Team:AFCM-Egypt/Description

Project Description

In Egypt, breast cancer is the most prevalent malignancy and leading cause of cancer death among women around the world. According to Globocan 2018, the number of new cancer cases is 128892 which represents a percentage of 0.1297% of the total population. In both sexes and all ages, the number of new breast cancer cases is 23081 with a percentage of (17.9%) of total cancer cases as it lies in the second rank of the most incident cancers in Egypt after liver cancer as shown in Graph (1). The number of new cases among females is 23081 with a percentage of 35.1% of total cancers among females as illustrated in Graph (2).(1),(3)

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR) and HER2/neu.(2). To get to know all the most common neoantigens related to TNBC in Egypt, we started by Dataset for all Cancer-Associated Antigens (TAAs). Neoantigens related to breast cancer were then selected and filtered to obtain a new dataset of neoantigens expressed specifically in TNBC which were then compared to the Egyptian population.

DNA Vaccination is a direct insertion of a plasmid containing a DNA sequence encoding the antigen(s) to which the immune response is obtained and depending on the in-situ generation of the target antigen into the appropriate tissues. This approach provides many possible advantages compared with conventional approaches, including activation of both B- and T-cell responses, increased vaccine safety, absence of any infectious agent and relative ease of manufacturing on a wide scale. That is why the team is concerned with innovating a multi-epitope DNA vaccine for TNBC. (4),(5),(6)

Notice: parts in our range for this season have been created as a part of our Phase I design of our project. These parts HAVE NOT been tested or characterized in the lab due to COVID-19-related precautionary measures. We have enriched our new parts pages with data from literature and results from our modeling and simulations. If you are intending on using this part or others in our range, please keep in mind these limitations and update these parts with data from your experimentation. Feel free to reach us at: igem.afcm@gmail.com for further inquiries.

Rationale

Immunotherapy is a promising alternative for traditional chemotherapies and treatment options for triple-negative breast cancer.(28) Continuous Ongoing studies and clinical trials may show more evidence for the effectiveness of Immunotherapeutic approaches. Immunotherapy can be very expensive we are talking about costs that may reach over than $100,000 per year is frequently secured by health insurance, but patients still need to deal with Surgery and other cancer medications which add more to the financial predicament of rising out-of-pocket costs.(23)

Reason behind choosing Breast cancer, and why TNBC in particular?

Breast cancer is not a new-born global health crisis. It has been a looming issue in the medical field for years, encouraging the researchers to go further with their studies in developing de-novo strategies. In 2018, there were an evaluated 2.1 million new cases of breast cancer around the world. Within the United States alone one life is lost each 15 minutes due to Breast cancer. CRI (Cancer Research Institute) anticipated that there will be 279,000 cases of breast cancer diagnosed in 2020 along with 42,000 deaths(22). This medical issue became more imminent in our country. Which was observed through assessing the incidence ratio and the survival outcomes through a study conducted on 520 Breast cancer patients treated between (2010-2011) in ASU as a single institute experience in Egypt. Among those 520 cases, 139 were diagnosed as TNBC(26).

Among breast cancer subtypes, TNBC has characteristics that may make it more responsive to treatment with immunotherapy, including:

  1. TNBC tends to show higher frequency of immunogenic mutations than HER2-positive Breast cancer.
  2. TNBC also exhibits higher tumour infiltrating lymphocytes (TILs) which is described as a potential biomarker of immunotherapy response(31).

Traditional Chemotherapy and other treatment options:

For years cytotoxic chemotherapy had been the only available systemic approach, beside the radiotherapy, invasive surgical interventions and mastectomy in managing Breast cancer.

Standard cancer chemotherapy deals with Tumours in two major ways:

  1. Inducing Tumor cell death either by autophagy or necroptosis with simultaneous unleashing of (TAAs) and (DAMP)
  2. disrupting cancer immune evasion techniques(27).

Our Aims and Goals

Our new Hope: A quick overview on Immunotherapy and its impact in TNBC

Cancer Immunotherapy, AKA ‘’biotherapy’’, offers a biological response modifier system for Breast cancer, including: (18), (19), (20), (24)

Figure: shows the different Anti-Cancer immunotherapeutic approaches .

The main aim of developing antitumor vaccination methods is to enhance the immune surveillance system of the body, by boosting the body's natural defence mechanisms achieved through the production of highly-efficient long lasting Memory cells to attenuate the tumour progression and avoid its recurrence. However, suppose we were able to create this’’immune memory’’, there is still a formidable obstacle that we will be facing to maintain and ensure the survival of these memory B and T-Cells and guarantee long-term cancer remission(21),(30).

The aforementioned issue is not the only concern we need to tackle through our journey in developing TNBC Vaccines, but in addition there are other aspects we have to be aware of. These quandaries due to complexity of tumor-host interactions are summarized as follows:

  1. Considering the source and proper choice of antigenic epitopes as well as the vector for their delivery method.
  2. Be that as it may, among all the advantages of immunotherapies regarding availability, relatively low cost, less side effects and utilization without any HLA restrictions but they require adequate monitoring of immune responses to the vaccine where these epitopes would have to be processed by APCs and DCs, which may be dysfunctional in patients with advanced stage of malignancy.(29)
  3. Enhancement of adequate T- lymphocyte infiltration that overlaps the immunosuppressive tumour environment(25)
Figure: Shows our pipeline for modular design of our DNA-launched RNA Replicons (DREP).

