Periodic Reporting for period 1 - RAS-AlphaTAC (KRAS-AlphaTACs: a novel class of biotherapeutics to target KRAS)
Okres sprawozdawczy: 2023-08-01 do 2025-01-31
RAS genes are among the most frequently altered oncogenes, driving approximately 15% of all cancers, including aggressive lung, pancreatic, and colorectal tumors. While the recent development of mutant-selective KRAS inhibitors, particularly those targeting KRAS-G12C, has marked a therapeutic milestone, these agents benefit only a subset of patients and are often limited by rapid resistance and insufficient efficacy in heterogeneous tumors. In contrast, targeted degradation of RAS proteins offers a more comprehensive strategy, potentially enabling sustained and mutation-independent silencing of oncogenic signaling.
The RAS-AlphaTAC project introduced a novel class of intracellular biologics designed to eliminate KRAS rather than inhibit its function. These fusion constructs consist of high-affinity KRAS-binding Alphabodies coupled to E3 ligase recruitment domains to trigger proteasomal degradation. Unlike small-molecule PROTACs, AlphaTACs offer enhanced specificity, modularity, and broader intracellular target access. Our aim was to generate pan-RAS AlphaTACs capable of targeting a spectrum of wild-type and mutant KRAS isoforms, particularly those refractory to existing inhibitors, and to lay the foundation for their preclinical development and translational application.
The RAS-AlphaTAC project introduced a novel class of intracellular biologics designed to eliminate KRAS rather than inhibit its function. These fusion constructs consist of high-affinity KRAS-binding Alphabodies coupled to E3 ligase recruitment domains to trigger proteasomal degradation. Unlike small-molecule PROTACs, AlphaTACs offer enhanced specificity, modularity, and broader intracellular target access. Our aim was to generate pan-RAS AlphaTACs capable of targeting a spectrum of wild-type and mutant KRAS isoforms, particularly those refractory to existing inhibitors, and to lay the foundation for their preclinical development and translational application.
The project focused on the design, engineering, and functional assessment of AlphaTACs. Initial work centered on recombinant protein-based AlphaTACs using CPAB (cell-penetrating Alphabody) technology. These constructs displayed high KRAS-binding affinity in vitro but exhibited limited intracellular activity due to poor cytoplasmic release and significant cytotoxicity at concentrations above 1 µM. Although the CPAB strategy enabled target engagement, it did not yield consistent KRAS degradation in cell-based assays.
To overcome these limitations, we shifted toward AlphaTACs fused to various E3 ligase domains. Constructs containing the SPOP domain consistently reduced KRAS protein levels and suppressed downstream RAS signaling in HEK293T, HeLa, and KRAS-mutant cancer cells. These effects were observed across multiple KRAS isoforms, including G12D, G12V, and G13D, supporting the feasibility of pan-KRAS degradation via intracellular biologics.
We then advanced to RNA-based delivery by engineering circular RNA (circRNA) constructs encoding SPOP-fused AlphaTACs. Transient transfection in cultured cells led to robust protein expression and KRAS degradation, validating RNA as a delivery format. In parallel, we initiated the development of PLGA and lipid nanoparticle (LNP) systems to enable future in vivo administration. The use of circRNA expands the platform’s flexibility, enabling tunable, transient intracellular expression with translational potential in oncology.
Mechanistically, we observed that some AlphaTACs paradoxically increased KRAS levels, consistent with a Hook effect at high concentrations, highlighting the importance of degrader stoichiometry and expression control. Half-life extended variants and hybrid constructs were deprioritized due to technical inefficiencies and limited functional advantage.
In total, the project produced and screened over 30 AlphaTAC constructs, identified SPOP as a robust E3 domain for KRAS degradation, and established circRNA as a viable modality for intracellular AlphaTAC expression. While recombinant AlphaTACs proved suboptimal, the RNA-delivered strategy showed functional promise and scalability.
To overcome these limitations, we shifted toward AlphaTACs fused to various E3 ligase domains. Constructs containing the SPOP domain consistently reduced KRAS protein levels and suppressed downstream RAS signaling in HEK293T, HeLa, and KRAS-mutant cancer cells. These effects were observed across multiple KRAS isoforms, including G12D, G12V, and G13D, supporting the feasibility of pan-KRAS degradation via intracellular biologics.
We then advanced to RNA-based delivery by engineering circular RNA (circRNA) constructs encoding SPOP-fused AlphaTACs. Transient transfection in cultured cells led to robust protein expression and KRAS degradation, validating RNA as a delivery format. In parallel, we initiated the development of PLGA and lipid nanoparticle (LNP) systems to enable future in vivo administration. The use of circRNA expands the platform’s flexibility, enabling tunable, transient intracellular expression with translational potential in oncology.
