Targeting intracellular target-binding determinants with intracellular antibodies
Inventors
Weisbart, Richard H. • Nishimura, Robert N.
Assignees
US Department of Veterans Affairs
Publication Number
US-10683363-B2
Publication Date
2020-06-16
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 by 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, while the second Fv fragment binds the intracellular target to inhibit its function.
The problem addressed is that current therapies often rely on small molecules to target intracellular sites because large molecules like antibodies typically cannot penetrate living cells. Small molecule inhibitors often bind unintended targets causing side effects, whereas antibodies have excellent binding specificity but are limited to extracellular targets. Intracellular antibodies generated by gene therapy pose potential dangers, and existing cell-penetrating peptides have limitations such as targeting endosomes and toxicity. The invention overcomes these challenges by employing a unique monoclonal anti-DNA antibody 3E10 Fv fragment that penetrates cells via an equilibrative nucleoside salvage pathway (hENT2), allowing delivery of therapeutic antibodies inside cells.
The invention demonstrates the feasibility of delivering antibodies intracellularly using bispecific single-chain Fv fragments where one fragment enables cell penetration (e.g., derived from 3E10) and the other binds and inhibits intracellular targets such as the oncoprotein MDM2. This approach allows for targeted inhibition of intracellular proteins involved in cancer pathways. The invention enables enhanced or synergistic therapeutic effects by targeting multiple components of regulatory pathways with combinations of bispecific antibodies. Additionally, the bispecific antibodies can be engineered with localizing signals or enzyme cleavage sites to improve intracellular targeting or functional separation.
Claims Coverage
The patent contains multiple independent claims focusing on methods of inhibiting tumor or cancer cells using bispecific antibodies with cell-penetrating and intracellular target-binding Fv fragments, defining their structural elements and binding specificities.
Method for inhibiting tumor or cancer cell growth using bispecific antibody with 3E10 cell-penetrating Fv and intracellular target-binding Fv
A method exposing tumor or cancer cells expressing Mdm2, MDC1, 53BP1, ATR, CHK1, CHK2, or p21 to a bispecific antibody composed of a first Fv fragment derived from 3E10 monoclonal antibody with defined CDR sequences enabling cell penetration and a second Fv fragment binding and inhibiting one of the intracellular targets to inhibit tumor or cancer cell growth.
Bispecific antibody having defined amino acid or nucleic acid sequences
The bispecific antibody used in the method comprises the amino acid sequence of SEQ ID NO:2 or 3 or is encoded by nucleic acid sequence SEQ ID NO:1, ensuring the antibody structure necessary for function.
Bispecific antibody can be chimeric, human or humanized
The bispecific antibody in the method may be of chimeric, human or humanized origin, facilitating therapeutic use and compatibility.
Intracellular target-binding Fv derived from anti-oncoprotein or anti-idiotypic antibody
The second Fv fragment binding the intracellular target is derived from an anti-oncoprotein antibody or an anti-idiotypic antibody thereof, preferably a monoclonal antibody that binds Mdm2 oncoprotein, such as mAb 3G5.
Second Fv fragment comprising complementarity determining regions of mAb 3G5
The second Fv fragment contains the light and heavy chain variable domains encompassing the CDRs of mAb 3G5, with specified amino acid sequences (e.g., SEQ ID NOs:17, 19, 21 for light chain and SEQ ID NOs:23, 25, 27 for heavy chain), ensuring binding specificity to Mdm2.
The claims cover a method of inhibiting tumor or cancer cells using bispecific antibodies composed of a cell-penetrating 3E10-derived Fv fragment and a second Fv fragment binding intracellular targets such as Mdm2 and related proteins, including defined sequences of the antibody components and possibilities for humanized or chimeric forms to enable therapeutic applications.
Stated Advantages
Delivery of antibodies into cells enables therapeutic regulation of intracellular targets which are otherwise inaccessible to conventional antibodies.
Use of antibody specificity reduces off-target binding and potential toxic side effects common to small molecule inhibitors.
The bispecific antibodies can target intracellular epitopes such as transcription factors and DNA repair proteins considered undruggable by small molecules.
Combination therapy with bispecific antibodies targeting multiple components of regulatory pathways may enhance or synergize inhibition of tumor or cancer cell growth.
Documented Applications
Treatment of tumors and cancers, including melanoma, soft tissue tumors, sarcomas, breast cancer, cervical carcinomas, ovarian carcinomas, testicular tumors, urothelial carcinomas, esophageal carcinomas, lung cancers, colorectal cancer, glioblastomas, astrocytomas, and other malignancies.
Inhibition of growth of MDM2-addicted tumor or cancer cells.
In vivo use for inhibiting melanoma tumor growth in xenograft mouse models.
Delivery of therapeutic antibodies into cells to regulate intracellular targets such as Mdm2, p53, RAD52, ATM, CHK1, CHK2, BCL2, BRCA1, MDC1, 53BP1, ATR, and p21.
Combination cancer therapy using bispecific antibodies targeting intracellular proteins of the same biochemical pathway or signaling network for enhanced effectiveness.
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