Team:NJTech China/Design

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• TEAM
  Team Members Attributions Collaborations
• Human Practices
  Silver HP Integrated HP Education & Public Engagement
• PARTS
  Parts Overview
• PROJECT
  Description Design Model Results Experiments

• TAT
• MODEL
• VALIDATION

DESIGN

TAT (T cells and Tumors) DESIGN

Here is NJT0401.

In order to improve immunity in patients and reduce the probability of breast cancer recurrence and metastasis, we designed a product of a brand new fusion protein, which is called NJT0401. Its structure is shown here.

NJT0401 has three components: heavy chain-only antibody (HCAb), linker and human Interleukin-2 (hIL-2). Under the combined action of these three components, NJT0401 can effectively promote immunity and prevent cancer recurrence and metastasis.

Production of NJT0401

In our project, the optimized DNA sequence of NJT0401 is placed in frame with α-factor secretion signal sequence under GAP promoter. The recombinant plasmid is integrated into the genome of the Pichia pastoris GS115. The His tag is added in order to greatly reduce the amount of separation and purification work.

How does NJT0401 enhance immunity?

Based on the research results of Human Practices, we chose improving immunity of patients as the starting point for design of our project. After consulting a large amount of literature, we decided to use IL-2 to achieve this goal. After binding to the receptor complex, IL-2 can stimulate the proliferation of activated T cells, while CD8+ CTL cells have excellent effects in killing tumor cells. With reference to the information provided in NCBI and some related literature, we designed the human IL-2 sequence. In our sequence, the original signal peptide sequence of human IL-2 is removed, and the 125th Cys is mutated to Ala, which is beneficial to the structural stability of human IL-2.

How to further inhibit cancer recurrence and metastasis?

Inhibition of the immune checkpoints in the tumor microenvironment can further enhance the body‘s resistance to residual tumor cells. In recent years, PD-1/PD-L1 enjoys great popularity in immunotherapy. Considering that blocking PD-L1 is safer than blocking PD-1, we selected PD-L1 as our target to block the PD-1/PD-L1 immune checkpoint. After Human Practices activities, we chose to use heavy chain-only antibody. Compared to traditional antibodies, heavy chain-only antibody is characterized by their high efficiency and ease of expression in none-animal cell hosts. After reviewing the relevant literature, the human PD-L1 nanobody that had been sequenced was selected and ligated to the Fc sequence in which human IgG removed CH1.

Why we design a fusion protein?

The combination of PD-L1 heavy chain-only antibody and human IL-2 can effectively prolong the half-life of human IL-2, enhance the targeting ability of drugs, and achieve a function like double antibody. The attachment of human IL-2 to the Fc region of sdAb is effective in maintaining VHH activity. Through crystal structure model analysis, it is found that the N-terminally linked sdAb exposed to human IL-2 did not affect human IL-2 activity. This makes the design of a double-antibody-like drug possible. Under the expert's suggestion, we selected a flexible linker and a rigid linker to test the effect of fusion protein.

MODEL

What is our model?

Hydrogel Model：We establish Stepwise Regression Model and Drug Release Kinetics Model to Optimize the release effects of NJT0401 with hydrogels physical and chemical parameters regulated.

Structure Model: We establish Structural Model to verify whether the NJT0401 sequence we have identified can construct a complete protein structure, and whether the protein structure is feasible.

Why we build it?

Drug delivery constitutes an indispensable part of achieving drug efficacy. Our drugs are highly susceptible to inactivation and degradation in vivo and in vitro, which tends to result in low bioavailability. Advanced drug carriers and delivery technologies are the primary methods to improve the bioavailability of drugs, increase the efficacy of drugs, reduce their side effects and improve patient tolerance. Therefore, we choose temperature-sensitive hydrogels as drug carriers, which can be locally injected into the lesion to improve long-term sustained release rate. How to screen out hydrogel samples that optimize the release effects of NJT0401 is the meaning of our model.

How can we build it?

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. Then calculate the sum of partial-square regression (i.e. contribution) of total regression equation and the variables which have been introduced into the regression equation. When the total regression equation is not significant, which indicates the linear relationship of the multiple regression equation does not hold; and when an independent variable X has no significant effect on Y, it should be removed and re-established. A multiple regression equation containing the factors with significant influence are selected as independent variables. And the other variables in the equation are tested in order from small to large in the sum of partial-square regression. All variables which are not significant will be removed, and all factors that have significant effects will be retained. We thus establish an optimal regression equation, obtain the optimal interleukins under the optimal physical and chemical indicators of the hydrogels finally.

The Drug Release Kinetic Model is designed to describe the stabilization time of NJT0401 release progress to the optimal concentration range. We fit the experimental data with the curve to obtain a series of kinetic constant K values, then filter out hydrogel samples that meet the sustained release mechanism. Considering the lag period, we introduce the modified Peppas-Sahlin Model and similarly screen out the qualified hydrogel samples.

The Structural model is 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 method.

