Aberrant cell-restricted immunoglobulins provided with a toxic moiety

Inactive Publication Date: 2013-07-18
APO T
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for creating a drug that targets specific cells in the body, such as cancer cells or cells associated with tumors. The drug is made by attaching a toxic substance to an immunoglobulin molecule, which is a type of protein. The immunoglobulin molecule has a specific structure that allows it to bind to specific cells, making it a target for the toxic substance. This method ensures that the drug is effective in treating the targeted cells while minimizing harmful effects on healthy cells. The toxic substance can be a protein, fused to the immunoglobulin molecule through a linker sequence. The linker should allow for flexibility and stability, and should not interfere with the immunoglobulin's natural functions.

Problems solved by technology

Many of the conjugates have a limited degree of efficacy.
For example, the maximum tolerated dose of immunotoxins is relatively low due to their toxicity towards healthy tissue.
Lowering the dose, however, hampers the delivery of an efficacious amount of conjugate at the site of for example a tumor.
Thus, insufficient specificity for aberrant cells over healthy cells hampers desired efficacy and hampers obtaining the desired safety profiles of the nowadays immunoglobulin-drug conjugates.
Insufficient tumor cell specificity however still limited the therapeutic usefulness.
Even when selecting tumor cell surface antigens that are (highly) over-expressed at aberrant cells, still the low expression levels at healthy cells gives rise to insufficient selectivity of the antibody-drug conjugates.
Antibiotic calicheamicin conjugated to an anti-human CD33 monoclonal antibody was approved and used in the clinic, but was withdrawn due to serious side effects.
All these immunoglobulin-drug conjugate development programs thus inherently bear the risk for unacceptable safety profiles and consequent poor efficacy due to low maximum tolerated doses.

Method used

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  • Aberrant cell-restricted immunoglobulins provided with a toxic moiety
  • Aberrant cell-restricted immunoglobulins provided with a toxic moiety
  • Aberrant cell-restricted immunoglobulins provided with a toxic moiety

Examples

Experimental program
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example 1

[0053]Non-exhaustive examples of immunoglobulins hereof comprising at least an immunoglobulin variable region that specifically binds to an MHC-peptide complex preferentially associated with aberrant cells or to an aberrant cell surface marker preferentially associated with aberrant cells, with domain topologies as outlined for example in FIG. 5B, are:

[0054]Antibodies hereof comprising immunoglobulin variable regions that specifically bind to:[0055](a) a complex comprising a T-cell epitope selected from 146-KLQCVDLHV-154 (SEQ ID NO:74), 141-FLTPKKLQCV-150 (SEQ ID NO:75), 154-VISNDVCAQV-163 (SEQ ID NO:76), 154-YISNDVCAQV-163 (SEQ ID NO:77) of PSA, presented by HLA-A2 and / or 162-QVHPQKVTK-170 (SEQ ID NO:78) of PSA, presented by HLA-A3, and / or 152-CYASGWGSI-160 (SEQ ID NO:79), 248-HYRKWIKDTI-257 (SEQ ID NO:80) of PSA, presented by HLA-A24, and / or 4-LLHETDSAV-12 (SEQ ID NO:81), 711-ALFDIESKV-719 (SEQ ID NO:82), 27-VLAGGFFLL-35 (SEQ ID NO:83) of PSMA, presented by HLA-A2, and / or 178-NYAR...

example 2

Selection of Human Antibody Fragments Specific for HLA-A0201 / Multi-MAGE-A

[0066]To obtain human antibody fragments comprising immunoglobulin variable regions specific for the HLA-A0201 presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:6) and FLWGPRALV (SEQ ID NO:23) a Human Fab phage display library was constructed according to the procedure previously described by de Haard et al.[2] and used for selections 1) essentially as described by Chames et al. using biotinylated MHC / p complexes,[3] or 2) on cells expressing the relevant antigen.

[0067]2.1: Selection of Human Antibody Fragments Specific for HLA-A0201 / YLEYRQVPV (SEQ ID NO:6) Using Biotinylated MHC-Peptide Complexes:

[0068]Human Fab phages (1013 colony forming units) were first pre-incubated for one hour at room temperature in PBS containing 2% non-fat dry milk (PBSM). In parallel, 200 μl Streptavidin-coated beads (Dynal™) were equilibrated for one hour in PBSM. For subsequent rounds, 100 μl beads were used. To deplete f...

example 3

Cell Binding and Internalization of an Immunoglobulin Provided with a Toxic Moiety

[0081]Binding capacity of an antibody hereof is analyzed by flow-cytometry. For example, an antibody comprising immunoglobulin variable regions specific for complexes of HLA-A0201 and the multi-MAGE-A peptide is analyzed. HLA-A0201 / multi-MAGE-A-positive tumor cells (Daju, MDN and mel 624) and HLA-A0201 / multi-MAGE-A-negative cells (BSM, G43 and 293) are incubated on ice with purified antibody and detected by addition of fluorescently labeled antibodies. Cells bound by the antibody are quantified and visualized by flow-cytometry. Internalization of antibody is analyzed by confocal microscopy. To this end, cells are incubated with the antibody, kept on ice for 30 minutes to allow binding but no internalization. Next, fluorescently labeled antibodies specific for the antibody are added. To induce internalization cells are transferred to 37° C. and fixed with 1% PFA after 5, 10 and 15 minutes.

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Abstract

Described are immunoglobulins provided with a toxic moiety, comprising at least an immunoglobulin variable region that specifically binds to an MHC-peptide complex preferentially associated with aberrant cells. These immunoglobulins provided with a toxic moiety are preferably used in selectively modulating biological processes. The provided immunoglobulins provided with a toxic moiety are of particular use in pharmaceutical compositions for the treatment of diseases related to cellular aberrancies, such as cancers and autoimmune diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit under 35 U.S.C. §119(e) to U.S. Ser. No. 61 / 586,568, filed on Jan. 12, 2012, the contents of which are incorporated herein by this reference.TECHNICAL FIELD[0002]The disclosure relates to the field of biotechnology and biotherapeutics. More specifically, it relates to immunoglobulins provided with a toxic moiety and human antibodies. It also relates to the use of these biotherapeutics in the treatment of a host suffering from a disease associated with aberrant cells, such as cancers and autoimmune diseases.BACKGROUND[0003]The development of immunoglobulin-drug conjugates is a drug development field that receives great attention nowadays. Humanized or human antibodies are the largest and most important class of immunoglobulins under investigation for use in antibody-drug conjugates (ADCs) and in immunotoxins and antibody-radionuclide conjugates. These antibodies target binding sites (over)expressed at abe...

Claims

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Application Information

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IPC IPC(8): A61K47/48C07K16/28
CPCA61K47/48369C07K2317/76C07K16/30C07K16/084C07K16/085C07K16/2884C07K16/3069C07K16/3092C07K16/32C07K16/40C07K2317/32C07K2317/34C07K2317/569A61K2039/505C07K16/2833A61K47/6809A61K47/6813A61K47/6849A61K47/6851A61K47/6883A61P35/00C07K2319/33
Inventor RENES, JOHANSTEVERINK, PAULUS J. G. M.WILLEMSEN, RALPH ALEXANDER
Owner APO T
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