De-immunized shiga toxin a subunit effector polypeptides for applications in mammals

Abstract

The present invention relates to Shiga toxin effector polypeptides with reduced antigenic and/or immunogenic potential. Immunogenicity can be a limitation for the repeated administration to mammals of proteins and polypeptides derived from Shiga toxins. The Shiga toxin effector polypeptides of the present invention have uses as components of therapeutics, diagnostics, and immunization materials. The cytotoxic proteins of the present invention have uses for selective killing of specific cell types and as therapeutics for the treatment of a variety of diseases, including cancers, immune disorders, and microbial infections. The proteins of the present invention also have uses for detecting specific cell types, collecting diagnostic information, and monitoring the treatment of a variety of diseases, such as, e.g., cancers, immune disorders, and microbial infections.

Description

FIELD OF THE INVENTION[0001]The present invention relates to de-immunized Shiga toxin effector polypeptides derived from A Subunits of naturally occurring Shiga toxins. Polypeptides of the invention are beneficial alone or as components of molecules, e.g. therapeutics, for administration to mammals where reducing associated immune responses is desirable. For example, the polypeptides of this invention may be used as components of specifically targeted molecules, e.g. immunotoxins and ligand-toxin fusions, for the targeted killing of specific cell types. Proteins of the invention have uses as, e.g., components of therapeutics and diagnostics for the diagnosis, prognosis, and treatment of a variety of diseases, disorders and conditions, including cancers, tumors, immune disorders, and microbial infections.BACKGROUND[0002]Shiga toxins have been synthetically engineered for medical applications by rational alterations to the toxin's structure, characteristics, and biological activities ...

AI Technical Summary

Benefits of technology

[0009]The present invention provides Shiga toxin effector polypeptides which have reduced antigenic and / or immunogenic potential in mammals (referred to herein as “de-immunized”). In addition, the present invention provides cytotoxic proteins and diagnostic proteins comprising de-immunized Shiga toxin effector polypeptides. De-immunized polypeptides derived from Shiga toxin A Subunits may be linked to one or more polypeptides that mediate cell targeting via specific extracellular binding interactions to enable the engineering of improved therapeutics for cell type specific targeting of cellular internalization and Shiga toxin cytotoxicity. The linking of detection promoting agents with de-immunized Shiga toxin effector region polypeptides enables the engineering of improved diagnostic molecules for detecting the presence of specific cell types. The polypeptides and proteins of the invention have uses for targeted cell killing, delivering exogenous materials into specific cell types, obtaining diagnostic information, and as therapeutics for the treatment of a variety of diseases, disorders, and conditions, including cancers, immune disorders, and microbial infections.

Problems solved by technology

Unwanted immunogenicity in protein therapeutics has resulted in reduced efficacy, unpredictable pharmacokinetics, and undesirable immune responses that limit dosages and repeat administrations.

Although there is a need for molecules comprising de-immunized Shiga-toxin-derived polypeptides that have reduced immunogenic potential to help avoid unwanted immune responses, there exists no predictable method to successfully identify and remove internal B-cell epitopes while maintaining Shiga toxin effector function(s).

Although some antigenic and / or immunogenic epitopes might be removed by truncation, the main challenge is silencing epitopes within the Shiga toxin polypeptide's effector domains, e.g. its enzymatic domain, while retaining the desired Shiga toxin effector functions, e.g., potent ribosome inhibition and directing subcellular routing.

While these internal epitopes may be diminished or abolished by mutation and / or chemical modification, the challenge is to do so while preserving Shiga toxin effector functions.

It is a significant challenge to disrupt antigenic sites by amino acid substitution in a protein while preserving protein function because functionally constrained residues and structures must be maintained and certain positions do not tolerate certain amino acid substitutions without impacting protein structure, stability, and / or function (see Cantor J et al., Methods in Enzymology 502: Ch.

For some samples, accurate values for either IC50 or CD50 might be unobtainable due to the inability to collect the required data points for an accurate curve fit.

The failure to detect activity in Shiga toxin effector function may be due to improper expression, polypeptide folding, and / or polypeptide stability rather than a lack of cell entry, subcellular routing, and / or enzymatic activity.

Certain Shiga toxin effector functions are not easily measurable, e.g. subcellular routing functions.

It may be difficult to gauge the immunogenicity of a weakly immunogenic molecule without a reference point, such as another more immunogenic molecule.

However, disrupting certain amino acids and polypeptide regions using certain disruptions are more likely to successfully reduce antigenicity and / or immunogenicity while retaining a Shiga toxin effector function.

Similarly, amino acid substitutions which remove charge, polarity, and / or reduce side chain length can disrupt an epitope while maintaining at least one Shiga toxin effector function.

In addition, B-cell epitopes often coincide or overlap with epitopes of mature CD4+ T-cells, thus the disruption of a B-cell epitope often simultaneously disrupts a CD4+ T-cell epitope.

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