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Selective UV crosslinking of peptides and functional moieties to immunoglobulins

a functional moiete and immunoglobulin technology, applied in the field of selective uv crosslinking of peptides and functional moieties to immunoglobulins, can solve the problems of reducing sensitivity and reproducibility, heterogeneous immunoglobulin population, and non-site-specific conjugation methods that have negative effects on the outcome of immunoglobulin-based detection assays

Inactive Publication Date: 2016-01-28
UNIV OF NOTRE DAME DU LAC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for site-specific photo crosslinking of an immunoglobulin molecule with a conserved nucleotide binding site. This is achieved by using a hetero-bifunctional crosslinker with a photo-reactive functional group and a non-photo-reactive functional group. The non-photo-reactive functional group can be a thiol group that is coupled to a surface or an effector molecule. The method can be used to create immunoglobulin molecules with specific functional groups attached, allowing for controlled modification and use in various applications such as drug delivery and labeling.

Problems solved by technology

However, it is not possible to control the number and sites of conjugation with this method, which results in a heterogeneous immunoglobulin population.
Therefore, non-site-specific conjugation methods often have a negative impact on the outcome of immunoglobulin-based detection assays by decreasing sensitivity and reproducibility.
However, these methods often require complicated chemical procedures with variable outcomes, and also risk denaturing the immunoglobulin and reducing its activity due to exposure to chemically harsh reaction conditions.
In addition, the complexity of these methods results in a high overall cost for preparation of immunoglobulin conjugates.

Method used

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  • Selective UV crosslinking of peptides and functional moieties to immunoglobulins
  • Selective UV crosslinking of peptides and functional moieties to immunoglobulins
  • Selective UV crosslinking of peptides and functional moieties to immunoglobulins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Nucleotide Binding Site Conjugation

[0184]The nucleotide binding site (NBS) provides a useful site for selective conjugation of immunoglobulins to small ligands that contain aromatic rings to selectively bind this site. To identify such small molecules with a high binding affinity and selectivity for the NBS, we performed an in silico screening by docking various small molecules from the ZINC database at the NBS. The top scoring molecules were then experimentally investigated for their binding affinity to the NBS with indole-3-butyric acid (IBA) emerging as the highest affinity binding nucleotide analogue, with Kd values ranging between 1 and 8 μM depending on the immunoglobulin. Consequently, the IBA conjugated versions of functional ligands (IBA-ligand) such as affinity tags, fluorescent molecules, peptides, and chemotherapeutics can be photocrosslinked to immunoglobulins site-specifically at the NBS.

[0185]In this example we have particularly demonstrated the site-specific function...

example 2

UV-NBS Photocrosslinking of Reactive Thiol Moieties

[0246]The UV-NBS photocrosslinking technique requires exposure to UV energy that some functional ligands may not be stable to. In this study, we demonstrate the utility of the UV-NBS immunoglobulin functionalization strategy for conjugation of reactive thiol ligands to immunoglobulins at their NBS. By synthesizing an IBA conjugated version of cysteine (IBA-Thiol) a reactive thiol group can be site-specifically photocrosslinked to immunoglobulins at the NBS (FIG. 32). This thiol group can then be used as an orthogonally reactive site to conjugate UV sensitive functional ligands that possess either a thiol reactive group resulting in disulfide bond formation or subsequent reaction with a maleimide functionalized ligand (FIG. 28). The results detailed here provide a universal technique for the site-specific conjugation of UV sensitive functional ligands to immunoglobulins at the NBS, while preserving immunoglobulin activity.

[0247]Mater...

example 3

UV-NBS Photocrosslinking of Biotin

[0267]Here, we describe an alternate photochemistry based NBS-specific immunoglobulin immobilization method that utilizes biotin for oriented immobilization to streptavidin-functionalized surfaces (UV-NBSBiotin, FIG. 40). We predicted that site-specifically conjugating a biotin molecule to the immunoglobulin NBS prior to immobilization would allow for nearly 100% immunoglobulin functionalization to overcome the poor immobilization efficiency of the previously reported UV-NBS method while still maintaining maximum immunoglobulin activity.

[0268]1. Materials

[0269]IBA, Biotin N-hydroxysuccinimide ester (NHS-Biotin), N,N-Diisopropylethylamine (DIEA), were purchased from Sigma-Aldrich (St. Louis, Mo.). Streptavidin-HRP and HRP-conjugated IgG Fcγ specific goat anti-mouse were purchased from Jackson ImmunoResearch (West Grove, Pa.). Heat shock isolated bovine serum albumin (BSA), Amicon Ultra centrifugal filters (0.5 mL, 10K) and Coomassie R-250 were purcha...

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Abstract

The invention provides for a method of crosslinking a hetero-bifunctional photo crosslinking compound to an immunoglobulin having at least one heterocyclic photo reactive group and at least one non-photo reactive group where the non-photo reactive group is coupled to an effector molecule and the photo reactive group is coupled to the nucleotide binding site of an immunoglobulin. Alternatively, the photo crosslinker contains an orthogonal reactive group such as a thiol, which can be coupled to an effector molecule or functionalized ligand.

Description

RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. §119(e) to co-pending U.S. Provisional Patent Application Nos. 61 / 851,962 and 61 / 851,981, both filed Mar. 14, 2013, which applications are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT[0002]This invention was made with government support under CBET-1263713 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Immunoglobulins are secreted by plasma cells and are used by the immune system to identify and neutralize objects foreign to the host. The immunoglobulin recognizes a unique part of the foreign object known as an antigen. Due to their exceptional specificity and nearly limitless diversity, immunoglobulins are extensively used in an array of diagnostic and therapeutic applications. When utilizing immunoglobulins, it is often necessary to conjugate them with various functional ligands to make them amenable for...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K31/337C07K16/28G01N33/532G01N33/533G01N33/543
CPCA61K47/48715C07K16/2887A61K47/48384A61K31/337G01N33/533G01N33/54353G01N33/532C07K16/00G01N33/531A61K47/6889A61K47/6803
Inventor BILGICER, ZIHNI BASARALVES, NATHAN
Owner UNIV OF NOTRE DAME DU LAC
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