Methods for making conjugates from disulfide-containing proteins

A protein and conjugate technology, applied in the field of immobilizing proteins, can solve problems such as stability issues

Active Publication Date: 2015-08-19
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conjugates formed by this method have stability problems because the Michael addition of the thiol on the maleimide is reversible

Method used

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  • Methods for making conjugates from disulfide-containing proteins
  • Methods for making conjugates from disulfide-containing proteins
  • Methods for making conjugates from disulfide-containing proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Treatment of CRM197 with TCEP(xx) (see: G. Giannini and R. Rappuoli, Nucleic Acids Res., 1984, 25, 4063.), resulted in the reduction of C201-C185 disulfides, but little or no reduction of C451- C471 disulfide (see Example 3). The reduced CRM197 was treated with 1,3-dichloroacetone to obtain activated protein with intact C451-C471 disulfide, and C201 passed -CH 2 -C(=O)-CH 2 -The linker is connected with C185. The activated protein is contacted with PL1 (the PL1 is an aminooxy group-containing aminated fatty acid derivative whose preparation method is as described above), thereby forming an oxime that links the fatty acid derivative with the protein. Conjugates with linked fatty acid groups are expected to reduce renal clearance, thereby prolonging the circulating half-life of the CRM197 protein and improving its usefulness as a carrier for conjugate vaccines. Figure 3 provides natural protein ( Figure 3A , Use method A), activated protein ( Figure 3B ) And protein con...

Embodiment 2

[0159] Raw material preparation: pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH (disulfide C 6 -C 12 )(8) Synthesis

[0160]

[0161] ●Preparation of Intermediate 8a

[0162] (Use Fmoc-F-OH to load 2-chlorotrityl chloride resin, remove Fmoc and determine the resin loading)

[0163] Wash the 2-chlorotrityl chloride resin (40.0 g, 64.0 mmol) with DCM (3x). A solution of Fmoc-F-OH (24.8 g, 64.0 mmol) in DCM (400 mL) and DIPEA (44.7 mL, 256 mmol) was added, and the suspension was shaken at room temperature for 22 h. The resin was washed thoroughly with DCM / MeOH / DIPEA (17:2:1) (3x), DCM (3x), DMA (3x), DCM (3x). The resin was then treated with a piperidine / DMA (1:4) mixture (400 mL) 4 times for 10 minutes, and then washed with DMA (2x 180 ml). Collect piperidine / DMA solution and DMA washing solution for determination of resin loading. 1 mL of the combined solution was diluted to 500 mL with MeOH, and the UV absorption at 299.8 nm was determined to be A=0.368. This corresponds to an Fmoc amount of 4...

Embodiment 3

[0180]

[0181] To CRM197 (200μg, 6.2ul, 0.0034μmol) in 50mM sodium phosphate buffer pH7.4 (10μl) was added TCEP HCl (5.89μg, 0.021μmol) aqueous solution. The reaction mixture was left at room temperature for 15 h. 1,3-Dichloropropan-2-one (4.58μg, 0.034μmol 10eq) was added to the mixture. The reaction was left at room temperature for 2h. Pass the crude product through Zeba TM Size exclusion column. LCMS; [M+1]=58465. This activated protein can react with an aminated payload such as a TLR agonist to form a carrier protein conjugated with a compound that can increase the immune response to any antigen added to the carrier protein.

[0182]

[0183] To the ketone-modified CRM197 solution (5mg / ml, sodium phosphate buffer, pH 6.0) (50μg, 0.00086μmol) was added N-(3-(4-(2-(4-(2-(5-amino- 8-methylbenzo[f][1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethyl)piperazin-1-yl)propyl)-2-( Aminooxy)acetamide (66.8μg, 0.064μmol) and aniline (0.0020μl, 0.021μmol). The reaction was placed a...

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Abstract

The invention provides methods to prepare protein conjugates from proteins having at least two cysteines. In one embodiment, a protein with a disulfide linkage is reduced to provide two free cysteines for reaction with a 1,3-dihaloacetone or similar reactant, linking the sulfur atoms of the two cysteines together. The ketone inserted between the sulfur atoms is then used to form a Schiff base to an aminated payload molecule, thus conjugating the protein to a payload. In another embodiment, two cysteine residues are tied together by reaction with a 1,3-dihaloacetone or similar reactant. The linkage between the sulfur atoms in each case holds the protein or peptide in a constrained conformation, while also providing a convenient place for attaching a payload with good specificity and efficiency.

Description

Background technique [0001] Various chemical moieties ("payloads") have been covalently linked to enzymes, antibodies, and other large polypeptides or proteins to form conjugates. Payloads can be used to locate the protein to which they are attached (e.g., tag), modify the physicochemical properties or stability of the protein (e.g., pegylation), connect the protein to another molecule or protein (for attaching a conjugate To another compound or another conjugate), or modify the function or activity of the payload or protein (e.g., vaccine conjugate). Proteins can also serve as carriers to deliver attached payloads to specific tissues or cell types, such as in antibody-drug conjugates (ADC). The categories of payloads that can be usefully attached to proteins include detectable moieties (tags), moieties that attach the protein to a surface or another compound, antigens that cause an immune response when conjugated to proteins, and chemical complements Conjugation is a coupling...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48
CPCA61K47/48261A61K47/48561A61K47/48038A61K47/48384A61K47/4833A61K47/542A61K47/6415A61K47/646A61K47/6803A61K47/6849A61C5/90A61F5/566A61K47/6845A61F5/0006
Inventor Q-Y·胡今濑英智
Owner NOVARTIS AG
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