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1.
ACS Bio Med Chem Au ; 3(6): 516-527, 2023 Dec 20.
Article in English | MEDLINE | ID: mdl-38144259

ABSTRACT

NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.

2.
Sci Immunol ; 6(57)2021 03 01.
Article in English | MEDLINE | ID: mdl-33649101

ABSTRACT

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.


Subject(s)
Antibodies, Bispecific/pharmacology , Antigens, Neoplasm , Biomarkers, Tumor/antagonists & inhibitors , Mutant Proteins/antagonists & inhibitors , ras Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , Antibodies, Bispecific/immunology , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/immunology , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , Cell Line , Cross Reactions , HLA Antigens/immunology , Humans , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mutant Proteins/chemistry , Mutant Proteins/immunology , Mutation , Peptide Fragments , Protein Binding/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , ras Proteins/chemistry , ras Proteins/genetics , ras Proteins/immunology
3.
Science ; 371(6533)2021 03 05.
Article in English | MEDLINE | ID: mdl-33649166

ABSTRACT

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.


Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neoplasm/immunology , Antigens, Neoplasm/immunology , HLA-A2 Antigen/immunology , Neoplasms/therapy , Tumor Suppressor Protein p53/immunology , Alleles , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/therapeutic use , Antibodies, Neoplasm/chemistry , Antibodies, Neoplasm/therapeutic use , Arginine/genetics , COS Cells , Chlorocebus aethiops , Female , HEK293 Cells , HLA-A2 Antigen/chemistry , HLA-A2 Antigen/genetics , Histidine/genetics , Humans , Immunization, Passive , Jurkat Cells , Lymphocyte Activation , Mice, Inbred NOD , Mutation , T-Lymphocytes/immunology , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
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