Mhc class i epitope delivering polypeptides and cell-targeted molecules for direct cell killing and immune stimulation via mhc class i presentation and methods regarding the same

Abstract

The present invention is directed to T-cell epitope delivering polypeptides which deliver one or more CD8+ T-cell epitopes to the MHC class I presentation pathway of a cell, including toxin-derived polypeptides which comprise embedded T-cell epitopes and are de-immunized. The present invention provides cell-targeted, CD8+ T-cell epitope delivering molecules for the targeted delivery of cytotoxicity to certain cells, e.g., infected or malignant cells, for the targeted killing of specific cell types, and the treatment of a variety of diseases, disorders, and conditions, including cancers, immune disorders, and microbial infections. The present invention also provides methods of generating polypeptides capable of delivering one or more heterologous T-cell epitopes to the MHC class I presentation pathway, including polypeptides which are 1) B-cell and / or CD4+ T-cell de-immunized, 2) comprise embedded T-cell epitopes, and / or 3) comprises toxin effectors which retain toxin functions.

Description

FIELD OF THE INVENTION[0001]The present invention relates generally to methods of modifying polypeptides to introduce the ability of the polypeptide to deliver a heterologous T-cell epitope for MHC class I presentation by a chordate cell and the polypeptides made using these methods. More specifically, the invention relates to methods of modifying polypeptides comprising proteasome delivery effector functions into heterologous, T-cell epitope delivering polypeptides that differ in their immunogenic properties from their parent molecules by the addition of one or more T-cell epitope-peptides which can be recognized by a MHC class I molecule and be presented on a cell surface by the MHC class I system of a chordate cell. Certain methods of the present invention relate to methods of modifying polypeptides to reduce antigenicity and / or immunogenicity via the introduction of one or more T-cell epitopes. In another aspect, the present invention relates to polypeptides created using method...

AI Technical Summary

Benefits of technology

[0025]In addition, the present invention provides embodiments of methods of generating novel polypeptides capable of delivering one or more heterologous T-cell epitopes to the MHC class I presentation pathway of a cell. The present invention also provides various embodiments of methods of generating variants of polypeptides by simultaneously reducing the probability of B-cell and / or CD4+ T-cell immunogenicity while increasing the probability of CD8+ T-cell immunogenicity. The present invention also provides certain embodiments of the methods of generating novel polypeptides capable of delivering one or more heterologous T-cell epitopes to the MHC class I presentation pathway of a cell, wherein the starting polypeptide comprises a toxin effector region and certain polypeptides produced by using the methods of the invention result in polypeptides which retain toxin effector functions, such as, e.g., enzymatic activity and cytotoxicity.

[0033]In certain embodiments of the methods of the present invention is a method of increasing CD8+ T-cell immunogenicity of a polypeptide capable of intracellular routing to a subcellular compartment of a cell in which the polypeptide is present selected from the group consisting of: cytosol, endoplasmic reticulum, and lysosome; the method comprising the step of: embedding or inserting a heterologous CD8+ T-cell epitope in the polypeptide. In certain further embodiments, the method comprises the embedding or inserting step wherein the embedding or inserting in an endogenous B-cell epitope, an endogenous CD4+ T-cell epitope, and / or a catalytic domain of the polypeptide. In certain further embodiments of the method, the polypeptide of the method is derived from a toxin. In certain further embodiments of the method, the polypeptide comprises a toxin effector polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a proteasome of a cell in which the toxin effector polypeptide is present, and the method comprises embedding or inserting the heterologous T-cell epitope in the toxin effector polypeptide. In certain further embodiments of the method, the embedding or inserting step results in a toxin effector polypeptide capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present.

[0034]In certain embodiments of the methods of the present invention is a method of increasing CD8+ T-cell immunogenicity of a polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a proteasome of a cell in which the polypeptide is present, the method comprising the step of: embedding or inserting a heterologous CD8+ T-cell epitope in the polypeptide. In certain further embodiments of the method, the polypeptide of the method is derived from a toxin. In certain further embodiments of the method, the polypeptide comprises a toxin effector polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a proteasome of a cell in which the toxin effector polypeptide is present, and the method comprises embedding or inserting the heterologous T-cell epitope in the toxin effector polypeptide. In certain further embodiments of the method, the embedding or inserting step results in a toxin effector polypeptide capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present.

[0035]In certain embodiments of the methods of the present invention is a method of increasing CD8+ T-cell immunogenicity of a polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the polypeptide is present, the method comprising the step of: embedding or inserting a heterologous CD8+ T-cell epitope in the polypeptide. In certain further embodiments of the method, the polypeptide of the method is derived from a toxin. In certain further embodiments of the method, the polypeptide comprises a toxin effector polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a proteasome of a cell in which the toxin effector polypeptide is present, and the method comprises embedding or inserting the heterologous T-cell epitope in the toxin effector polypeptide. In certain further embodiments of the method, the embedding or inserting step results in a toxin effector polypeptide capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present.

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.

In efforts to de-immunize therapeutics, one main challenge is silencing or disrupting immunogenic epitopes within a polypeptide effector domain, e.g. its cytosolic targeting domain, while retaining the desired polypeptide effector function(s), such as, e.g., proteasome delivery.

In addition, it is a significant challenge to disrupt immune epitopes by amino acid substitution in a polypeptide structure while preserving its function while simultaneously adding one or more T-cell epitopes that will not be recognized by the immune system until after cellular internalization, processing, and cell-surface presentation by a target cell.

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.

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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