INTERGRATED HUMAN PRACTICES

As our project design and laboratory part progressed, we encountered a very large number of practical difficulties, including three main areas: schematic design, experimental implementation and clinical application. For this purpose, we have interviewed three different groups, including: experts in proteomics to help us with design aspects; pharmaceutical companies of recombinant proteases and protein production centers to help us with difficulties encountered in experimental production; and experts in clinical aspects to help us with difficulties that we may encounter when the product is put into use.

To achieve this goal, we communicate and interact with different stakeholders through seminars, expert interviews, and public questionnaires. We believe that our solution will not only bring significant benefits to the community, but will also address the issues prevalent in our final product development phase.

1. Pre-project research

Cancer, has always been a major challenge on the road to human survival. This includes cure and prognosis, both of which are very difficult to work with. In particular, many in situ cancers also have complicated prognostic difficulties after resection and treatment, resulting in low 5-year as well as 10-year survival cycles, to the extent that it makes the quality of survival of patients severely reduced.

Among others, we observed in our reading of the literature that overexpression of the c-myc gene, which is associated with a variety of cancers, has a strong impact on the recurrence aspect of prognosis in particular. For example, in the literature: c-myc gene amplification in breast, bladder and kidney cancer tissues, it was mentioned that the c-myc gene is very strongly associated with the amplification of breast, bladder and kidney cancers.

In a 2012 CELL review, "MYC on the Path to Cancer", a systematic explanation of the molecular functions, signaling pathways and roles of MYC in cancer development is provided.

The MYC gene is one of the most widely studied intranuclear oncogenes, and the MYC gene family consists of three major members, C-Myc, N-Myc and L-Myc, of which C-Myc was first identified as the cellular homolog of the avian myeloma virus transformation sequence. The role of L-Myc is still poorly understood, and the expression of N-Myc is tissue-specific and can replace c-Myc in mouse development. proto-oncogene MYC is a crossover point for a number of growth-promoting signaling pathways and an immediate early response gene downstream of many ligand-membrane receptor complexes (Figure 1). MYC expression is highly controlled and its expression levels are tightly regulated by a number of transcriptional regulatory modality-related mechanisms within the proximal promoter region.

Early studies of oncogenic retroviruses causing fulminant chicken tumors paved the way for the discovery of MYC, which led to the identification of v-myc oncogenes causing myelomonocytosis. The homologous sequence of v-myc in the host genome is c-myc. Although the search for similar human retroviruses could not generalize retroviral oncogenes in human cancers, it was also found that human MYC is usually altered in Burkitt lymphoma (Burkitt lymphoma) due to balanced chromosomal translocations and is therefore a true human oncogene. MYC is frequently found in MYC is frequently translocated in multiple myeloma and is also one of the most highly amplified oncogenes in a variety of different human cancers. It has been shown that defective Wnt-APC signaling in human colon cancer promotes transcriptional activation of MYC by TCF, and in T-cell leukemia MYC is downstream of the uncontrolled Notch signaling pathway. Thus alterations in MYC are most commonly seen on the pathway to cancer.

In addition to its role in tumor formation, MYC can also work with Sox2, Oct4 and KLF4 to reprogram fibroblasts to a pluripotent stem cell state. Based on its central role in cell growth, proliferation, tumor formation and stem cells, author Chi V. Dang states in the article that he takes these key questions such as what is the molecular function of Myc, the protein product of MYC, how does MYC contribute to tumor formation, the differences between the MYC proto-oncogene and the runaway forms found in various human cancers, and whether MYC or Myc's Can target genes be targets for cancer therapy?

Summary: c-Myc and Cancer

Dysregulation of the proto-oncogene MYC has a key role in human tumorigenesis. Unlike other proto-oncogenes that are active due to mutation or truncation, MYC is dysregulated due to loss of transcriptional control, resulting in protein overexpression.

For example, in human Burkitt's lymphoma, translocation of the MYC gene to the immunoglobulin heavy or light chain site results in c-Myc overexpression.

Amplification of the MYC gene was also found in several cases of human epithelial cell tumors and diffuse large B-cell lymphomas, directly correlating with poor prognosis of the disease.

At this point, we also found the desired research direction, i.e., can target genes of MYC or Myc become targets for cancer therapy?

