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Tumor necrosis factor superfamily and tnf-like ligand muteins and methods of preparing and using the same

a tumor necrosis factor and superfamily technology, applied in tumor necrosis factor, metabolism disorder, immunological disorders, etc., can solve the problems of degrading and removal of soluble tnfsf ligands from the body, and achieve the effect of avoiding adverse side effects

Pending Publication Date: 2022-03-31
THE GENERAL HOSPITAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention features a polypeptide that includes a change in a surface-exposed amino acid residue of a TNFSF or TNF-like ligand with a cysteine residue or the insertion of one or more cysteine residues within this region. These changes can lead to the formation of quaternary structures with other TNFSF or TNF-like ligands, resulting in potential therapeutic effects that can be achieved without causing adverse side effects. The "therapeutically effective amount" refers to the amount of the polypeptide needed to achieve the desired therapeutic effect.

Problems solved by technology

However, because of the high dissociation rate of soluble TNFSF ligands at low concentrations, soluble TNFSF ligands are often degraded and removed from the body.

Method used

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  • Tumor necrosis factor superfamily and tnf-like ligand muteins and methods of preparing and using the same
  • Tumor necrosis factor superfamily and tnf-like ligand muteins and methods of preparing and using the same
  • Tumor necrosis factor superfamily and tnf-like ligand muteins and methods of preparing and using the same

Examples

Experimental program
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Effect test

example 1

and Methods

[0323]Reagents

[0324]Recombinant human and mouse TNF-α was purchased from Leinco Technologies (St. Louis, Mo.), and recombinant human IL-2 was purchased from Sigma-Aldrich (St. Louis, Mo.). Human monoclonal antibodies against TNFR2 were from internal sources and external commercial vendors. Intracellular staining of FOXP3 and CD25 were performed using either FOXP3 Fix / Perm Buffer set (Biolegend) or Human FOXP3 Buffer set (BD Biosciences).

[0325]Human Subjects

[0326]Subjects used in this study were either Type-1 diabetic subjects or non-affected control subjects. They were recruited from Massachusetts General Hospital with full institutional approval and informed consent (MGH-2001P001379). Each subject donated 4 tubes of blood and the blood was used fresh in the morning for the preparation of either CD8 T cells for the WST-1 proliferation / death assay or CD4 T cells for the T regulatory assays. For the WST-1 assays of cell proliferation or death, each autoimmune subject was si...

example 2

ion of Wild-Type Soluble TNF-α and the TNF-α Mutein

[0329]In this example, the wild-type soluble TNF-α is a TNF-α monomer and the TNF-α mutein is a disulfide-bonded homo-trimer of TNF-α muteins in which each monomer contains S171C and G224C cysteine substitutions. Construction of wild-type soluble TNF-α and the TNF-α mutein involved original gene synthesis of the DNA fragments into the His6-thrombin-site-TNF-α gene-fusion and these were subcloned into pDEST42 expression vector. Positive clones were confirmed by restriction enzyme digestion and sequence validation. Wild-type soluble TNF-α and the TNF-α mutein were expressed in E. coli strain BL21 DE3 pLys S using the following conditions: a seed culture was incubated at 37° C. for 16 hours and was used to inoculate 4 liter of tissue culture media, which was incubated at 37° C. until A600=0.8. Protein expression was induced with IPTG (0.1 mM) for 16 hours at 18° C. Cells were harvested by centrifugation and stored at −80° C.

[0330]Cell ...

example 3

lot Analysis of the TNF-α Mutein

[0333]In this example, the wild-type soluble TNF-α is a TNF-α monomer and the TNF mutein is a disulfide-bonded homo-trimer of TNF-α muteins in which each monomer contains S171C and G224C cysteine substitutions. Wild-type soluble TNF-α and TNF-α mutein were analyzed on a Peggy Automated Western Assay Platform in size separation mode (ProteinSimple, Santa Clara, Calif.). Samples (0.5 μg / ul) were mixed at a ratio of 3:1 with a 4× Mastermix containing fluorescent molecular weight standards and sample buffer, but without DTT (Dithiothreitol) reducing agent. To further prevent reduction, the samples were not subjected to high (95° C.) temperature as is customary, but rather were kept on ice until use. All running parameters were left at their defaults, except for loading time (12 seconds instead of default of 8 seconds) and for first antibody incubation time (240 min instead of default of 120 min). Detection of wild-type soluble TNF-α and the TNF-α mutein w...

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Abstract

The invention features homo-multimers, e.g., homo-trimers, of TNFSF or TNF-like ligand muteins in which each TNFSF ligand or TNF-like ligand mutein monomer contains at least one cysteine residue substitution or insertion that promotes the formation of a disulfide bond with a cysteine residue on a neighboring TNFSF or TNF-like ligand mutein monomer. The invention features methods of producing such TNFSF and TNF-like ligand muteins, pharmaceutical compositions containing such muteins, and methods of using such muteins in cancer immunotherapy, in treating autoimmune and neurological diseases, and in reducing or eliminating the complications and risks of rejection in organ transplantation or tissue or organ repair or regeneration.

Description

BACKGROUND[0001]The tumor necrosis factor superfamily (TNFSF) refers to a group of ligands, e.g., cytokines, that have diverse functions in the immune system. The TNFSF ligands, such as TNF-α, lymphotoxin (e.g., LT-α and LT-β), CD40L, CD70, CD153, OX40 ligand (OX40L), Fas ligand (FasL), 4-1BB ligand (4-1BBL), TRAIL, RANKL, TWEAK, APRIL, BLys, LIGHT, TL1, GITRL (also known as TL6), and EDA (e.g., EDA-A1 and EDA-A2), are structurally related ligands that bind to one or more receptors of the TNFSF. Ligands of the TNFSF are typically expressed as type II transmembrane proteins, which can be proteolytically cleaved so that the extracellular domain is released as a soluble protein (FIG. 1A). Both the transmembrane and soluble portions of the TNFSF ligands form non-covalent homo-multimers, e.g., homo-trimers, in order to bind to their respective receptors to exert their biological functions. However, because of the high dissociation rate of soluble TNFSF ligands at low concentrations, solu...

Claims

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Application Information

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IPC IPC(8): C07K14/525A61K38/19
CPCC07K14/525A61K38/00C07K14/5255A61K38/191A61K38/1793A61K38/22A61P1/16A61P19/08A61P21/02A61P25/00A61P25/08A61P25/14A61P25/16A61P25/18A61P27/02A61P3/00A61P3/04A61P31/00A61P31/06A61P31/08A61P31/12A61P31/18A61P35/00A61P35/02A61P37/06A61P37/08A61P3/10A61K45/06C07K14/575C07K14/70575C07K14/70578
Inventor FAUSTMAN, DENISE L.VANAMEE, EVA
Owner THE GENERAL HOSPITAL CORP
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