Targeting intracellular target-binding determinants with intracellular antibodies
Inventors
Weisbart, Richard H. • Nishimura, Robert N.
Assignees
US Department of Veterans Affairs
Publication Number
US-12116416-B2
Publication Date
2024-10-15
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 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. The first Fv fragment causes the bispecific antibody to enter the cell, and the second Fv fragment binds and inhibits the intracellular target under suitable conditions.
Current therapies primarily use small molecules to target intracellular sites since cells are generally impermeable to large molecules like proteins. Small molecule inhibitors can cause undesirable side effects by binding unintended targets, while antibodies have high specificity but typically cannot penetrate cells. The invention addresses the limitation that therapeutic antibodies are generally restricted to extracellular or membrane targets by developing bispecific antibodies that combine cell-penetrating and intracellular target-binding functionalities to access and regulate intracellular proteins.
Specifically, the invention utilizes the Fv fragment of the unique cell-penetrating monoclonal anti-DNA antibody 3E10, which enters cells via the hENT2 nucleoside salvage pathway and localizes in the nucleus without apparent harm. This Fv fragment is combined with a second Fv fragment targeting intracellular proteins such as the oncoprotein MDM2 or other intracellular regulatory proteins. The bispecific antibodies can inhibit growth of tumor cells addicted to MDM2 and other intracellular pathways, expanding therapeutic options to previously undruggable intracellular targets.
Claims Coverage
The patent includes two main inventive features based on the independent claims: the composition of specific bispecific antibodies and their pharmaceutical compositions with optional conjugations.
Composition of bispecific antibody having specified amino acid sequences
A bispecific antibody comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:30.
Pharmaceutical and conjugate compositions of the bispecific antibody
A pharmaceutical composition comprising the bispecific antibody of the invention, optionally including a pharmaceutically acceptable carrier or conjugated to a chemotherapeutic agent, toxin, radioisotope, or detectable label.
The claims principally cover bispecific antibodies defined by specific amino acid sequences and their pharmaceutical compositions including conjugates with therapeutic or diagnostic agents.
Stated Advantages
Improved therapeutic indices by delivering antibodies intracellularly to inhibit targets with high specificity, thereby avoiding off-target effects associated with small molecules.
The bispecific antibody retains cell-penetrating and intracellular target-binding activities, enabling targeting of previously undruggable intracellular proteins such as transcription factors and DNA repair proteins.
Demonstrated inhibition of tumor cell growth in vitro and in vivo without apparent toxicity, indicating potential for effective cancer therapy.
Combination therapies using multiple bispecific antibodies targeting different components of dysregulated intracellular pathways may have enhanced or synergistic effectiveness against tumors.
Documented Applications
Treatment of tumors and cancers including melanomas, soft tissue tumors, sarcomas, brain tumors, gliomas, breast cancer, ovarian carcinoma, lung cancers, colorectal cancer, and more by targeting intracellular proteins such as MDM2 and mutant p53.
Inhibition of MDM2-addicted tumor or cancer cells to increase p53 tumor suppressor protein levels and inhibit tumor growth.
Use in combination therapies to regulate and inhibit dysregulated intracellular processes, signaling pathways, or regulatory networks in cancer cells.
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