Periodic Reporting for period 1 - TargetRANK (Targeting RANK receptor as a novel therapeutic strategy in triple negative breast cancer)
Periodo di rendicontazione: 2023-03-01 al 2024-08-31
Breast cancer is still a significant cancer with over 1/2 million deaths per year, mainly due to metastatic disease. Triple negative breast cancers (TNBC) are responsible for around 20% of all breast cancers and are particularly aggressive and with high mortality rates. To date there are only few drugs in the market to treat TNBC, leaving patients with little treatment options other than surgery, radiotherapy and chemotherapy. Despite its sensitivity to chemotherapy, TNBC remains a clinical challenge due to high rate of relapse, a propensity to form visceral metastasis and limited targeted therapies.
RANK signaling pathway has emerged as a novel target for breast cancer prevention and treatment. The work of my lab and others evidenced that RANK protein over expression may be responsible for a number of these cancers and can also facilitate metastasis. Current antibodies targeting RANK ligand (RANKL) prevent tumor initiation but do not reduce tumor growth. In TargetRANK we aim to target the receptor RANK, instead of the ligand. This value proposition is based on: (1) RANKL expression is gradually lost during breast cancer progression, whereas RANK is expressed in a subset of luminal and TNBC human mammary adenocarcinomas. (2) RANK overexpression in breast cancer cells leads to constitutive activation of the pathway in a RANKL-independent manner, promoting stemness, tumorigenesis and metastasis
In addition, a worrying problem is identifying the RANK+ patients that could benefit from anti-RANK therapies. The monoclonal anti-human RANK antibodies available in the market are neither well characterized nor specific to detect RANK in breast tumor samples, whereas the best and widely accepted antibody developed to date from Amgen, is not sensitive enough and not commercialized. Thus, there is a need to identify novel targeted therapies for the treatment of TNBC and to develop diagnostic tools to identify RANK+ patients.
The general objective is to validate RANK as a new target in breast cancer therapy, develop molecules that bind and inhibit RANK signaling and diagnostic antibodies to identify patients who can benefit from the treatment. The outcomes of the present study can provide patients with triple-negative breast cancer with another, much-needed therapeutic option. Furthermore, the project also aims to develop a new diagnostic tool to identify RANK+ patients who will benefit from the treatment. This could result in both better treatments and improved diagnostic testing.
RANK signaling pathway has emerged as a novel target for breast cancer prevention and treatment. The work of my lab and others evidenced that RANK protein over expression may be responsible for a number of these cancers and can also facilitate metastasis. Current antibodies targeting RANK ligand (RANKL) prevent tumor initiation but do not reduce tumor growth. In TargetRANK we aim to target the receptor RANK, instead of the ligand. This value proposition is based on: (1) RANKL expression is gradually lost during breast cancer progression, whereas RANK is expressed in a subset of luminal and TNBC human mammary adenocarcinomas. (2) RANK overexpression in breast cancer cells leads to constitutive activation of the pathway in a RANKL-independent manner, promoting stemness, tumorigenesis and metastasis
In addition, a worrying problem is identifying the RANK+ patients that could benefit from anti-RANK therapies. The monoclonal anti-human RANK antibodies available in the market are neither well characterized nor specific to detect RANK in breast tumor samples, whereas the best and widely accepted antibody developed to date from Amgen, is not sensitive enough and not commercialized. Thus, there is a need to identify novel targeted therapies for the treatment of TNBC and to develop diagnostic tools to identify RANK+ patients.
The general objective is to validate RANK as a new target in breast cancer therapy, develop molecules that bind and inhibit RANK signaling and diagnostic antibodies to identify patients who can benefit from the treatment. The outcomes of the present study can provide patients with triple-negative breast cancer with another, much-needed therapeutic option. Furthermore, the project also aims to develop a new diagnostic tool to identify RANK+ patients who will benefit from the treatment. This could result in both better treatments and improved diagnostic testing.
Work Package 1. Development of first-in-class drugs against RANK.
High throughput virtual and cellular screenings successfully identified three potential RANK inhibitors (ETP-33, ETP-68 and ETP-18). These compounds selectively inhibit RANK signaling in breast cancer cells without affecting cell viability. Current analyses of interaction profiles indicate potential direct binding to RANK although further experiments are underway to confirm this observation and determine their affinity (Kd). On the other hand, five known antitumoral compounds also showed selective RANK inhibition in breast cancer RANK overexpressing cells. We are currently studying their mechanism of action to determine whether they are direct binders of RANK or modulators of RANK signaling by inhibition of other targets/pathways.
Additional deliverables include throughput cell-based assays to specifically test RANK agonist/antagonist activity.