With this question in mind we sought to learn more about the literature, among which we found Professor Gerard Evan from the University of Cambridge, whose seminal contributions include the discovery of Myc's involvement in apoptosis and the development of early tools for studying Myc, such as the 9E10 antibody against the Myc tag. In one of his interviews, he mentioned that "the commonality of cancer is an important clue that people avoid ...... "...... [We need] to come up with a cure, no matter who you are or what you have. Now, a lot of people think this is science fiction, but I think all the data clearly shows it's not .

" ...... This is one of the great mysteries in cancer biology, and no one knows why it is. No one knows why tumors are susceptible to disruption of these signals and die."

Interview Professor Gerard Evan Video

In our next literature study, we found a paper published in Nature Biotechnology by researchers from Lund University in Sweden entitled "A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer.

The researchers found that children with acute pyelonephritis (APN) had significantly lower MYC expression levels during the acute infection period. Through in vitro infection experiments, it was confirmed that infection with uropathogenic Escherichia coli (UPEC), such as E. coli CFT073 and E. coli 536, rapidly reduced c-MYC protein expression levels. To investigate the mechanism of c-MYC degradation, the investigators screened the E. coli 536 mutant for activity, identified the region responsible for c-MYC inhibition located on pathogenicity islands (PAIs) PAI I, and found that c-MYC was inhibited by the supernatant of E. coli 536 cultures. Proteomic analysis of the bacterial culture supernatants of wild-type E. coli 536 and PAI I deletion mutant strain PAI I536 identified the MYC inhibitor as the protease Lon. Lon protease is an ATP-dependent serine protease that interacts with c-MYC by directly cleaving the serine-rich repeat sequence of c-MYC, depriving c-MYC of essential functional domains, such as, DNA binding site and C-terminal MAX binding site.

At the same time, the investigators further demonstrated the mechanism by which pathogenic E. coli 536 regulates the expression level of c-MYC through the pore-forming toxin (PFT) α-hly. α-hly induced Ca2+ flux activates casein kinase 1 alpha 1 (CK1α1), and the activated CK1α1 is reported to be associated with c-MYC. CK1α1 binds to c-MYC, phosphorylates c-MYC, and is degraded by proteases. cK1α1 is also involved in the degradation of the c-MYC transcriptional enhancer c-MYB, which presumably further attenuates MYC expression. In addition, it was found that pathogenic E. coli 536 targets the transcriptional regulator of c-MYC: CCAAT enhancer-binding protein delta (CEBPD), which in turn accelerates the degradation of c-MYC.

In vivo experiments with Lon, an α-hly-independent inhibitor that acts directly and alone on c-MYC, delivered to mice either intravesically or orally, found that Lon protease delayed tumor development in MYC-dependent bladder and colon cancers and improved survival in cancer model mice, respectively. These results suggest that bacteria have evolved strategies to control c-MYC levels in the host and that Lon proteases hold promise as therapeutic cancer drugs targeting c-MYC.

This has greatly stimulated our interest, and we believe that Lon proteins may become potentially excellent targeting agents for c-MYC that could effectively improve the survival of patients with cancers due to c-MYC overexpression, including bladder and kidney cancers.

So we planned to use experimental techniques to produce Lon protease, to judge the use and application value of Lon protease, and to be able to package it as a product with the standard of a biological drug and to improve this product according to the needs of actual stakeholders.

2.Interview

2.1 Project design phase.

In the project design phase, since our project today is a protease-based anti-cancer drug, we interviewed proteomics experts and clinical experts for this purpose to get a preliminary understanding of the product that will be used in the future and to have a clear goal of how the product will be used. My design goal is to design a drug that can be used as an adjunct to surgical treatment of bladder cancer with TUR-BT, which can be followed by adjuvant perfusion therapy to achieve a reduction in recurrence of bladder cancer after surgery.

2.1.1 Proteomics Expert

Since our goal this year is to make a drug that can protein, we interviewed Lotro Technologies, for which James Dewey Watson is the general scientific advisor, and Lotro Technologies was happy to accept our interview, arranging for Dr. Yanjing Su and his scientific team to be interviewed by us. Jeffrey Su, B.Sc., M.Sc., Nankai University, Ph.D., Carleton University, Canada, and Postdoctoral Fellow, National Protein Engineering Center (PENCE), Canada.

