Immunogenic peptide carrier conjugates and methods of producing same

A peptide immunogen and carrier technology, which is applied in the field of immunogenic peptide carrier conjugates and their production, can solve the problems of sensitivity to the destructive effect of chemical treatment and destruction of the function of the conjugate.

Inactive Publication Date: 2007-06-20
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of using a binding reagent is that the active site, if not neutralized, is free to associate with any undesired molecules in vitro (thus negatively affecting the function or stability of the conjugate) or in vivo (thus affecting the human or animal immunized with the preparation). Potentially dangerous to bring negative events) reaction, the binding reagent introduces the active site to the side chain of the active amino acid molecule on the carrier and / or hapten molecule
This excess active site can be reacted or "capped" using various known chemical reactions to inactivate these sites, but these reactions can disrupt the function of the conjugate
May be particularly problematic when attempting to introduce an active site into a carrier molecule, since its larger and more complex structure (relative to haptens) may make it more susceptible to the damaging effects of chemical treatment
In fact, there are no known examples of conjugates prepared by first activating the carrier, then reacting with the hapten in a conjugation reaction, and finally "capping" the remaining active site, while retaining the resulting conjugate as a Capability of Immunogenic Compositions with Desired "Vehicle Effect" Properties

Method used

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  • Immunogenic peptide carrier conjugates and methods of producing same
  • Immunogenic peptide carrier conjugates and methods of producing same
  • Immunogenic peptide carrier conjugates and methods of producing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0253] CRM 197 Binding to Aβ peptide

[0254] Activation vector CRM with 39 lysine residues 197 Hapten / antigenic peptide conjugation was carried out by reaction with a hapten / antigenic peptide having a pendant thiol group using the method described below (Fig. 1). All A[beta] peptides contain a cysteine ​​residue at the carboxy terminus to facilitate conjugation of these peptides to carrier proteins via cysteinyl sulfhydryl groups. These peptides were produced by solid phase synthesis.

[0255] I. Activation

[0256] CRM 197 The free amino group of ® was bromoacetylated by reaction with an excess of N-hydroxysuccinimide bromoacetate (Sigma Chemical Co., St. Louis, MO) (Bernatowicz and Matsueda, 1986). To the cold CRM 197 (about 15mg) solution, add 10% (v / v) of 1.0M NaHCO 3 (pH8.4). will be used with the CRM 197 An equal weight of N-hydroxysuccinimide bromoacetate was dissolved in 200 μL of dimethylformamide (DMF) and slowly added to the CRM 197 and stirred gently for...

Embodiment 2

[0272] Aβ peptide-CRM 197 Preparation of conjugates and purification by ultrafiltration

[0273] CRM 197 bromoacetylation

[0274] CRM 197 (100mg) of 0.01M sodium phosphate buffer, 0.9% NaCl, pH7.0 solution and N-hydroxysuccinimide bromoacetate (dissolved in DMSO to 20mg / mL) in an argon atmosphere at a weight ratio of 1:1 The next reaction. The reaction was titrated as needed to maintain the pH at 7.0. The mixture was stirred at room temperature in the dark for 1.5 hours. The reaction mixture was filtered through a 1.2 μm filter into the retentate reservoir of a UF / DF system (Millipore Labscale TFF, Billerica, MA). Purification was accomplished by diafiltration (30-fold) in 0.01 M sodium phosphate buffer / 0.9% NaCl, pH 7.0 using 10K or 30K UF membranes. Bromoacetylated CRM 197 Filtration was performed by passing through a 0.2 μm filter. The degree of bromoacetylation is determined by the activation of the CRM 197 Reaction with cysteine ​​followed by determination by a...

Embodiment 3

[0279] Conversion to aminoacetyl by capping unreacted bromoacetyl group

[0280] Bromoacetylated CRM prepared as described in Example 2 197 (50 mg) was transferred to a reaction vessel. To the stirred solution maintained at 2-8°C was added 1M sodium carbonate / sodium bicarbonate. Titration was performed under an argon atmosphere to achieve a target pH of 9.0. Weigh 50 mg of Aβ peptide individually and dissolve in WFI to 20 mg / mL. To this solution was added 1 M sodium carbonate / bicarbonate until pH 9.0 was obtained. CRM to bromoacetylation 197 To the solution was added the peptide solution and the mixture was stirred at 2-8°C for 14-18 hours. The remaining bromoacetyl groups were capped using 8% ammonium bicarbonate solution for 4 hours at 2-8°C.

[0281] The reaction mixture was filtered through a 1.2 μm filter into the retentate reservoir of the UF / DF system (Millipore XL), passed through 0.01M sodium phosphate buffer / 0.9% NaCl, pH7. The conjugate was purified by 30-fol...

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Abstract

The present invention is directed to methods of producing conjugates of peptide immunogens with protein / polypeptide carrier molecules, which are useful as immunogens, wherein peptide immunogens are conjugated to protein carriers via activated functional groups on amino acid residues of the carrier or of the optionally attached linker molecule, and wherein any unconjugated reactive functional groups on amino acid residues are inactivated via capping, thus retaining the immunological functionality of the carrier molecule, but reducing the propensity for undesirable reactions that could render the conjugate less safe or effective. Furthermore, the invention also relates to such immunogenic products and immunogenic compositions containing such immunogenic products made by such methods.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. §119(e) to US Provisional Patent Application 60 / 530,480, filed December 17, 2003, which is hereby incorporated by reference in its entirety. Background technique [0003] The essence of adaptive immunity is the organism's response to the presence of foreign substances and the production of components (antibodies and cells) capable of specifically interacting with the host and protecting it from aggression. An "antigen" or "immunogen" is a substance capable of eliciting such an immune response and capable of interacting with sensitized cells and antibodies produced against the antigen. [0004] Antigens or immunogens are generally large molecules that contain unique antigenic sites or "epitopes" that are recognized by and interact with various components of the immune system. They may exist as separate molecules consisting of synthetic organic chemical products, proteins, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/38A61K39/385C07K
CPCA61K39/385A61K2039/627A61K39/0007A61K2039/645A61K2039/6031A61P37/00A61P37/02A61P37/04Y02A50/30C07K1/06A61K38/00C07K14/47A61K39/00A61K2039/6068
Inventor 拉萨帕·G·阿鲁穆格哈姆A·克利须那·普拉萨德
Owner WYETH LLC
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