAS, BRAF and Other Biomarkers RHOA, IRF8, MYD88, ID3, NRAS, SF3B1 and EZH2 Kunwar Singh 1,*,† , Sumanth Gollapudi 2,† , Sasha Mittal 2, Corinn Small 2, Jyoti Kumar 1 and Robert S. Ohgami 2,* 1 Department of Pathology, Stanford University, Stanford, CA 94063, USA; [email protected] 2 Department of Pathology, University of California, San Francisco, CA 94143, USA; [email protected] (S.G.); [email protected] (S.M.); [email protected] (C.S.) * Correspondence: [email protected] (K.S.); [email protected] (R.S.O.); Tel.: +1-347-856-7047 (K.S.); +1-415-514-8179 (R.S.O.) † These authors contributed equally. Abstract: B-cell and T-cell lymphomas and leukemias often have distinct genetic mutations that are diagnostically defining or prognostically significant. A subset of these mutations consists of specific point mutations, which can be evaluated using genetic sequencing approaches or point mutation Citation: Singh, K.; Gollapudi, S.; specific antibodies. Here, we describe genes harboring point mutations relevant to B-cell and T-cell Mittal, S.; Small, C.; Kumar, J.; malignancies and discuss the current availability of these targeted point mutation specific antibodies. Ohgami, R.S. Point Mutation Specific We also evaluate the possibility of generating novel antibodies against known point mutations Antibodies in B-Cell and T-Cell by computationally assessing for chemical and structural features as well as epitope antigenicity Lymphomas and Leukemias: of these targets. Our results not only summarize several genetic mutations and identify existing Targeting IDH2, KRAS, BRAF and point mutation specific antibodies relevant to hematologic malignancies, but also reveal potential Other Biomarkers RHOA, IRF8, underdeveloped targets which merit further study. MYD88, ID3, NRAS, SF3B1 and EZH2. Diagnostics 2021, 11, 600. https://doi.org/10.3390/ Keywords: single nucleotide polymorphisms; hematopathology; cancer; diagnostics; point mutation diagnostics11040600 specific antibodies; precision medicine Academic Editor: Chung-Che (Jeff) Chang 1. Introduction Received: 4 February 2021 In 2016, the World Health Organization (WHO) revised the fourth edition of the WHO Accepted: 24 March 2021 Classification of Tumours of Haematopoietic and Lymphoid Tissues based on new molecular and Published: 27 March 2021 morphological findings [1]. The revised criteria for numerous hematopoietic malignancies indicate that genetic mutations play a significant role in the diagnosis of these entities. Publisher’s Note: MDPI stays neutral Many of these mutations are well-defined recurrent point mutations in specific genes. with regard to jurisdictional claims in While such mutations can be detected using genetic sequencing methodologies, another published maps and institutional affil- strategy for identification is to develop antibodies that specifically target mutated sites in iations. proteins present within patient tissue samples [2,3]. Monoclonal or polyclonal antibodies can also be developed against these mutations by conjugating the relevant peptide to a carrier protein and injecting the peptide-protein complex into a host animal (Figure1). Many biotechnology companies produce antibodies that target commonly expressed Copyright: © 2021 by the authors. proteins but there is a paucity of antibodies available on the market that are designed Licensee MDPI, Basel, Switzerland. to target point mutations related to T-cell and B-cell malignancies. The ability to use This article is an open access article antibodies in hematologic malignancies would provide benefit both in research and clinical distributed under the terms and settings. Compared to next-generation sequencing (NGS) assays, in which results and conditions of the Creative Commons interpretation can take weeks to finalize, in situ immunohistochemistry (IHC) assays Attribution (CC BY) license (https:// are a sensitive and often more efficient approach. In order to expedite the diagnosis creativecommons.org/licenses/by/ 4.0/). and treatment of hematologic malignancies, a tiered system consisting of early IHC and Diagnostics 2021, 11, 600. https://doi.org/10.3390/diagnostics11040600 https://www.mdpi.com/journal/diagnostics Diagnostics 2021, 11, 600 2 of 14 Diagnostics 2021, 11, 600 follow up NGS is used in many clinical settings and has been proposed in some published2 of 14 studies [4]. However, a limitation to this rapid protocol, in certain settings, is the availability of mutation-specific antibodies. Figure 1. Example of a conventional targeted monoclonal and polyclonal antibody development process using conju- Figure 1. Example of a conventional targeted monoclonal and polyclonal antibody development process using conjugated gated peptides. peptides. In this study, we aim to describe both current and potential biomarker targets in T-cell Many biotechnology companies produce antibodies that target commonly expressed and B-cell neoplasms, highlighting several point mutations for their suitability in antibody proteins but there is a paucity of antibodies available on the market that are designed to development based on their predicted antigenicity in relation to multiple chemical and targetstructural point mutations parameters. related to T-cell and B-cell malignancies. The ability to use anti- bodies in hematologic malignancies would provide benefit both in research and clinical settings.2. Materials Compared and to Methods next-generation sequencing (NGS) assays, in which results and in- terpretation2.1. 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Peptide and Antibody Evaluation 2. 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A main factor considered for crosslinker selection was the ability to bind to residues 2.2.without Peptide and reacting Antibody to nearby Evaluation side chains, which could lead to incorrect conjugation to the carrier protein. MBS is a crosslinker compound with a maleimide reactive group at one end andThe an potential ester group for onpoint the mutation other end. specific The maleimide antibody group production reacts with of the sulfhydryl selected groupsgenes to wasform assessed thioester by using linkages, peptide therefore sequences the 21-mer from shouldthe associated not contain proteins cysteine that residues. contained EDC the is a mutatedcarbodiimide, amino acids, and itswhich reactivity were toidentified carboxyl using groups NCBI’s leads GenBank to direct conjugation and UniProt. to aThe peptide’s se- quencesfree amineused for group. analysis As consisted a result, of the the side targeted chains point of aspartic mutation and site, glutamic the 10 amino acid residues,acids upstreamboth of and which the contain10 amino carboxylate acids downstream ions, can of react the with