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Hla-restricted epitopes encoded by somatically mutated genes

A cell and cell receptor technology, applied in receptor/cell surface antigen/cell surface determinant, translation product of oncogene, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc.

Active Publication Date: 2018-06-08
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Intracellular proteins do not have access to large molecules such as antibodies, but unfortunately, the vast majority of abnormal epitopes encoded by mutant genes are not on the cell surface(2)

Method used

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  • Hla-restricted epitopes encoded by somatically mutated genes
  • Hla-restricted epitopes encoded by somatically mutated genes
  • Hla-restricted epitopes encoded by somatically mutated genes

Examples

Experimental program
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Embodiment 1

[0047] Materials and methods

[0048] cell line. T2 cells (ATCC, Manassas, VA) were incubated at 37°C in 5% CO 2 were cultured in the presence of 10% FBS (GEHyclone, Logan, Utah, USA), 1% penicillin streptomycin (Life Technologies) and 20IU / mL recombinant human IL-2 (Proleukin TM , Prometheus Laboratories) in RPMI-1640 (ATCC). T2A3 cells (a gift from Eric Lutz and Liz Jaffee, Johns Hopkins University (JHU)) were grown under the same conditions as T2 cells, but with the addition of 500ug / mL Geneticin (Life Technologies) and 1 × Non-essential amino acids (Life Technologies).

[0049] Phage display library construction. Oligonucleotides were synthesized at DNA 2.0 (Menlo Park, CA) using mixed and split pooldegenerate oligonucleotide synthesis. Oligonucleotides were incorporated into pADL-10b phagemid (Antibody Design Labs, San Diego, CA). The phagemid contains an F1 origin, and a transcriptional repressor unit consisting of a lactose operator and a lactose repressor to limi...

Embodiment 2

[0073] Design and construction of scFv-based phage display scFv libraries. The inventors initiated these studies in an attempt to generate antibodies against the mutant KRAS peptide in mice (the basis for this selection is described below). Using conventional methods to obtain monoclonal antibodies after immunization of mice, these efforts failed because no antibodies specifically reactive with MANA were identified. The inventors therefore turned to a phage display method for generating MANAbodies ( figure 1 ). The design of phage display libraries follows principles used in published studies (22) and includes some specific features. The framework of the library is based on the scFv sequence of the humanized 4D5 antibody (Trastuzumab) raised against the protein encoded by ERBB2 (23). This framework was chosen for its stability on phage and its ease of conversion into soluble scFv, Fab or antibody (22, 24). A high-resolution crystal structure of humanized 4D5 has identified...

Embodiment 3

[0076] Target selection and competition strategies for identifying selectively responsive phage clones. The inventors selected MANAbody targets based on the frequency of specific mutations and their predicted strength of binding to HLA alleles. KRAS is one of the most frequently mutated genes in human cancers, with mutations particularly prevalent in pancreatic, colorectal and lung adenocarcinomas. The inventors chose the G12V mutation as a target because a related peptide containing it is predicted to bind with high affinity to HLA-A2, the most common HLA allele in many human populations (32). This bioinformatics (in silico) prediction was performed using the NetMHC v3.4 algorithm (33-35). Additionally, based on structural studies of other peptide-HLA complexes, a key mutated residue (V at codon 12) was expected to be exposed on the surface of HLA proteins (36). The peptide KLVVVGAVGV (SEQ ID NO: 4), in which the valine residue (V) at position 8 represents the G12V mutation...

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Abstract

Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We generated single chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by human leukocyte antigen (HLA) molecules. These scFvs can be converted to full-length antibodies, termed MANAbodies, targeting "Mutation Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for peptides bound to pre-defined HLA types. In this way, we obtained scFvs, including one specific for a peptide encoded by a common KRAS mutant andanother by a common EGFR mutant. Molecules targeting MANA can be developed that specifically react with mutant peptide-HLA complexes even when these peptides differ by only one amino acid from the normal, wild-type form.

Description

[0001] This invention was made with government support under CA 43460 and CA 062924 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0002] The present invention relates to the field of antibody production. In particular, it relates to constructs containing antibody variable regions in single chain or other types of antibody molecules. Background technique [0003] Cancer is the result of sequential mutations in oncogenes and tumor suppressor genes (1). In theory, somatic mutations are ideal therapeutic targets because they are hardly found in any normal cells (2). Even though these mutated protein products usually differ only slightly from the wild-type (wt) form, often by a single amino acid, this difference is sufficient for effective targeting. When the protein is an enzyme, such as that encoded by BRAF, the resulting structural changes can provide pockets for binding specific enzyme inhibitors (3-5)....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/32C07K14/74C07K19/00G01N33/574A61K47/68A61P35/00
CPCC07K16/32C07K19/00G01N33/6854G01N2333/7051C07K14/71C07K14/82C07K16/18C07K16/2863C07K7/00C07K16/40C07K14/47C07K14/4746C07K2317/34C07K2317/56C07K2317/565C07K2319/00C07K2319/03C07K2319/41C07K2319/50C07K16/2833C07K2317/24C07K2317/32C07K2317/622C07K2317/92A61P35/00C07K16/005C07K2317/55C07K2317/734G01N33/574G01N2333/70539
Inventor B·沃格尔斯坦K·W·肯斯勒S·周L·迪亚斯N·帕帕佐普洛斯A·斯科拉J·多格拉斯M·S·赫望
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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