Work Package 2. Development of human anti-RANK monoclonal antibodies
In this work package, we have successfully generated monoclonal antibodies targeting both the extracellular and intracellular domains of the RANK protein. Some of these antibodies demonstrate enhanced sensitivity compared to commercial alternatives in Western Blot assays. However, their capacity to detect endogenous RANK remains limited. Notably, one of the antibody clones shows potential for detecting human RANK through immunohistochemistry (IHC), particularly in patient-derived breast cancer samples. Further validation is required to fully establish its utility in clinical diagnostics.
Additional deliverables include:
- The crystal structure of the extracellular domain of human RANK (XTU Unit)
- Generation of recombinant intracellular and extracellular RANK proteins (XTUnit)
Work Package 3.
1. Preliminary patentability and infringement analysis of TargetRANK-related data. Specifically, on the 8 compounds identified as potential RANK inhibitors. The analysis revealed that one of the hits, ETP-68, is protected under CNIO´s patent family WO2014140644, so a third party might infringe if it produces, possesses, offers for sale or uses a compound with ETP-68 structure.
2. Regarding antibody development some clones were found exhibiting higher sensitivity than the commercial one. Clones 593E and 383B will be further explored in order to add it to CNIO´s monoclonal antibodies portfolio.
3. Market analysis of TNBC treatment. Market sizing data was gathered, together with an analysis of the state-of-the-art of the TNBC systemic treatment, from the standard of care to the advanced and early-stage developments. Main players and business operations were also reviewed.
High throughput virtual and cellular screenings successfully identified three potential RANK inhibitors (ETP-33, ETP-68 and ETP-18). These compounds selectively inhibit RANK signaling in breast cancer cells without affecting cell viability. Current analyses of interaction profiles indicate potential direct binding to RANK although further experiments are underway to confirm this observation and determine their affinity (Kd). On the other hand, five known antitumoral compounds also showed selective RANK inhibition in breast cancer RANK overexpressing cells. We are currently studying their mechanism of action to determine whether they are direct binders of RANK or modulators of RANK signaling by inhibition of other targets/pathways.
Additional deliverables include throughput cell-based assays to specifically test RANK agonist/antagonist activity.
Work Package 2. Development of human anti-RANK monoclonal antibodies
In this work package, we have successfully generated monoclonal antibodies targeting both the extracellular and intracellular domains of the RANK protein. Some of these antibodies demonstrate enhanced sensitivity compared to commercial alternatives in Western Blot assays. However, their capacity to detect endogenous RANK remains limited. Notably, one of the antibody clones shows potential for detecting human RANK through immunohistochemistry (IHC), particularly in patient-derived breast cancer samples. Further validation is required to fully establish its utility in clinical diagnostics.
Additional deliverables include:
- The crystal structure of the extracellular domain of human RANK (XTU Unit)
- Generation of recombinant intracellular and extracellular RANK proteins (XTUnit)
Work Package 3.
1. Preliminary patentability and infringement analysis of TargetRANK-related data. Specifically, on the 8 compounds identified as potential RANK inhibitors. The analysis revealed that one of the hits, ETP-68, is protected under CNIO´s patent family WO2014140644, so a third party might infringe if it produces, possesses, offers for sale or uses a compound with ETP-68 structure.
2. Regarding antibody development some clones were found exhibiting higher sensitivity than the commercial one. Clones 593E and 383B will be further explored in order to add it to CNIO´s monoclonal antibodies portfolio.
3. Market analysis of TNBC treatment. Market sizing data was gathered, together with an analysis of the state-of-the-art of the TNBC systemic treatment, from the standard of care to the advanced and early-stage developments. Main players and business operations were also reviewed.
Work package 1
Candidate Compounds that can act as novel RANK inhibitors have been identified. Hit optimization and modification is required for validation of the assets and patentability.
Next Steps: If we confirm any of the described compounds as truly direct binders of RANK, we plan to deploy medicinal chemistry exploration (SAR) to optimize their direct binding properties, avoiding their primary targets (as priority) as well as the generation of intellectual property. These activities are subjected to new funding.
Work package 2
Clones for anti-RANK detection by Western Blot and IHC have been selected. Upon further experimental validation licensing will be considered.
Work Package 3.
Market analysis of TNBC treatment lays the foundations on which the competitive analysis and potential market evaluation will be based for the business roadmap of TargetRANK assets.
Candidate Compounds that can act as novel RANK inhibitors have been identified. Hit optimization and modification is required for validation of the assets and patentability.
Next Steps: If we confirm any of the described compounds as truly direct binders of RANK, we plan to deploy medicinal chemistry exploration (SAR) to optimize their direct binding properties, avoiding their primary targets (as priority) as well as the generation of intellectual property. These activities are subjected to new funding.
Work package 2
Clones for anti-RANK detection by Western Blot and IHC have been selected. Upon further experimental validation licensing will be considered.
Work Package 3.
Market analysis of TNBC treatment lays the foundations on which the competitive analysis and potential market evaluation will be based for the business roadmap of TargetRANK assets.