Polymer Conjugates with Decreased Antigenicity, Methods of Preparation and Uses Thereof

Abstract

Methods are provided for the preparation of conjugates of a variety of bioactive components, especially proteins, with water-soluble polymers (e.g., poly(ethylene glycol) and derivatives thereof), which conjugates have reduced antigenicity and immunogenicity compared to similar conjugates prepared using poly(ethylene glycol) containing a methoxyl or another alkoxyl group. The invention also provides conjugates prepared by such methods, compositions comprising such conjugates, kits containing such conjugates or compositions and methods of use of the conjugates and compositions in diagnostic and therapeutic protocols.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation-in-part of U.S. application Ser. No. 10 / 317,092 filed Dec. 12, 2002, which claims the benefit of the filing date of U.S. Provisional Application No. 60 / 414,424, filed Sep. 30, 2002. The disclosures of the above-referenced applications are incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention is in the fields of protein biochemistry and the pharmaceutical and medical sciences. In particular, the invention provides methods for the production of conjugates between water-soluble polymers (e.g., poly(ethylene glycol) and derivatives thereof) and bioactive components, which conjugates have reduced antigenicity and immunogenicity compared to standard polymer-bioactive component conjugates. The invention also provides conjugates produced by such methods, compositions comprising such conjugates, kits comprising such conjug...

AI Technical Summary

Benefits of technology

[0027]In one aspect, the invention provides a conjugate comprising one or more bioactive components covalently linked to at least one linear or branched monofunctionally activated polyalkylene glycol, wherein the mono-functionally activated polyalkylene glycol does not comprise a methoxyl group, another alkoxyl group or an aryloxyl group at any terminus. In certain such embodiments, the conjugate has reduced or substantially reduced antigenicity compared to a conjugate prepared using an alkoxypoly(ethylene glycol), e.g., mPEG, or a branched polymer containing mPEG, such as di-mPEG-lysine.

[0031]The invention also provides methods of producing conjugates between a bioactive compound and a monofunctionally activated polyalkylene glycol, for example comprising: (a) obtaining or preparing a linear or branched polyalkylene glycol comprising at least one unreactive blocking group that can be subsequently removed, such as one or more triphenylmethyl groups (“trityl groups”); (b) producing a derivative of the polyalkylene glycol by reacting it with at least one derivatizing compound under conditions such that the polyalkylene glycol is derivatized with one derivatizing group (such as one carboxyl group) at an end that lacks the blocking group(s); (c) removing the blocking group(s) without removing the derivatizing group to produce, in one or more steps, a monofunctionally activated polyalkylene glycol; and (d) contacting the monofunctionally activated polyalkylene glycol with at least one bioactive component under conditions that favor the covalent binding of the bioactive component to the monofunctionally activated polyalkylene glycol. Preferably, the conjugates produced by such methods are of reduced, substantially reduced or undetectable antigenicity and immunogenicity, when compared to conjugates derivatized to the same extent with mPEG of similar size, structure and linkage to the bioactive agent. The invention also provides conjugates produced by such methods.

Problems solved by technology

However, the half-lives of small proteins following i.v. administration are usually extremely short (see examples in Mordenti, J., et al., (1991) Pharm Res 8:1351-1359; Kuwabara, T., et al., (1995) Pharm Res 12:1466-1469).

As a result, maintenance of therapeutically useful concentrations of small recombinant proteins in the circulation is problematic following i.v. administration.

The high dose rate increases the cost of therapy, decreases the likelihood of patient compliance and increases the risk of adverse events, e.g., immune reactions.

Both cellular and humoral immune responses can reduce the circulating concentrations of injected recombinant proteins to an extent that may preclude the administration of an effective dose or may lead to treatment-limiting events such as anaphylaxis (Pui, C.-H., et al.

However, the bioavailability can be quite low, making it difficult to achieve effective circulating concentrations of such drugs.

A further problem that may be related to the poor bioavailability of drugs administered s.c. or i.m. is the increased probability of degradation of the therapeutic protein at the site of injection.

On the other hand, as the number of strands of PEG is increased, so is the probability that an amino group in an essential region of the bioactive component (particularly if the bioactive component is a protein) will be modified, impairing its biological function (e.g., catalysis by an enzyme or receptor binding by a cytokine).

For smaller proteins, such as polypeptide hormones and cytokines, however, a relatively high degree of substitution is likely to decrease the functional activity to the point of negating the advantage of an extended half-life in the bloodstream (Clark, R., et al., supra).

The use of this method has disadvantages however, such as the toxicity of cyanuric chloride and its non-specific reactivity for proteins having functional groups other than amines, such as solvent-accessible cysteine or tyrosine residues that may be essential for function.

There was a concomitant loss in ability to agglutinate human erythrocytes.

Excessive conjugation of polymers and / or conjugation involving the active site of a therapeutic moiety where groups associated with bioactivity are found, however, Can often result in loss of activity and, thus, in loss of therapeutic efficacy.

To date, however, there has been no disclosure of a mechanism for rendering the PEG in such polymers or conjugates non-antigenic.

Such conjugates will have the benefits provided by the polymer component of increased stability and bioavailability in vivo, but will not elicit a substantial immune response in an animal into which the conjugates have been introduced for therapeutic or diagnostic purposes.

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