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Compositions monovalent for CD40L binding and methods of use

A monovalent, single technology, applied in drug combination, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve weak affinity, can not obtain ideal antibody drugs Metabolic kinetics, etc.

Active Publication Date: 2015-07-01
DORMANTIS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Davies and Riechmann (1995, Biotechnology N.Y. 13:475-479) also reported camelized human V H Production of repertoires for domain phage display, and selection of clones that bind haptens with affinities in the 100-400 nM range, but clones selected for binding protein antigens have weaker affinities
[0022] Although many antibodies and their derivatives are useful for diagnosis and therapy, ideal antibody pharmacokinetics are often not available for specific applications

Method used

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  • Compositions monovalent for CD40L binding and methods of use
  • Compositions monovalent for CD40L binding and methods of use
  • Compositions monovalent for CD40L binding and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0492] Example 1. Biotinylation of recombinant CD40L

[0493] Recombinant human soluble CD40L (PeproTech) was biotinylated and used in the phage selection process. Reagents, devices and their available sources are provided in Table 1.

[0494] According to the product instructions, by mixing CD40L (0.5mg / ml) with EZ-Link TM Sulfo-NHS-LC-biotin[sulfosuccinimidyl-6-(biotinylamino)hexanoate] (Pierce) was incubated at a molar ratio of 5:1 for 2 h on ice for CD40L of biotinylation. Then, the biotinylation reaction mixture was incubated at 4°C in Dialysis was performed in the dialysis cassette against 3 changes of PBS (1000 x sample volume) to remove unbound biotinylated reagent.

[0495] Biotinylated-CD40L was detected by a receptor binding assay binding to CD40 / Fc to determine its biological activity. The quality of biotin-CD40L was also monitored by analysis on a NuPaGE 4-12% Bis-Tris gel, and by Simply Blue Safe-staining (Invitrogen) ( Figure 1a ), by Western blot prob...

Embodiment 2

[0498] Example 2. Phage selection using biotinylated antigen

[0499] Domain antibody (dAb) library based on V H (DP47 and JH4b) and VK (DPK9 and JK1) a single human framework with side chain diversity incorporated at antigen binding site positions that make contact with antigen of known molecular structure and reflect the diversity of residues in the human antibody repertoire . Using the phage vector pDOM4(Fd-Tet), which encodes the Fd-phage genome, dAb expression is controlled by the gene III promoter, and the antibody is displayed as a fusion protein covalently linked to the N-terminus of the Fd-phage protein pill. The dAb cassette consists of the following (5' to 3'): eukaryotic leader, dAb, myc tag, gIII. The vector contains M13 and colE1 origins of replication and can be selected using tetracycline. V H and V κ Libraries each have more than 1×10 10 The calculated size of a molecule. Reagents, devices and their available sources are provided in Table 2.

[0500]...

Embodiment 3

[0513] Example 3. Cloning the enriched phage selection results into the soluble dAb expression vector pDOM5

[0514] After the second and third rounds of selection, E. coli cells infected with the enriched dAb-displaying fd-phage population were obtained. An aliquot of these cells was used to prepare phage DNA, and the enriched V-genes were excised by digestion with the restriction endonucleases SalI and NotI. The purified V-gene was connected to the corresponding site of pDOM5 (expression vector derived from pUC119, with LacZ promoter, eukaryotic leader sequence, dAb cloning site, myc marker), and the connected DNA was used for electrotransformation in a cell containing E. coli HB2151 cells grown overnight on agar plates with the antibiotic carbenicillin. Resulting clones were induced to express dAb protein in 200 μl microcultures or 50 ml cultures. The resulting dAbs were analyzed for inhibitory activity using a CD40L receptor binding assay.

[0515] Following phage sel...

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Abstract

The invention relates to antibody polypeptides that monovalently bind CD40L. Antibody polypeptides that are monovalent for binding of CD40L can inhibit CD40L activity while avoiding potential undesirable effects that can occur with antibodies capable of divalent or multivalent binding of CD40L. In one aspect, a monovalent anti-CD40L antibody polypeptide consists of or comprises a single immunoglobulin variable domain that specifically binds and antagonizes the activity of CD40L, preferably without substantially agonizing CD40 activity. In another aspect, the monovalent anti-CD40L antibody polypeptide is a human antibody polypeptide. The invention further encompasses methods of antagonizing CD40 / CD40L interactions in an individual and methods of treating diseases or disorders involving CD40 / CD40L interactions, the methods involving administering a monovalent anti-CD40L antibody polypeptide to the individual.

Description

Background of the invention [0001] CD40 is a 50kD cell surface glycoprotein molecule that is present on the surface of mature and immature B cells, macrophages, follicular dendritic cells, thymic epithelial cells, normal basal epithelial cells, and some tumor-derived cell lines on the expression. The CD40 molecule is a member of the TNF receptor family and has important signaling functions that elicit various downstream effects in many cell types. Early studies have shown that cross-linking of CD40 on the surface of B cells with antibodies leads to proliferation and activation of B cells. Antibodies crosslinked to CD40 in the presence of IL-4 induce in vitro proliferation and class switching, B cell recruitment by LFA-1 (Gordon et al., 1988, J. Immunol. 140:1425), and serine of many intracellular substances / threonine and tyrosine phosphorylation (Gordon et al., 1988, supra; Uckun et al., 1991, J. Biol. Chem. 266:17478). Anti-CD40 monoclonal antibodies also elicit B cell re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61P37/06A61K39/395
Inventor 史蒂芬·格兰特刘海群凯文·莫尔德
Owner DORMANTIS LTD
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