Compositions comprising aav expressing dual antibody constructs and uses thereof

a technology of dual antibody and composition, which is applied in the field of compositions comprising aav expressing dual antibody constructs, can solve the problems of inconvenient re-administration, limited duration of efficacy per administration, and inconsistent serum levels

Active Publication Date: 2019-01-31
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]Still other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.

Problems solved by technology

Current antibody therapy often involves repeat administration and long term treatment regimens, which are associated with a number of disadvantages, such as inconsistent serum levels and limited duration of efficacy per administration such that frequent re-administration is required and high cost.
One limitation to the widespread clinical application of antibody technology is that typically large amounts of antibody are required for therapeutic efficacy and the costs associated with production are significant.
Due to the limited transgene packaging capacity of AAV, it has been a technical challenge to have a tightly regulated system to express heavy and light chains of an antibody using a single AAV vector in order to generate full length antibodies.

Method used

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  • Compositions comprising aav expressing dual antibody constructs and uses thereof
  • Compositions comprising aav expressing dual antibody constructs and uses thereof
  • Compositions comprising aav expressing dual antibody constructs and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

n of Vectors Containing Full-Length Antibody Co-Expression Cassettes

[0090]A series of cis-plasmids were prepared for use in generating an AAV viral particle containing a nucleic acid molecule for delivery to a host target cell. The nucleic acid molecules comprise AAV2 5′ and 3′ ITR sequences at each terminus, a shared CMV enhancer flanked by two expression cassettes in opposite orientations, where a first expression cassette is controlled by a first minimal CMV promoter and a second expression cassette is controlled by a second minimal CMV promoter. All sequences located between AAV2 ITRs were de novo synthesized by a commercial vendor (GeneArt). All coding sequences for immunoglobulin variable domains were flanked with the unique restriction enzymes to allow convenient shuttling of the desired variable domains. To create constructs with heterologous light chain sequence (kgl), a coding sequence encoding germline light chain (IGKV4-1*01) was de novo synthesized and used to replace F...

example 2

ization of Products Expressed from AAV8 Vectors Co-Expressing F16 Monoclonal Antibody (MAB) and IA6 MAB

[0095]A series of ELISA assays were performed to characterize expression levels and to assess binding of the FI6 MAB co-expressed with the IA6 MAB from the cis plasmid generated as described in Example 1 after transfection into HEK 293 cells. TSG101 peptide was synthesized using f-Moc chemistry by Mimotopes. All flu antigens were procured from a commercial supplier, ImmuneTechnologies, Inc. ProteinA was purchased from Sigma-Aldrich and was used to monitor expression of total human IgG1. Detection of human IgG1 in tissue culture supernatants was measured by either antigen-specific or proteinA capture ELISA. High binding ELISA plates were coated with 2 μg / ml of HA proteins or peptides, or with 5 μg / ml proteinA diluted in PBS and incubated overnight at 4° C. Wells were washed 5-8 times and blocked with 1 mM EDTA, 5% heat inactivated PBS, 0.07% Tween 20 in PBS for one hour at room temp...

example 3

ization of Products Expressed from AAV8 Vectors Co-Expressing Fi6 Monoclonal Antibody (MAB) and Pandemic Flu MAB C05

[0098]The possibility of differential detection of two different monoclonal antibodies was assessed in a capture assay. Monoclonal antibodies FI6 and C05 co-expressed from a cis-plasmid prepared as described in Example 1 and transfected into HEK293 cells were assessed for binding. FI6 is expected to bind to full-length HA and to HA stem, but not to the head only region. The results of the binding study illustrated in FIG. 3 demonstrate that the co-expressed antibodies retain their characteristic binding. More particularly, binding to full-length HA and the HA stem characteristic of FI6 is observed and binding to HA and HA head only (no stem) characteristic of C05 is also observed. ELISA assays were performed as described in Example 2.

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Abstract

A recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses two functional antibody constructs in a cell is described. Also described are antibodies comprising a heavy chain and a light chain from a heterologous antibody. In one embodiment, the antibodies are co-expressed from a vector containing: a first expression cassette which encodes at least a first open reading frame (ORF) for a first immunoglobulin under the control of regulatory control sequences which direct expression thereof; and a second expression cassette which comprises a second ORF, a linker, and a third ORF under the control of regulatory control sequences which direct expression thereof, wherein the second and third ORF for a second and third immunoglobulin construct. The vector co-expressing these two antibody constructs is in one embodiment an AAV, in which the 5′ and 3′ ITRs flank the expression cassettes and regulatory sequences.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This is a continuation of U.S. patent application Ser. No. 15 / 310,555, filed Nov. 11, 2016, now pending, which is a national stage application under 35 USC 371 of PCT / US2015 / 030533, filed May 13, 2015, now expired, which claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 61 / 992,649, filed May 13, 2014, now expired. Each of these applications is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with government support under grant number ARO No. 64047-LS-DRP awarded by Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM[0003]Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “14-7032PCT_Seq Listing_ST25.txt” and dated May 13, 2...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/10A61K48/00C12N15/86C12N7/00
CPCA61K48/0008C12N2830/30C12N2750/14143C07K2317/51C12N2840/203C07K16/1018C12N2750/14121C12N15/86C12N2830/205C07K2317/31C12N7/00C07K2317/622C07K2317/14C12N2830/50A61K2039/507C07K2317/515C07K2317/52A61P19/02A61P25/00A61P29/00A61P31/00A61P31/04A61P31/10A61P31/12A61P31/16A61P33/00A61P35/00A61P37/06A61P43/00A61K35/761C07K16/00A61K39/395
Inventor WILSON, JAMES M.TRETIAKOVA, ANNA
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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