Emerging Proteolysis Targeting Antibodies (PROTABs) for Application in Cancer Therapy

Abstract

The emerging proteolysis targeting antibodies (PROTABs) offer an attractive technology that circumvents some of the challenges of small-molecule intracellular degraders with limited bioavailability and cell permeability. PROTABs present an improved approach to target degradation of membrane-bound and cell surface proteins and use multispecific binding proteins such as multispecific antibodies that bind to at least one transmembrane E3 ubiquitin ligase as well as cell surface proteins intended for degradation.

Important Compound Classes

Title

Multispecific Binding Protein Degrader Platform and Methods of Use

Patent Publication Number

WO 2022/169872 A1 (URL: https://patents.google.com/patent/WO2022169872A1/en?oq=WO+2022%2f169872)

Publication Date

August 11, 2022

Priority Applications

US63/145,336, US63/217,470, and US63/274,288

Priority Dates

February 3, 2021, July 1, 2021, and November 1, 2021

Inventors

Agard, N. J.; De Sousa e Melo, F.; Li, J.; Marei, H. M. G. M. F. E.; Tsai, W. K.; De Sauvage, F. J.; He, J.; Ke, Y. S.

Assignee Company

Genentech, Inc. [US/US]; 1 DNA Way, South San Francisco, California 94080-4990, United States

Disease Area

Cancer

Biological Target

Proteolysis targeting antibodies (PROTABs)

Summary

Cancer is a complex, multistep process that often begins with minor preneoplastic changes, which under certain conditions could progress to neoplasia. It is a disease that is characterized primarily by an uncontrolled division of abnormal cells, leading to uncontrolled cellular proliferation that could derive from a given normal tissue and the invasion of adjacent tissues by these malignant cells. Endogenous transporters such blood or the lymphatic system can spread cancer cells to other parts of the body, which may lead to regional lymph nodes and to metastasis. There are more than 100 different types of cancer (main categories include carcinoma, myeloma, leukemia, sarcoma, lymphoma, and central nervous system cancers) and the incidence of cancer continues to climb as it is exacerbated by the general aging population and development of new cancers. Consequently, a tremendous demand therefore exists for new methods and compositions that can be used to treat patients with cancer.

The proteolysis-targeting chimeric molecules (PROTACs) have been successful in rational drug design therapeutics among the heterobifunctional molecules. An improved approach to target degradation of membrane-bound and cell surface proteins uses multispecific binding proteins, such as multispecific antibodies that bind to at least one transmembrane E3 ubiquitin ligase, or other cell surface ligases, as well as cell surface proteins intended for degradation. The multispecific binding proteins use the transmembrane E3 ubiquitin ligase to target the cell surface protein of interest and tag the lysosomes for degradation. Example of transmembrane E3 ubiquitin ligases include zinc Zing and RING finger 3 (ZNRF3) and its homologue RING finger 43 (RNF43) that may be activated in certain types of cancers, which promote the degradation and turnover of the Frizzled (FZD) and LRP6 receptors on cell surfaces. RNF43 and ZNRF3 are members of the Wnt signaling pathway that may be found in tumor cells from various cancers and expressed in relatively high levels in comparison to normal cells. A number of other transmembrane E3 ubiquitin ligases can be used as targets for multispecific binding proteins.

The patent disclosure in WO 2022/169872 A1 provides a variety of antibody heavy- and light-chain variable regions targeting RNF43 or ZNRF3 that may be useful in constructing multispecific antibodies and binding proteins. The binding proteins can therapeutically degrade proteins such as the Writ pathway when activated and express transmembrane E3 ubiquitin ligases that contribute to the disease progression. The multispecific binding proteins can target one or both of those ligases and may be selectively targeted to cancer cells due to the cell’s overexpression of these proteins. Reported are the optimal attributes of the multispecific antibodies, including optimized binding affinities and the multispecific formats. Some of the multispecific binding proteins are called PROTABs (proteolysis targeting antibodies).

An attractive advantage of the PROTAB technology is that it may overcome the challenges shared by small-molecule intracellular degraders with limited bioavailability and cell permeability that have limited their in vivo activity. The multispecific binding proteins bind to at least a first cell surface target protein and a second cell surface protein, wherein the first cell surface target protein is a transmembrane E3 ubiquitin ligase. The multispecific binding protein reduces the level of the second cell surface protein on the surface of a cell compared to the level observed in the absence of the multispecific binding protein. The transmembrane E3 ubiquitin ligase may or may not have a catalytic activity; in either case, catalytic activity is determined in a cell surface degradation assay. The binding proteins may also be used in vitro to degrade a particular membrane protein in cell culture or tissue assays and thereby reduce its level and knock down its associated signaling.

Biological Assay

Western blot and immunoprecipitation, flow cytometry to assess target cell-surface clearance. TOPBrite reporter assay, Wnt-dependent reporter Nano-Glo Dual luciferase system, and NanoLuc luciferase complementation assay. The biological consequence of degradation on cell signaling and growth was assessed by looking at modulation of IGF1R signaling.

Biological Data

The table below show exemplary surface IGF1R clearance by bispecific RNF43-IGF1R, ZNRF3-IGF1R, and new format bispecific antibodies.

The author declares no competing financial interest.