In 2023, SYPHU-China focuses on targeted therapy for HER2-positive solid tumors. Protein engineering techniques were used to construct chemoreceptor eTlpC that can target tumor microenvironments with high lactate concentration, and ADC(Antibody-drug conjugate) drugs were used as a means to attack tumors. A synthetic biology approach was utilized to ensure the safety of LBP(Live Biotherapeutic Products) drugs using gene circuits to construct population sensing modules and suicide switch modules. Based on the chassis cell E.coli Nissle1917 , its genome was edited by CRISPR-Cas9 to make it more suitable to be the chassis cell for this project. The final construction of eCancerKiller that can target the tumor microenvironment and kill HER2-positive tumors using ADC drugs.We have also created some useful synthetic biology elements, such as sensitive, high-intensity promoters that sense lactic acid.
During the brainstorming process, we came up with many novel ideas, such as non-alcoholic beer, fungus brewing factory, cultured meat production, and treatment of seborrheic dermatitis. There is already a large mature market for non-alcoholic beer, and it is difficult for us to find new entry points. This is very difficult for project innovation, so we gave up this project. Fungal brewing factories and cultured meat both rely on mushroom chassis fungi, which cannot be achieved due to limited conditions in our laboratory. For the treatment of seborrheic dermatitis, we initially wanted to choose Bacillus subtilis as the base organism, but it is rarely used on the skin and needs further gene editing. A nitrogen-fixing bacterium suitable for application to the skin attracted our attention, but we lacked corresponding editing tools, but we have already done some research on this aspect. (Click here!!!) As a result, different ideas have different prospects.
An idea from our PI attracted us. We focus on the receptors on the surface of different bacterial species, which can cause the bacteria to flow into their own suitable environment. Nissle 1917 is one of the safest bacteria currently known. It has four receptors on its surface. The serine sensor is a transmembrane sensor protein, and other sensor proteins are intramembrane sensors or are difficult to modify. Therefore, this transmembrane sensor protein became the breakthrough point of the project.[1] [2].
There is a lactic acid sensor in Helicobacter pylori, which can prompt Helicobacter pylori to accumulate in places with high lactic acid. However, Helicobacter pylori is not a widely safe bacterium, so we wondered whether the lactic acid sensor protein on Helicobacter pylori could be Moved to Nissle1917.[3].
After having this initial idea, we began to study the structures of the two proteins. It is clever that the serine sensor protein of Niisle1917 and the lactate chemoattractant protein of Helicobacter pylori have the same three structures of extramembrane, transmembrane and intramembrane. . But if the entire protein is replaced, will it be impossible to transmit the signal to the flagellum of Nissle1917? Therefore, we want to make a fusion protein targeting these three regions and retain the intramembrane or intramembrane part of Nissle1917, so as to ensure the integrity of the signal transmission. This was the prototype of our earliest project.
We first used E.Coli Nissle1917 as the chassis cell and knocked out the serine sensor protein of Nissle1917 to avoid the serine sensor protein in the wild type from competing with the transferred fusion protein for ligands in the signal transduction pathway.
CRISPR-Cas9 Plasmid
pEcGRna Plasmid
Then, we fused the sensor part of the TlpC lactate chemotactic protein in Helicobacter pylori with the intramembrane and transmembrane parts of Nissle1917, respectively, to form two sensor proteins, eTlpC-A and eTlpc-B, to obtain lactate chemotaxis.
Then, the project referred to trastuzumab. We retained the heavy chain variable region and light chain variable region of HER2-targeted trastuzumab and connected them using different linking peptides to construct HER2 Targeting single-chain antibodies. A single-chain antibody-conjugated drug was constructed by fusing the single-chain antibody with the PE38 protein through a connecting peptide, so that it can specifically target and kill HER2-positive cells.
First, we transferred the leghemoglobin plasmid and laccase plasmid with nirB detection plasmid into E. coli BL-21 to induce expression. Regularly detect the fluorescence intensity and OD value reflecting the strain concentration, draw a curve for analysis and modeling.
Then we introduced the gene circuit composed of the single-chain antibody conjugated drug and the lactate-initiated regulatory gene LldR into EcN-SY so that it can secrete the single-chain antibody conjugated drug under high lactate conditions to achieve the killing effect.
Final Results:
chemotaxis Module: After the lactate chemotaxis fusion protein was introduced into E. coli, Nissle1917 did possess lactate chemotaxis ability. By comparing with the wild type, we found that it would spontaneously aggregate to places with high lactate, which is beneficial to surge to the high lactate microenvironment of tumors.
ADC module: Through cell experiments, we confirmed that the killing module of the ADC drug we constructed has a killing effect. At the same time, we established the response curve of the lactate-inducible promoter, confirming the regulatory effect of the lactate-inducible promoter.
Since our project is targeted tumor killing, the modification of sensor proteins has great potential and may form a complete set of biological components in the future. We hope that the chemotaxis module we have modified now can be applied to the research of related scientific principles or products need to realize lactate chemotaxis. This sensor protein may not only be suitable for chemotaxis in the tumor microenvironment, but may also be used as a biosensor in the future. ADC drugs are hot today. Our modification of trastuzumab and its conjugation with Pseudomonas aeruginosa chlorotoxin have provided a lot of data for the development of ADC drugs. We have reached a consensus with many experts, scholars and companies that drug development will not turn off. In the future, after our team form the development of a complete set of chemotaxis modules, our project can evolve into a multi-track directional device.
[1] Ortega DR, Yang C, Ames P, Baudry J, Parkinson JS, Zhulin IB. A phenylalanine rotameric switch for signal-state control in bacterial chemoreceptors. Nat Commun. 2013;4:2881. doi: 10.1038/ncomms3881. PMID: 24335957; PMCID: PMC4310728.
[2] Salah Ud-Din AIM, Roujeinikova A. Methyl-accepting chemotaxis proteins: a core sensing element in prokaryotes and archaea. Cell Mol Life Sci. 2017 Sep;74(18):3293-3303. doi: 10.1007/s00018-017-2514-0. Epub 2017 Apr 13. PMID: 28409190.
[3] Machuca MA, Johnson KS, Liu YC, Steer DL, Ottemann KM, Roujeinikova A. Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate. Sci Rep. 2017 Oct 26;7(1):14089. doi: 10.1038/s41598-017-14372-2. PMID: 29075010; PMCID: PMC5658362.