In the first interview, we introduced our project background and experimental design to Dr. Su, who gave positive comments on our project design idea and thought that our design idea was very clear.

At the same time, Dr. Su gave his opinion that the molecular weight of rLon protein was 89Kda, which is a very large amount of protein and may be a challenge for protein drugs, and he gave his opinion as a proteomics expert to try to further optimize the molecular structure of this protein to see if some key structures can be found to optimize a large protein into a smaller peptide structure. . At the same time, we talked about an idea that if we can validate the protein potency, then can we try to refer to the way of mRNA drugs, and make the mRNA corresponding to the protein combined with LNP and other carriers to make targeted drugs to deliver to the cancer lesions, one can increase the potency, and the other can reduce the possible side effects.

2.1.2 Clinical experts

Meanwhile, at the beginning of the project design, in order to get medical clinical information related to bladder cancer triggered by C-myc gene overexpression, we interviewed Dr. Zhou from Beijing Chaoyang Hospital, who graduated from Peking University School of Medicine and is also a senior who participated in the iGEM competition.

We met at a Starbucks for an appointment. At Dr. Zhou's request, we did not take photos or recordings, but only kept the corresponding transcripts.

In the interview, Dr. Zhou introduced us to the current clinical treatment of bladder cancer.

There are two common types of bladder cancer at present, treatment of non-muscle-invasive bladder cancer and surgical treatment of muscle-invasive bladder cancer, of which non-muscle-invasive bladder cancer (non muscle-invasive bladder cancer) or superficial bladder cancer (superficial bladder cancer) accounts for 75% to 85% of all bladder tumors.

Certain factors are closely associated with the prognosis of non-muscle-invasive bladder cancer. Among these factors, the number of tumors, frequency of tumor recurrence, especially at 3 months postoperatively, tumor size, and tumor grade are strongly associated with recurrence. The factors most associated with tumor progression include pathologic grading of the tumor and tumor stage. The prognosis for tumors at the bladder neck is poor.

The main treatment options are surgical resection and postoperative adjuvant therapy. The surgical resection treatment is based on an assessment of the nature of the tumor, the depth of infiltration, and the choice of conservative transurethral resection of bladder tumor (TUR-BT) with postoperative radiation and chemotherapy, and close postoperative follow-up is required. An alternative, more radical surgical treatment is radical cystectomy, in which the patient's entire bladder is directly removed.

Recurrence occurs in 10% to 67% of patients within 12 months after TUR-BT and 24% to 84% of patients within 5 years after surgery. There are two peak periods of recurrence after TUR-BT for non-muscle invasive bladder cancer, 100 to 200 days after surgery and 600 days after surgery. Because of the concern about recurrence, more and more patients are choosing the radical treatment of radical cystectomy.

However, from Dr. Zhou's conversation with us, we learned that although recurrence is lower with this type of resection, the quality of patient's survival is namely lower, such as the need for abdominal wall stoma, lifelong wearing of urinary collection bags, urinary incontinence and other inconveniences.

Clinically speaking, in order to reduce the problem of high recurrence and progression after surgery, adjuvant bladder perfusion therapy is generally used for bladder cancer patients after surgery. The perfusion drugs include chemotherapeutic drugs such as epirubicin or mitomycin, etc. It has been clinically proven that bladder perfusion chemotherapy can reduce the tumor recurrence rate by 40%. Another is postoperative bladder perfusion immunotherapy with BCG, for example, for intermediate-risk non-muscle-infiltrating bladder uroepithelial carcinoma, the probability of tumor recurrence after surgery is 45% and the probability of progression is 1.8% with BCG perfusion. However, BCG instillation has more side effects and needs to be used with caution.

At the time of the interview, we had a very good idea to use Lon protein as a perfusion drug to assist postoperative treatment of TUR-BT surgery, which is used to reduce the risk of cancer recurrence. At the same time, this approach can also circumvent one of the risks of injecting protease into the body and worrying about generating an immune response, which we initially considered in the design of the project.

Dr. Zhou also feels that this idea is very promising and hopes to have the opportunity to see the efficacy of this method in the clinic.

