Targeting intracellular target-binding determinants with intracellular antibodies
Inventors
Weisbart, Richard H. • Nishimura, Robert N.
Assignees
US Department of Veterans Affairs
Publication Number
US-12195550-B2
Publication Date
2025-01-14
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 intracellular targets in cells using bispecific antibodies comprising two Fv fragments: one with a cell-penetrating determinant and another with an intracellular target-binding determinant. The first Fv fragment enables the bispecific antibody to enter the cell, while the second binds and inhibits the intracellular target. This approach specifically includes antibodies such as the anti-DNA monoclonal antibody 3E10 for cell penetration and antibodies like 3G5 that bind intracellular targets such as MDM2, an E3 ubiquitin ligase involved in cancer pathways.
The problem addressed by the invention arises from current therapeutic limitations where small molecule inhibitors target intracellular sites but often bind unintended targets causing side effects. Antibodies offer higher specificity but generally do not penetrate living cells, restricting their use to extracellular or membrane-bound targets. Previous attempts at intracellular antibody delivery, including gene therapy and cell-penetrating peptides, have challenges including toxicity and endosomal sequestration. The invention overcomes these limitations by using the 3E10 antibody fragment as a delivery system exploiting the nucleoside salvage pathway to penetrate cells, potentially expanding therapeutic intracellular targeting.
The invention also demonstrates that bispecific antibodies with cell-penetrating and intracellular target-binding fragments can modulate intracellular regulatory pathways, such as increasing p53 tumor suppressor levels by inhibiting MDM2 in cancer cells. Moreover, combination therapy with multiple bispecific antibodies targeting different proteins in the same pathway can enhance therapeutic effects. The invention includes recombinant production methods of such bispecific antibodies, incorporation of linker sequences, and potential for targeting various intracellular proteins including oncoproteins, DNA repair proteins, and tumor suppressor-interacting proteins.
Claims Coverage
The patent includes two independent claims directed to bispecific antibodies with specified complementarity determining regions (CDRs) in their heavy and light chain variable domains.
Defined bispecific antibody comprising two Fv fragments with specific CDR sequences
The bispecific antibody comprises a first Fv fragment with heavy chain variable domain CDRH1, CDRH2, CDRH3 of SEQ ID NOS: 11, 13, 15 respectively and light chain variable domain CDRL1, CDRL2, CDRL3 of SEQ ID NOS: 5, 7, 9 respectively; and a second Fv fragment with heavy chain variable domain CDRH1, CDRH2, CDRH3 of SEQ ID NOS: 23, 25, 27 respectively and light chain variable domain CDRL1, CDRL2, CDRL3 of SEQ ID NOS: 17, 19, 21 respectively.
Bispecific antibody having the amino acid sequence of SEQ ID NO: 3
The bispecific antibody as defined having the specified amino acid sequence corresponding to SEQ ID NO: 3, encoding the linked Fv fragments with the above mentioned CDRs.
The claims cover bispecific antibodies composed of two specific Fv fragments each defined by precise heavy and light chain CDR sequences, demonstrating the invention's focus on antibody structures that combine cell penetration and intracellular target binding functionalities for therapeutic applications.
Stated Advantages
The use of bispecific antibodies allows highly specific targeting of intracellular proteins, reducing off-target effects present with small molecule inhibitors.
The cell-penetrating capability of the antibody facilitates intracellular delivery without the toxicity and cellular sequestration issues of previous transport methods.
Combination therapies using bispecific antibodies targeting multiple components of regulatory pathways may enhance or synergize therapeutic efficacy against tumors or dysregulated processes.
The bispecific antibodies are largely degraded within 4 hours after nuclear localization, minimizing potential toxicity.
Documented Applications
Treatment of tumors and cancers including melanoma, soft tissue tumors, sarcomas, Ewing's sarcoma, brain tumors, glioblastomas, astrocytomas, breast cancer, cervical carcinomas, ovarian carcinomas, lung cancers, non-small cell lung carcinoma, colorectal and colon cancer.
Inhibiting growth of MDM2-addicted tumor or cancer cells by intracellular inhibition of MDM2, increasing p53 tumor suppressor activity.
Regulating activity of MDM2-interacting proteins to control intracellular signaling relevant to cancer and dysregulated cellular processes.
Combination therapies with bispecific antibodies targeting different proteins within the same biochemical or regulatory pathway to treat tumors, cancers, or intracellular dysregulated conditions.
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