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

a technology of effector polypeptides and shiga toxin, which is applied in the field of deimmunization of shiga toxin effector polypeptides, can solve the problems of unfavorable immune responses, unpredictable pharmacokinetics, and reduced efficacy, and achieve the effect of reducing antigenic and/or immunogenic potential

Inactive Publication Date: 2016-11-24
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides de-immunized Shiga toxin effector polypeptides that can be used for targeted cell killing and delivering exogenous materials into specific cell types. These polypeptides have reduced antigenic and immunogenic potential in mammals, making them safer for use in various treatments for cancers, immune disorders, and microbial infections. The invention also provides diagnostic proteins for detecting the presence of specific cell types.

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.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

example 1

Predicting Immunogenic and Antigenic Epitopes in Shiga Toxin A Subunits

[0308]The antigenic and / or immunogenic sites within the A Subunits of Shiga toxins had never been systematically mapped. Computational methods were utilized to predict antigenic and / or immunogenic epitopes in various Shiga toxin A Subunits. Both B-cell epitopes and CD4+ T-cell epitopes with potential to elicit responses in mammalian immune systems were predicted in silico.

[0309]Linear B-cell epitopes were predicted for the mature A Subunit of Shiga-like toxin 1 (SLT-1A; SEQ ID NO: 1) from the polypeptide sequence and 3D structural data of Shiga-Like Toxin Chain A (PDB ID: 1DMO0_A) by ProImmune Inc. (Sarasota, Fla., U.S.) using their REVEAL® system.

[0310]In parallel, B-cell epitopes were predicted from the amino acid sequences of the A Subunits of Shiga toxin (StxA; SEQ ID NO:2), Shiga-like toxin 1 (SLT-1A; SEQ ID NO: 1), and Shiga-like toxin 2 (Stx2A; SEQ ID NO:3) using the BcePred webserver (Saha S, Raghava G, L...

example 2

De-Immunization of Shiga Toxin Effector Polypeptides

[0316]Deletions and / or amino acid substitutions were made in the putative B-cell and T-cell epitopes of Shiga toxin effector polypeptides derived from the A Subunit of Shiga-like Toxin 1 (SLT-1A). In addition to the described point mutations that disrupted predicted epitopes, some constructs comprised one or more point mutations that had no apparent effect on Shiga toxin effector enzymatic activity or cytotoxicity, such as, e.g., R223A in SLT-1A, C242S in SLT-1A, and / or C261S in SLT-1A.

[0317]In this example, a Shiga toxin effector polypeptide region was derived from the A Subunit of Shiga-like Toxin 1 (SLT-1A). A polynucleotide that encoded amino acids 1-251 of SLT-1A was used as a template to create various polynucleotides encoding various Shiga toxin effector polypeptides with one or more disruptions of a predicted B-cell epitope(s). Shiga toxin effector polypeptides comprising one or more epitope disruptions were expressed from ...

example 3

Empirically Testing De-Immunized Shiga Toxin Effector Polypeptides for Retention of One or More Shiga Toxin Effector Functions

[0329]Various de-immunized Shiga toxin effector region polypeptides were empirically tested for retention of enzymatic activity and cytotoxicity.

[0330]The retention of enzymatic activity of Shiga toxin effector polypeptides after de-immunization was tested using a ribosome inhibition assay in the context of the Shiga toxin effector polypeptide as a component of a cytotoxic protein. In certain experiments, the full-length coding sequence of the cytotoxic protein of this example began or ended with a polynucleotide encoding a Strep-tag® II to facilitate detection and purification.

[0331]The ribosome inactivation capabilities of de-immunized cytotoxic proteins were determined using a cell-free, in vitro protein translation assay using the TNT® Quick Coupled Transcription / Translation kit (L1170 Promega Madison, Wis., U.S.). The kit includes Luciferase T7 Control D...

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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 ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/25C07K16/28C07K16/32C12N9/24C07K16/00C07K16/10C12N9/10C07K14/245C07K16/08
CPCC07K14/25C07K2319/55C07K16/2887C07K16/32C07K16/085C07K16/00C07K16/286C07K16/2863C07K16/2866C07K16/1063C07K16/088C12N9/1077C12N9/2497C12Y204/02036C12Y302/02022C07K2319/33C07K14/245C12N15/62A61K38/00A61K2039/6037C07K2319/40C07K2319/04A61P1/04A61P11/06A61P17/00A61P17/06A61P19/02A61P25/00A61P29/00A61P31/00A61P31/04A61P31/18A61P35/00A61P37/00A61P37/02A61P37/04A61P37/06A61P43/00A61P5/14A61P9/00A61P3/10C07K16/089C07K2317/22C12N15/63Y02A50/30
Inventor POMA, ERICWILLERT, ERINROBINSON, GARRETT LEERAJAGOPALAN, SANGEETHABRIESCHKE, BRIGITTE
Owner MOLECULAR TEMPLATES
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