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Compositions and methods for binding antibodies and inhibiting neutralizing antibodies

An antibody, binding site technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the moderate increase in antibody escape, insufficient resistance to polyclonal anti-AAV serum, low efficacy, etc. question

Pending Publication Date: 2022-05-17
THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

AAV capsid engineering is an innovative approach but ultimately insufficient against polyclonal anti-AAV sera, with a modest 10-fold increase in antibody escape observed in vivo
In addition, modification of the capsid structure to alter the NAb recognition epitope often results in less potent vectors and defective vector production, since these altered surface regions are multifunctional
Currently, no method successfully overcomes pre-existing anti-AAV NAbs above typical thresholds excluded from clinical trials, or allows repeated administration of the same AAV vector

Method used

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  • Compositions and methods for binding antibodies and inhibiting neutralizing antibodies
  • Compositions and methods for binding antibodies and inhibiting neutralizing antibodies
  • Compositions and methods for binding antibodies and inhibiting neutralizing antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0362] Example 1: Method

[0363] AAV virus production: AAV vectors were produced in HEK293 cells by the standard method of three-plasmid transfection. Briefly, the AAV transgenic plasmid pTR CBA-Luc was co-transfected with the AAV Rep / Cap helper plasmid (pXR2 or pXR8) and the adenovirus helper plasmid pXX6-80. After 72 hours, cell cultures were harvested and lysed by freeze-thawing and sonication. Clarified cell lysates were DNase-treated and ultracentrifuged with a 15% / 25% / 40% / 60% iodixanol gradient and purified by anion exchange Q-columns. Purified AAV vectors were titrated by qPCR with primers used to amplify the packaged AAV transgene segment.

[0364] Protein production: Rajesh widely provided the plasmid pET-28b(+), which encodes protein M, a truncated M. genitalium protein MG281, lacking the transmembrane domain (amino acids 74 to 479) and carrying an N-terminal His-tag and Thrombin cleavage site. Plasmids were propagated in electrocompetent DH10B cells and purifie...

Embodiment 2

[0371] Example 2: Protein M abolishes the inhibitory activity of IVIG on AAV transduction

[0372] To investigate the antibody-blocking function of protein M, a human intravenous immunoglobulin gamma (IVIG) in vitro AAV neutralization assay was established. AAV neutralization assays were performed using serial dilutions of IVIG to determine the amount of IVIG that would neutralize a given amount of AAV2. Luciferase activity resulting from transduction of cells with AAV luciferase vector serves as a functional readout of gene expression. The neutralization was determined as a percentage of luciferase activity normalized to the no IVIG control. 12.5 μg of IVIG was found to neutralize 75% (+ / - 5%) of AAV2 ( figure 1 ), select this amount of IVIG for experimental design to test that protein M blocks IVIG from neutralizing AAV. Next, a dose dilution series was performed on protein M (SEQ ID NO:2), collected by thrombin cleavage to remove the His-tag, and its ability to block 12....

Embodiment 3

[0373] Example 3: Interaction of protein M with AAV vector virions enhances AAV transduction

[0374] Previous studies by the inventors have shown that the interaction of serum proteins with AAV virions enhances AAV transduction. To further characterize the enhancing function of protein M on AAV transduction, a dose-response assay was performed without the use of IVIG, in which protein M was incubated with AAV at serial 2-fold dilutions for 1 h prior to cell culture transduction. exist Figure 4 found that in the absence of IVIG, an 8:1 ratio of protein M (33 μg of protein M) in previous experiments was able to dose-dependently enhance AAV transduction, and for 2x10 8 virus particles, loss of enhancement at dilutions below 2 μg of protein M. The equivalent molar ratio of protein M molecules that lost the enhancement effect to AAV particles was lower than 40000:1. Next, it was demonstrated that the enhanced transduction of protein M was dependent on the incubation of protein...

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Abstract

The present invention relates to methods and compositions for binding antibodies. The methods may be used to isolate antibodies, treat conditions associated with excess antibodies, acute block antibodies to prevent autoimmune or inflammatory responses, and inhibit neutralizing antibodies. In an embodiment, the present invention relates to a method of inhibiting a neutralizing antibody against a heterologous agent when the heterologous agent is administered to a subject, comprising administering to the subject an effective amount of Mycoplasma Protein M, or a functional fragment or derivative thereof, thereby inhibiting neutralization of the heterologous agent. The invention also relates to a modified Mycoplasma protein M or a functional fragment thereof having increased thermal stability relative to the wild-type protein M and its use in the method of the invention.

Description

[0001] priority statement [0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 881,765, filed August 1, 2019, the entire contents of which are incorporated herein by reference. technical field [0003] The present invention relates to methods and compositions for binding antibodies. These methods can be used to isolate antibodies, treat conditions associated with excess antibodies, acutely block antibodies to prevent autoimmune or inflammatory responses, and inhibit neutralizing antibodies. In an embodiment, the invention relates to a method of inhibiting neutralizing antibodies against a heterologous agent upon administration to a subject, comprising administering to the subject an effective amount of Mycoplasma protein M or functional fragments or derivatives thereof, thereby inhibiting the neutralization of the xenobiotic. The present invention also relates to modified Mycoplasma protein M or functional fragments thereof having increased...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/30C07K16/00C12N15/31C12N15/70C12N15/85C12N1/21C12N5/10A61K38/16A61P37/02A61P35/00A61P9/00G01N33/569G01N33/564
CPCC07K14/30C07K16/00C12N15/70C12N15/85A61P37/02A61P35/00A61P9/00G01N33/56933G01N33/564C12N2800/22C12N2800/101C12N2800/107A61K38/00G01N2800/24A61P37/00G01N33/57426C07K2319/036
Inventor 李成文查尔斯·艾斯丘布赖恩·库尔曼戴维·福里斯特·蒂克
Owner THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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