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Validation Design

In order to test the anti-tumor activity of NJT0401, we conduct a series of functional tests for its two ends functional analysis, both in vitro and in vivo.

1. To test the activity of the IL-2 moiety, T cell proliferation experiment is designed by comparison the proliferation efficiency of pure IL-2 and NJT0401.

2. We design a binding assay to evaluate fusion protein ability to specifically bind surface PD-L1 of tumor cells.

3. Given the short half-life of IL-2, we make a sustained-release preparation by loading it in hydrogel. By optimizing hydrogel physicochemical property, we design a drug release assay to analyze the release speed of bovine serum protein (BSA) with different crosslinking degrees hydrogel in vitro, and to explore whether hydrogels loaded with drugs can achieve the effect of slow release.

4. Previously the project is designed to evaluate anti-tumor effect and long-term immune memory effect of NJT0401 in vivo. However, due to the animal use permission, this part won’t be performed.

Validation of NJT0401:

1.Validation of NJT0401:

To determine proliferation effect of the IL-2 moiety of our fusion protein, we design T cell proliferation experiment in vitro and in vivo. In the in vitro proliferation experiment, we treat CD3+T cell with various concentration of fusion protein, IL-2 (positive control) and blank control, respectively. Next, we evaluate the proliferation effect by CCK8 assay and CFSE staining proliferation assay. As for in vivo experiment, we plan to inject hydrogel filling with NJT0401 into tumor bearing mice and then detect the proliferation by using a BrdU proliferation ELISA kit. However, without animal experiments permission, the in vivo experiments won’t be performed.

2. Specifically binding of NJT0401 and PD-L1 on tumor cells

To evaluate the ability of fusion protein to specifically bind to surface PD-L1 of tumor cells, we design a binding assay to analyze the activity of anti-PD-L1 in vitro and in vivo. For the in vitro binding assay, we group the MDA-MB-231 tumor cells into different treatment groups: NJT0401 groups (treated with different concentrations of NJ0401), anti-PDL1 group (treated with anti-PDL1) and control group (untreated cells). All groups were treated with fluorescent dyes, which bind to the antibody. To indicate the PDL1 binding ratio of the tumor surface, the fluorescence intensities of each group are detected by flow cytometry subsequent to incubation. For in vivo binding experiment, we plan to inject fusion protein into tumor bearing mice and then repeat the procedure indicated in the in vitro binding assay. Again, we won’t perform the in vivo experiment without permission.

3. Validation of hydrogel

We design the drug release assay to analyze the release of bovine serum protein (BSA) by hydrogels with different crosslinking degrees in vitro environment, and to explore whether hydrogels loaded with drugs can achieve the effect of slow release. We immerse BSA-loaded hydrogels in 80 mL PBS and place them in a circular mold, which occasionally shaking at 37 °C. We take out the release medium (0.2 mL) and replace with an equal volume of PBS every 4 hours. We finally determine the concentration of BSA by using a BCA assay kit.

4. Anti-tumor effect and long-term immune memory effect of fusion protein

Moreover, we plan to explore the anti-tumor effect and long-term immune memory effect of fusion protein stimulating T cell proliferation. In the long-term immune memory experiment, we plan to treat tumor bearing mice with PBS, fusion protein, γ-Glu+ fusion protein, γ-Glu and then the primary tumors are surgically resected on day 28. After the mice recovered, they are re-challenged with the same tumor cells in the opposite flank. At the end of the experiments, the primary and distant tumors are imaged by IVIS Spectrum.

References

[1] Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65, 1357-1369, doi:10.1016/j.addr.2012.09.039 (2013).

[2] Zhang, F. et al. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade. Cell Discov 3, 17004, doi:10.1038/celldisc.2017.4 (2017).

[3] Liao, W., Lin, J. X. & Leonard, W. J. IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol 23, 598-604, doi:10.1016/j.coi.2011.08.003 (2011).

[4] Sun, X. et al. PD-L1 Nanobody Competitively Inhibits the Formation of the PD-1/PD-L1 Complex: Comparative Molecular Dynamics Simulations. Int J Mol Sci 19, doi:10.3390/ijms19071984 (2018).

[5] Andre, M. C. et al. Impaired tumor rejection by memory CD8 T cells in mice with NKG2D dysfunction. Int J Cancer 131, 1601-1610, doi:10.1002/ijc.26191 (2012).

[6] Verma, V. et al. PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nat Immunol 20, 1231-1243, doi:10.1038/s41590-019-0441-y (2019).

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[8] Knudson, K. M. et al. M7824, a novel bifunctional anti-PD-L1/TGFbeta Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine. Oncoimmunology 7, e1426519, doi:10.1080/2162402X.2018.1426519 (2018).

[9] Ma, X. et al. Injectable hydrogels based on the hyaluronic acid and poly (gamma-glutamic acid) for controlled protein delivery. Carbohydr Polym 179, 100-109, doi:10.1016/j.carbpol.2017.09.071 (2018).

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