Mechanistically, we observed that some AlphaTACs paradoxically increased KRAS levels, consistent with a Hook effect at high concentrations, highlighting the importance of degrader stoichiometry and expression control. Half-life extended variants and hybrid constructs were deprioritized due to technical inefficiencies and limited functional advantage.
In total, the project produced and screened over 30 AlphaTAC constructs, identified SPOP as a robust E3 domain for KRAS degradation, and established circRNA as a viable modality for intracellular AlphaTAC expression. While recombinant AlphaTACs proved suboptimal, the RNA-delivered strategy showed functional promise and scalability.
This project demonstrated for the first time that biologic degraders targeting KRAS can be expressed intracellularly from RNA and induce proteasomal degradation of both wild-type and mutant KRAS, including variants not addressed by current drugs. The use of Alphabody scaffolds enabled selective KRAS engagement, while fusion to SPOP yielded consistent degradation and signaling inhibition. These results extend beyond existing small-molecule approaches, offering a mutation-agnostic strategy to target one of cancer’s most intractable oncoproteins.
The shift to RNA-based delivery represents a major innovation. Circular RNA constructs enabled controlled intracellular expression of AlphaTACs, addressing limitations in solubility, delivery, and stability that hampered recombinant formats. This approach not only positions AlphaTACs within the fast-evolving RNA therapeutic landscape but also facilitates adaptation to diverse intracellular targets.
AlphaTACs may provide a mutation-independent solution to overcome KRAS-driven oncogenesis, particularly in treatment-resistant or heterogeneous tumors. The platform is modular and could be extended to other undruggable targets beyond RAS, amplifying its relevance in oncology and other disease areas. RNA-delivered AlphaTACs align with current trends in transient, programmable biologics and are compatible with emerging nanoparticle delivery platforms.
To ensure uptake and clinical translation, several key steps are needed. First, further research is required to optimize construct potency, minimize off-target effects, and refine expression kinetics. Second, robust in vivo studies are essential to demonstrate therapeutic efficacy, tolerability, and pharmacokinetics. Third, RNA encapsulation technologies must be improved to achieve tumor-specific delivery. Although an initial patent covering the CPAB-based approach was withdrawn, the RNA-based modality creates new opportunities for IP generation and licensing. Early regulatory dialogue is also important, given the novel nature of RNA-delivered intracellular biologics. Commercial success will depend on strong preclinical data, a clear clinical indication, and engagement with biotechnology investors.
In conclusion, the RAS-AlphaTAC project established proof-of-principle that intracellular degradation of KRAS can be achieved using engineered Alphabody-based biologics expressed from RNA. These findings represent a step-change in the targeting of KRAS and pave the way for next-generation RNA-encoded therapeutics directed against previously inaccessible intracellular cancer drivers.
The shift to RNA-based delivery represents a major innovation. Circular RNA constructs enabled controlled intracellular expression of AlphaTACs, addressing limitations in solubility, delivery, and stability that hampered recombinant formats. This approach not only positions AlphaTACs within the fast-evolving RNA therapeutic landscape but also facilitates adaptation to diverse intracellular targets.
AlphaTACs may provide a mutation-independent solution to overcome KRAS-driven oncogenesis, particularly in treatment-resistant or heterogeneous tumors. The platform is modular and could be extended to other undruggable targets beyond RAS, amplifying its relevance in oncology and other disease areas. RNA-delivered AlphaTACs align with current trends in transient, programmable biologics and are compatible with emerging nanoparticle delivery platforms.
To ensure uptake and clinical translation, several key steps are needed. First, further research is required to optimize construct potency, minimize off-target effects, and refine expression kinetics. Second, robust in vivo studies are essential to demonstrate therapeutic efficacy, tolerability, and pharmacokinetics. Third, RNA encapsulation technologies must be improved to achieve tumor-specific delivery. Although an initial patent covering the CPAB-based approach was withdrawn, the RNA-based modality creates new opportunities for IP generation and licensing. Early regulatory dialogue is also important, given the novel nature of RNA-delivered intracellular biologics. Commercial success will depend on strong preclinical data, a clear clinical indication, and engagement with biotechnology investors.
In conclusion, the RAS-AlphaTAC project established proof-of-principle that intracellular degradation of KRAS can be achieved using engineered Alphabody-based biologics expressed from RNA. These findings represent a step-change in the targeting of KRAS and pave the way for next-generation RNA-encoded therapeutics directed against previously inaccessible intracellular cancer drivers.