Targeting intracellular target-binding determinants with intracellular antibodies
Inventors
Weisbart, Richard H. • Nishimura, Robert N.
Assignees
US Department of Veterans Affairs
Publication Number
US-9283272-B2
Publication Date
2016-03-15
Expiration Date
2033-03-15
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Abstract
The invention provides a method for inhibiting an intracellular target in a cell with a bispecific antibody comprising contacting the cell with a bispecific antibody having a first Fv fragment with a cell-penetrating determinant and a second Fv fragment with an intracellular target-binding determinant under suitable conditions so that the first Fv fragment causes the bispecific antibody to enter the cell and the second Fv fragment binds the intracellular target in the cell and thereby inhibiting the intracellular target.
Core Innovation
The invention provides a method for inhibiting an intracellular target in a cell using a bispecific antibody that comprises a first Fv fragment with a cell-penetrating determinant and a second Fv fragment with an intracellular target-binding determinant. The first Fv fragment facilitates entry of the bispecific antibody into the cell, and the second Fv fragment binds the intracellular target to inhibit its activity. Examples of intracellular targets include proteins such as MDM2, myc, ras oncoproteins, and DNA repair proteins including RAD52, ATM, CHK1, CHK2, BCL2, BRCA1, MDC1, 53BP1, p53, ATR, and p21.
The problem addressed by the invention is that current therapeutic antibodies cannot penetrate living cells to target intracellular molecules, limiting their use to extracellular or membrane targets. Small molecule inhibitors can target intracellular sites but often have undesirable side effects due to lack of specificity. Existing cell-penetrating peptides pose limitations such as toxicity and endosomal targeting. The invention utilizes the Fv fragment of the unique monoclonal anti-DNA antibody 3E10, which penetrates living cells via a nucleoside salvage pathway mediated by hENT2 without apparent harm. By combining 3E10 with an intracellular target-binding antibody fragment, the bispecific antibody enables intracellular delivery and specific inhibition of intracellular targets.
The invention demonstrates the feasibility of transporting antibodies into cells for therapeutic regulation of intracellular targets and provides novel reagents for treatment of tumors, cancers, diseases, and dysregulated processes. It allows for enhanced or synergistic inhibition of tumor cell growth by targeting multiple components of a regulatory pathway using combination therapies with different bispecific antibodies, each targeting distinct intracellular targets within the same or different pathways. The bispecific antibodies may incorporate features such as linkers, localizing signals, or enzyme cleavage sites to facilitate delivery and functionality within cells.
Claims Coverage
The patent includes two independent claims relating to a method for inhibiting an intracellular target in a cell and a bispecific antibody composition. These claims focus on the use of bispecific antibodies comprising two Fv fragments: one with a cell-penetrating determinant and the other with an intracellular target-binding determinant directed specifically to Mdm2 oncoprotein.
Method for inhibiting an intracellular target using bispecific antibody
A method comprising contacting a cell with a bispecific antibody having a first Fv fragment with a cell-penetrating determinant and a second Fv fragment with an intracellular target-binding determinant, wherein the first Fv fragment causes the bispecific antibody to enter the cell and the second Fv fragment binds the intracellular target, specifically directed to Mdm2 oncoprotein, thereby inhibiting the intracellular target.
Bispecific antibody with cell-penetrating and intracellular target-binding Fv fragments
A bispecific antibody comprising a first Fv fragment derived from an anti-DNA monoclonal antibody 3E10 or an antibody competing with it that mediates cell penetration, and a second Fv fragment with an intracellular target-binding determinant that inhibits biological, biochemical, regulatory activity or cellular signaling associated with the determinant or macromolecule, wherein the second Fv fragment is directed to the Mdm2 oncoprotein.
The claims cover both a therapeutic method using bispecific antibodies to inhibit intracellular targets by enabling intracellular delivery and specific binding of Mdm2, and a bispecific antibody composition combining a 3E10-derived cell-penetrating fragment with an anti-Mdm2 3G5-derived target-binding fragment.
Stated Advantages
The use of cell-penetrating bispecific antibodies enables delivery of therapeutic antibodies into cells, expanding antibody targeting to intracellular molecules that were previously inaccessible.
The high specificity of antibodies for their targets allows for improved therapeutic indices by avoiding off-target binding and associated toxic side effects common with small molecule inhibitors.
Bispecific antibodies can be designed to bind intracellular epitopes such as transcription factors and DNA repair proteins currently considered undruggable, broadening the spectrum of treatable intracellular targets.
Combination therapies using multiple bispecific antibodies targeting different components of the same or related intracellular pathways may provide enhanced or synergistic inhibition of tumor growth.
Documented Applications
Treatment of tumors and cancers including melanoma, colon adenocarcinoma, colorectal cancer, glioblastoma, astrocytoma, breast cancer, ovarian carcinomas, cervical carcinomas, non-small cell lung carcinoma (NSCLC), nasopharyngeal carcinoma, and other solid tumors.
Targeting Mdm2-addicted tumor or cancer cells to inhibit growth by increasing p53 tumor suppressor protein levels using bispecific antibodies penetrating cells and binding Mdm2.
Regulation of activity of Mdm2-interacting proteins implicated in intracellular signaling pathways relevant to cancer and dysregulated intracellular processes.
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