Human-derived Anti-(poly-ga) dipeptide repeat (DPR) antibody

A repetitive sequence, VH-CDR2 technology, applied in the direction of antibodies, dipeptides, radioactive carriers, etc., can solve the potential immunogenic properties of inherently unstable compounds in RNA, etc.

Pending Publication Date: 2021-04-09
BIOGEN MA INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] However, RNA-based therapeutics, small organic compounds, and gene therapies suffer from several disadvantages, such as the inherent instability of RNA, the potentially immunogenic properties of the compounds, and the need for delivery vehicles that are efficiently transported to target cells, and regarding Ethical questions about the use of gene therapy remain

Method used

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  • Human-derived Anti-(poly-ga) dipeptide repeat (DPR) antibody
  • Human-derived Anti-(poly-ga) dipeptide repeat (DPR) antibody
  • Human-derived Anti-(poly-ga) dipeptide repeat (DPR) antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0600] Example 1: Isolation and identification of anti-(poly-GA) dipeptide repeat (DPR) protein antibodies

[0601] Human antibodies targeting poly-GA dipeptide repeat (DPR) proteins, fragments thereof, C9orf72-DPR and / or fragments thereof were identified based on the methods described in international application WO 2016 / 050822 A2, which The disclosure of is incorporated herein by reference. In particular, the poly-GA dipeptide repeat protein GA was synthesized and purified by Schafer-N (Copenhagen, Denmark) 15 : H-CHHHHHH(GA) 15 -OH) (SEQ ID NO: 61). The poly-GA dipeptide repeat protein was then conjugated to bovine serum albumin (BSA) via a bifunctional linker (SMCC). Subsequently, direct ELISA was performed directly using a 96-well microplate (Corning) in coating buffer (15 mM Na 2 CO 3 , 35mM NaHCO 3 Coated with non-conjugated or BSA-conjugated poly-GA dipeptide repeat protein or BSA (Sigma-Aldrich, Buchs, Switzerland) at a concentration of 5 μg / ml in , pH 9.42). A...

Embodiment 2

[0602] Example 2: Determination of antibody sequences

[0603] The amino acid sequences of the variable regions of the anti-(poly-GA)DPR antibodies identified above were determined based on their mRNA sequences, see figure 1 A-F. Briefly, live B cells of selected non-immortalized memory B cell cultures were harvested. Subsequently, mRNA was extracted from cells producing the selected anti-(poly-GA)DPR antibody and converted to cDNA, and the sequence encoding the variable region of the antibody was amplified by PCR and cloned into a plasmid vector and sequenced. Briefly, primer combinations representing all sequence families of the human immunoglobulin germline repertoire were used for amplification of leader peptides, V-segments and J-segments. A first round of amplification was performed using leader peptide specific primers at the 5' end and constant region specific primers at the 3' end (Smith et al., Nat Protoc. 4 (2009), 372-384). For heavy and kappa light chains, a ...

Embodiment 3

[0607] Example 3: ELISA EC for C9orf72 dipeptide repeat protein 50 analyze

[0608] In order to determine the binding specificity and half maximal effective concentration (EC) of recombinant human C9orf72 antibody NI-308.5J10 to C9orf72 poly-GA DPR 50 ), ELISA EC 50 analyze. Briefly, dipeptide repeat proteins were synthesized and purified by Schafer-N (Copenhagen, Denmark): (GA) 15 : H-CHHHHHH(GA) 15 -OH (SEQ ID NO: 61); (GP) 15 : H-C(GP) 15 -OH (SEQ ID NO: 62); (GR) 15 : H-C(GR) 15 -OH (SEQ ID NO: 63); (PA) 15 : H-C(PA) 15 -OH (SEQ ID NO: 64); (PR) 15 : H-C(PR) 15 -OH (SEQ ID NO: 65). A 96-well microplate (Corning Incorporated, Corning, USA) was coated with coating buffer (15 mM Na 2 CO 3 , 35mM NaHCO 3 , pH 9.42) at a concentration of 5 μg / ml or 20 μg / ml dipeptide repeat protein peptide coating. At room temperature with PBS / 0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland) -20 to block non-specific binding sites for 1 hr. NI-308.5J10 was diluted t...

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Abstract

Provided are novel dipeptide repeat (DPR) specific antibodies (e.g., human-derived antibodies) as well as synthetic variants and biotechnological derivatives thereof, capable of binding C9orf72 poly-glycine-alanine DPRs, as well as methods and uses related thereto. The antibody of the present invention can be used in pharmaceutical and diagnostic compositions for DPR protein-targeted immunotherapy and diagnostics.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to European Patent Application No. 18169888.7, filed April 27, 2018, and U.S. Provisional Application No. 62 / 772,809, filed November 29, 2018. The contents of both applications are incorporated herein by reference in their entirety. [0003] sequence listing [0004] This application contains a Sequence Listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 24, 2019, is named 13751-0309WO1_SL.txt and is 102,178 bytes in size. technical field [0005] The present invention relates generally to antibody-based therapeutics and diagnostic methods. In particular, the present invention relates to novel human antibodies and their fragments, derivatives and biotechnological variants that specifically bind to unconventional non-ATG translations, especially as in chromosome 9 open reading frame 72 (C9o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/06C07K16/44
CPCC07K16/44C07K2317/56C07K2317/565C07K2317/55C07K2317/92C07K2317/94A61K2039/505A61P25/28C07K2317/33G01N33/6893A61P21/00C07K2317/24A61K47/6835A61K49/0002A61K51/10
Inventor J.格里姆F.蒙特拉西奥I.达尔基利克-利德尔M.M.拉什J.W.阿恩特
Owner BIOGEN MA INC
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