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Methods and Compositions for Localized Secretion of Anti-CTLA-4 Antibodies

an antictla4 antibody and localized secretion technology, applied in the field of methods and compositions for localized secretion of antictla4 antibodies, can solve problems such as unsatisfactory toxicities

Active Publication Date: 2017-04-27
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes an approach to create an anti-CTLA-4 antibody with reduced effector function. This can be achieved by engineering the antibody to have reduced binding to Fc receptors, lack an Fc region, or be engineered to have reduced dimerization. The antibody can also be integrated into a vector, like a retroviral, lentiviral, or AAV vector, for stable transformation of human cells. The technical effect of this approach is to create an anti-CTLA-4 antibody with improved safety and reduced side effects.

Problems solved by technology

Ongoing clinical investigations utilizing systemic administration of these antibodies have provided dramatic successes, but have also produced undesirable toxicities.

Method used

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  • Methods and Compositions for Localized Secretion of Anti-CTLA-4 Antibodies
  • Methods and Compositions for Localized Secretion of Anti-CTLA-4 Antibodies
  • Methods and Compositions for Localized Secretion of Anti-CTLA-4 Antibodies

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0120]The 9D9 mouse anti-mouse CTLA4 antibody was typed using the IsoStrip kit from Roche diagnostics. The 9D9 antibody was determined to be IgG2b-κ (data not shown).

example 2

[0121]The 9D9 antibody was cloned into Ig: FIG. 1 shows the cloning strategy.

[0122]Redundant primers for the 9D9 light chain and heavy chain were designed based on Wang, Z and Ratner, D (J Immunol Methods, 2000 Jan. 13; 233(1-2):167-77) to amplify the light chain (VL) and heavy chain (VH) variable regions. PCR was performed using the Advantage 2 PCR kit from Clontech to generate products with T-A ends, and using a 3:1 mix of Vent (NEB) and Pfu (Stratagene) polymerases to generate blunt-ended fragments. FIG. 2 shows the sequences of the light and heavy chain primers.

[0123]A low, 45 degree annealing temperature was used to promote hybridization of partially mis-matched primer / template sequences. Primers were designed to contain restriction sites to facilitate subsequent cloning of the VH and VL PCR producers as well as to append a (Gly4Ser)3 linker at the tail of the VL and head of the VH for later joining.

[0124]Blunt ended PCR products were cloned using the Zero Blunt PCR Cloning Kit...

example 3

[0129]MLV-based retroviral vectors expressing the Myc-HIS tailed and the IgG1 tailed versions of the 9D9 scFv were used to make 293T cells stably expressing each of the scFv molecules. FIG. 5 shows the expression vector used for production of the myc-HIS tailed 9D9 scFv molecule, and FIG. 6 shows the expression vector used for production of the IgG1 tailed version of the 9D9 scFv molecule.

[0130]Supernatants from these cells were collected and used to stain DT230 cells (a mouse L-cell line which expresses high levels of surface CTLA4). scFv bound to the surface of these cells was quantitated using an anti-mouse-PE secondary antibody and analyzed by flow cytometry on a Cyan-LX (Dako-Cytomation).

[0131]FIGS. 12 and 13 show CD specta of scFv antibodies against DT230 cells expressing surface CTLA-4 protein.

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Abstract

The present invention provides compositions and methods for effectuating the localized expression of anti-CTLA-4 antibody proximal to a target tissue in a patient.

Description

[0001]The disclosure claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60 / 787,972 filed Mar. 30, 2006, and U.S. Provisional Application Ser. No. 60 / 787,987 filed Mar. 31, 2006, each of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The disclosure relates to methods and compositions for the localized secretion of anti-CTLA-4 antibodies and their use in immunotherapy.BACKGROUND[0003]Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a member of the CD28-B7 immunoglobulin superfamily of immune regulatory molecules. Greenwald at el., Ann. Rev. Immunol. 23:515-548 (2005). Although initially mischaracterized as a positive regulator based on homology to its co-stimulatory counterpart CD28, CTLA-4 has now been recognized as one of the key negative regulators of adaptive immune responses in general, and T cell proliferation and effector functions in particular. Peggs et al., Curr. Opin. Immunol. 18:206-213 (2006). Unlike constitu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12C12N5/071A61P35/00C12N5/0783A61K31/7088C12N15/63C12N5/09A61K39/00
CPCC12N15/86C07K16/2818A61K2039/505C07K2317/622C07K2317/73C07K2317/74C12N2740/13043A61P35/00A61P43/00
Inventor ALLISON, JAMESCURRAN, MICHAEL
Owner RGT UNIV OF CALIFORNIA
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