Team:NJTech China/Description
Inspiration
Breastfeeding is the most important reason why all mammals can thrive from generation to generation. The breast, as a unique feature of women, is one of the primary bonds between the mother and the child, which portrays the curves and beauty of women and reflects the greatness of them as mothers. Sadly, the emergence of breast cancer has tarnished this gloomy flower. Breast cancer is the malignant tumor owning the highest incidence and mortality among female diseases worldwide. Accounting for 30% of all malignant tumors and as the rate increasing at a rate of 2% per year, breast cancer causes serious harm to women's life and health. In recent years, the incidence of breast cancer in women has shown a trend of rejuvenation, and even most patients are younger than 35 years old. With such a severe situation, we hope to contribute our own strength to the treatment of breast cancer. During discussion with the doctors in the Gynaecology department, they introduced the current clinical treatment of breast cancer to us. In particular, they all mentioned the current state for triple-negative breast cancer which is lack of targeted drugs. After effective communication with other doctors, we decided to design an effective treatment for triple-negative breast cancer. Based on all of the above, we decide to conduct brave exploration as the first movers.
DESCRIPTION
GENETIC
Pichia pastoris has been recognized as one of the most important hosts for heterologous protein production, and over 1,000 proteins have been expressed using this yeast. Unlike Escherichia coli or Saccharomyces cerevisiae, P. pastorishas an efficient secretion system which is suitable for heterologous expression. Specifically, the P. pastoris expression system is convenient for the expression of eukaryotic foreign proteins because of its potential for post-translational modifications and protein folding. Compared with AOX promoter, the pGAP (glyceraldehyde-3-phosphate dehydrogenase) avoids the use of methanol and a shift from the biomass growth phase to the methanol induction phase, which is favored in continuous fermentation processes. Thus, it is featured by a high-level constitutive expression and is more suitable for continuous cultivation due to simpler process controls.
Interleukin-2 (IL-2) is a pleiotropic cytokine, and involves in immune regulation by controlling the differentiation and homeostasis of both Pro-inflammatory and anti-inflammatory T cells. PD-L1 is a programmed death ligand that is highly expressed on the surface of tumor cells. It appears that upregulation of PD-L1 may allow cancers to evade the host immune system. Use of an inhibitor that blocks the interaction of PD-L1 with the PD-1 receptor can prevent the cancer from evading the immune system. Our product targets both IL-2 and PD-L1, implementing a trap between Tumor and T cells.
In our project, NJT0401 is composed of optimized IL-2 and anti PD-L1 is placed in frame with α-factor secretion signal sequence under GAP promoter. The recombinant plasmid pGAP-IL-2-anti PD-L1 is integrated into the genome of the P. pastoris. Considering codon usage bias, optimization of the codons of P. pastoris is imperative to achieve high levels of expression.
MODEL
For the Hydrogel Model , we establish Stepwise Regression Model and Drug Release Kinetics Model to Optimize the release effects of NJT0401 with hydrogels physical and chemical paraments regulated. The Stepwise Regression Model is to establish the total regression equation of the physical and chemical properties of the hydrogels as the independent variable X and optimal effect of the interleukins Y. The Drug Release Kinetic Model is designed to describe the stabilization time of NJT0401 release progress to the optimal concentration range.
For the Homology Model, we establish Structural model to verify whether the NJT0401 sequence we have identified can construct a complete protein structure, we computerized the protein structure of the sequence. The sequence composition is divided into Nanobody region, linker region and IL-2 region, wherein the Nanobody region is composed of VHH region and Fc region. The linker adopts rigid and flexible methods to analyze the rationality of the two connection methods through simulation.
VALIDATION
Here, we utilized a bifunctional fusion protein designed to block PD-L1 and activate T cells proliferation in the tumor microenvironment. To evaluate the anti-tumor activity of NJT0401, we employ a series of functional study for its two ends functional analysis, both in vitro and in vivo. In this study, we evaluate the mechanisms of action and anti-tumor efficacy of NJT0401 in combination with hydrogel and in murine tumor models (a perspective).