2.2 Experimental phase

During the experimental phase, we encountered a number of problems, among which insufficient protein yield and protein purification were the more troubling ones. For these two problems, we not only continued to demand the research team from LOTUS that we interviewed during the design phase of the project, but we also interviewed Dr. Han from UW-Lihe, who came to specialize in protein purification.

2.2.1 Recombinant protein drug company

We finished the construction of the plasmid of rLon protein in our experiment and expressed it in BL21 (DE3) system, but the expression amount was very low and the bands shown on SDS-page were very slight, which was still not obvious after we tried many times.

For this reason, we interviewed Dr. Han of UW-Lihe, a professional company in protein preparation and purification, which is a subsidiary of UW Genetics.

Dr. Han gave us some conditions that they usually experience in protein preparation and let us try to experiment with different situations.

Among them, 3 different directions to try were given.
1. change strains: Rosetta, BL21 plys S, Codon plus
2. change the temperature: the effect of overnight expression at 16°.
3. change the system: yeast or 293 cell line in a different species expression

2.2.2 Proteomics expert (second time)

We interviewed the R&D team of LOTUS again on the problem of difficult protein expression, and combined with the experience given by Dr. Han, the scientific team of LOTUS gave us some new suggestions.

The PhD's of Lotro R&D team gave some possibilities to explore based on the situation we encountered.

1. It may be because the protein is too large and there is difficulty in expression, if this is the reason, protein structure optimization is needed.
2. Lon protein itself is the gene that BL21 was knocked out, combined with the characteristics of Lon protein, this protein may affect the growth and division of the strain, and the timing of induced expression can be considered.
3. Overnight expression can only increase the activity of the protein and does not improve the protein output, a shorter time can be chosen for expression.

2.2.3 Proteomics expert (third time)

After our experimental mapping, we succeeded in increasing the protein expression. We gave feedback on this one to the Lakeland research team and they gave a good evaluation. The conditions we figured out were that we used Rosetta strain, OD600 was induced at 0.6-0.8, .IPTG amount 0.5mM, centrifuged after 4h incubation at 37°C, and we could get high yield of protein.

However, the new problem encountered was that the purification was difficult and the purity was not even high, while in SDS-page as well as Western Blot, we found 2 bands at around 35-40 as well.

We are using his tag, histidine for protein extraction technique.
Optimization advice was also given by the Lotro team in several directions.
1. change the purification method method, sec anion cation purification.
2. check the sequence design
3. experimental operation problem
4. 2 bands around 35 may be lon protein breakage

In the end, after our follow-up attempts, we confirmed that there was no problem with the sequence design, and it might be the problem of purification due to our unskilled operation at the beginning of the experiment. The 2 bands could not be ruled out either, so it was probably due to some Lon protein breakage.

Meanwhile the Lotro team suggested us to perform lon protease expression by many different conditions to its finding excellent production conditions.

Following this suggestion, we carried out protein expression under 12 different conditions and finally also found the expression conditions with better results. And this result was fed back with the members of the Lotro team.

Several experts from Lotro praised our experimental results as fruitful and in line with the scientific spirit of a research team.

2.3 Future plans: increase the yield.

After successfully obtaining the rLon protein, we interviewed a company that produces human recombinant protease drugs in Shunyi District, Beijing, in order to have the opportunity to actually prepare this protein into a drug in the future, from which we hope to learn about the production and manufacturing process of protein drugs and to compare the differences between laboratory production and drug plant production.

2.3.1 Recombinant protease production pharmaceutical companies

The target of our interview was Edelweiss Pharmaceuticals, a subsidiary of the Yibai Pharmaceutical Group. Dr. Wang, the general manager of Aide Pharmaceuticals, and a drug development engineer were interviewed by us. Their company's main product is rteplase for injection: recombinant human tissue-type fibrinogen kinase (rt-PA), which is close to our other Lon protease selection to be produced, and we use this interview to understand some knowledge and considerations in the use and production of synthetic proteases.

Also for the fact that we have completed a small-scale production and purification in the laboratory, Dr. Wang believes that we have completed a small stage of the drug development phase, which is very rare. The next step is to validate the drug in in vitro cellular and animal experiments to verify efficacy and safety, and to consider how to proceed to batch production.