A number of publications claimed that RSPO is overexpressed in a range of cancer types and has oncogenic functions¹, however, there is still a big gap between research and targeted drugs against RSPO family.
See more in “Description”

We sought to develop a novel protein that can successfully neutralize all four RSPOs in order to antagonize RSPO in malignancies. This protein, as compared to the RSPO3-specific antibody, has the therapeutic potential to treat both colorectal cancers caused by RSPO2 and RSPO3, as well as other cancers caused by RSPO1 or RSPO4. After the multiple steps of upgrading, we finalized the mature chimeric protein, RTAC (RSPO-Targeting Anti-cancer Chimeric protein).

To achieve sufficient affinity towards RSPOs, we fused the extracellular domain of ZNRF3 and LGR4 to specifically and effectively bind RSPOs.

Leucine rich repetitive G-protein coupled receptors (LGRs) are unique G-protein coupled receptors, characterized by large extracellular domains that recognize ligands and regulate many important developmental processes. LGR4-6 receptors are characterized by a long extracellular domain (ECD) containing 17 LRR repeats, which is used to bind R-spondin family proteins to stimulate Wnt signals². Thus, we choose LGR4 ECD as the first domain to trap RSPOs.

In order to bind RSPO more effectively, we also added the ECD of ZNRF3. ZNRF3 is a pivotal E3 ubiquitin ligase that regulates multiple biological processes. Upon binding to RSPO with the ECD, ZNRF3 forms tertiary structure with RSPO and LGR ECD, which eventually leads to the plasma membrane clearance of ZNRF3³. Our results showed RTAC exhibited pan-RSPO binding capacity towards all RSPOs, which was stronger than LGR4 or ZNRF3 ECD alone.
See more in “Results”

To ensure that the protein has sufficient flexibility and solubility for RSPO binding, we added an appropriate linker region between LGR4 and ZNRF3 ECDs.

The successful construction of the recombinant fusion protein requires two indispensable elements: function domains and proper linker. The selection of functional domains is based on the expected function of the fusion protein product, which is relatively simple in most cases. On the other hand, it may be complicated to select appropriate linkers to connect the protein domains. Direct fusion functional domains without linkers may lead to many undesirable results, including misfolding of fusion proteins, low protein production, or impaired biological activity⁴. Therefore, we chose a linker in between LGR4 and ZNRF3 ECD that was enriched in Gly and Ser to satisfy the flexibility and solubility. The linker can provide the proper three-dimensional space between domains and reduce their interference, thus improving the binding efficiency of the fusion protein and the target protein.

Through the analysis of the results, we decided to use the linker with twelve “Gly-Ser” repeats (total twenty-four amino acids) in our finalized fusion protein.
See more in “Engineering”

We used the signaling peptide from Frizzled5 for optimized protein secretion.

Aiming for optimized secretion efficiency of the recombinant protein consisting LGR4 and ZNRF3 ECDs, we picked four common and effective mammalian signal peptides from various proteins (IL-2, Albumin, LGR4 and Frizzled5) to detect them. By the means of biochemistry, we compared the effectiveness on the secretion efficiency of recombinant proteins with different signaling peptides. It was finally found the peptide from Frizzled5 could maximize the yield of the recombinant protein in the medium among these signal peptides. Therefore, we chose the signal peptide of Frizzled5 as the signal peptide of the recombinant protein.
See more in “Engineering”

At last, we added an IgG Fc region to the carboxyl terminal of the chimeric protein to provide further stability.

The presence of the Fc domain can significantly increase the plasma half-life of antibody drugs and prolong the therapeutic activity. Fc domain folds independently, which can improve the solubility and stability as a chaperone molecule in vitro and in vivo. From the technical point of view, Fc domain allows simple, cost-effective purification through protein affinity chromatography during the manufacturing process⁵.

The result showed that the addition of Fc region to our complexes significantly increased the stability of the structure and potentially elongated the activity.
See "Proof of concept" for the effect of whole iteration process

After selecting the brewer’s yeast (Saccharomyces cerevisiae) as the carrier，we next aimed to employ engineered microorganism to create a self-tunable machinery to deliver RTAC to tumor cells. The mating pathway of the engineered yeast was reprogrammed to sense the tumor environment-specific molecules (eATP)⁶, thus expressing RTAC in a self-tunable manner. And in consideration of safety, the kill switch was designed to allow the engineered yeast only functions in the intestinal tract without causing potential environmental hazard. We finally named this eATP-sensitive RTAC-expressing yeast as R-yeast.

The mating pathway refers to the combination of the pheromone ligand and the GPCR to activate the associated G proteins, a process that amplifies the signal through the MAPK (mitogen-activated protein kinase) cascade and finally activates gene expression^7,8.

We first reprogrammed the mating pathway by removing the receptor ste2 through gene editing to ensure the pheromone α could no longer stimulate the downstream pathway. Also, the mating inhibiting factor sst2 was removed to intensify the signal output. This double-knocked out yeast strand was used in our further experiments. Meanwhile, we introduced modified human P2Y2 receptor and a chimeric Gα^6,9. The introduction of these two components will enable the yeast to sense eATP and transduce the signal to the native MAPK signaling in the yeast.

After the upstream of the mating pathway was reprogrammed, we started for the downstream part. Since pFUS1 promoter target gene expression is the final output of the mating pathway, we constructed a plasmid with RTAC following the pFUS1 promoter. To maximize the secretion efficiency, we adapted the signaling peptide of RTAC to that of α-factor (MFα1) particularly for yeast expression⁹.

After this validation, we eventually transformed P2Y2-P2A-Gα and pFUS1-RTAC together into the engineered yeast. Upon eATP treatment, RTAC can be expressed and secreted into the culture in a dose-dependent manner as expected.

Considering the possible leakage and unexpected hazards, we decided to add a self-killing switch to completely resolve this issue. Since there was no well-recognized kill-switch in yeast, we decided to design and construct one on our own.

First, we proposed to pick the essential gene HSF1 in yeast as our target. By manipulating the expression of this gene, the survival of the yeast can be controlled. Copper ion was abundant in the intestine but scarce in the environment, and the copper ion can activate copper-ion dependent promoter CUP1 for target gene expression^10-12. As a result, we replaced the original promoter of HSF1¹³ in R-yeast’s genome to CUP1 by gene editing.