Dataset gathering

TNBC Patients' DNA sequences were retrieved from NCBI database consequently, all the candidate epitopes were analysed using Custommune analysis tools then filtered and Screened for highly ranked recognition score by multiple HLA alleles. Eventually, we Perform docking (DFIRE score) of the HLA-epitope binding to provide a final antibody epitope prediction. (7)

Figure: shows the sequential steps for Neoantigens gathering from Datasets and Literature and how we compared them with the Egyptian population.

DNA vaccine circuit

Multi-epitope DNA vaccine to combat the TNBC is designed using immunoinformatics approach, through the fusion of multiple dendritic cell epitopes presenting them to CD4 (+) T cells that once become activated will produce cytokines and can help activate antibody producing B cells, therefore Dendritic cells sequester antigenic epitopes acting as a bridge between innate and adaptive immunity, helper, cytotoxic and B-cell epitopes and adjuvant.(8),(9),(10). These crucial components of the newly constructed TNBC DNA vaccine were conjugated using proper Linkers as simulated in fig (1). This vaccine was subsequently tested for different physicochemical characteristics and examined by molecular dynamics simulation tools for structure refinement, safety insurance, efficacy, and Validity.

Figure: Showing the structure of our DNA-launched RNA Replicons Vaccine Versions.

Methodology Guidelines

This year, our team is creating novel DNA vaccination platform aims as an enhanced immunotherapeutic approach targeting (TNBC) using a combination of neatly selected multiple neo-epitopes conjugated using proper linkers that were optimized according to certain linker selection parameters constructing a SynBio design that relies on natural self-replicating viral abilities of RNA replicons delivered in logically optimized DNA circuits, therefore creating a DNA-launched RNA Replicons (DREP).(13)

The platform utilizes the inherent self-amplification ability of RNA-replicons to ensure enhanced neo-antigen uptake and presentation leading to the mounting of efficient cellular and humoral immune responses against TNBC. Embedded with optimized subgenomic regulation and linker-peptides alongside glycine-alanine repeats (GAr), a miRNA-based immune evasion mechanism and an OFF-switch, our platform is predicted to be highly safe and efficient through experimentally-driven mathematical simulations.(14),(15).

Immune-modulating adjuvant (Beta-Defensin), Pan HLA-DR epitopes (PADRE) and Heat Shock protein (HSP) sequence were also added with epitopes sequence to enhance the immunogenicity. All the epitopes, adjuvants and PADRE sequence were joined by linkers. In the construction of multiepitope vaccines EAAK linker is found to increase stability and reduce connection with other protein areas with efficient detachment. There is a possibility that the immunogenicity could increase with an adjuvant. Based on the interactions’ compatibility HTL, CTL and B-Cell epitopes were merged together sequentially with GPGPG, AAY and KK linkers, respectively. (16)

Mathematical modelling-based simulation proofed potential therapeutic immunity of our approach. Eventually, our vaccine will be sequestered by DCs to present the hotspots of epitopes predicted by Custommune to CTLs and T-Helper cells that once become activated, will produce cytokines and activate antibody producing B cells, thus acting as a bridge between innate and adaptive immune response to combat TNBC. (17) Using various sets of computational validation tools, we were able to assess the immunogenicity of our final proposed Vaccine models.

Conclusion

Narrowing the scope to choose the frequent neoepitopes in Egyptian patients with TNBC shows that there is no need for the traditional methods of whole exome sequencing to save money and time. In addition to the advantages in using them in Multi-epitope DNA-replicon based vaccine which has a significant therapeutic role in stimulating the immune response to the neoplastic cells and linking between innate and adaptive immune system through presenting the antigen on DCs surface which activates the adaptive immune system especially CTLs.

Literature Cited

  1. World Health Organization. Global Health Observatory.
    Geneva: World Health Organization; 2018. who.int/gho/database/en/. Accessed June 21, 2018.

  2. Uscanga-Perales, G., Santuario-Facio, S., & Ortiz-López, R. (2016, October 19). Triple negative breast cancer: Deciphering the biology and heterogeneity. Retrieved October 25, 2020, from https://www.sciencedirect.com/science/article/pii/S1665579616300667

  3. Ferlay J, Colombet M, Soerjomataram Iet al. Global and Regional Estimates of the Incidence and Mortality for 38 Cancers: GLOBOCAN 2018. Lyon: International Agency for Research on Cancer/World Health Organization; 2018.

  4. Thun MJ, Henley SJ, Travis WD. Lung cancer. In: Thun MJ, Linet MS, Cerhan JR, Haiman CA, Schottenfeld D, eds. Cancer Epidemiology and Prevention. 4th ed. New York, NY: Oxford University Press; 2018:519‐542.

  5. Garmory HS, Brown KA, Titball RW. DNA vaccines: improving expression of antigens. Genetic vaccines and therapy. 2003 Dec 1;1(1):2.

  6. Hobernik, Dominika, and Matthias Bros. “DNA Vaccines-How Far From Clinical Use?.” International journal of molecular sciencesvol.19,11 3605. 15 Nov. 2018, doi:10.3390/ijms19113605

  7. Tarek, M. (2020, January 1). Custommune: a web tool to design personalized and population-targeted vaccine epitopes. MedRxiv.
    https://www.medrxiv.org/content/10.1101/2020.04.25.20079426v1

  8. Nezafat, Navid, et al. "Designing an efficient multi-epitope oral vaccine against Helicobacter pylori using immunoinformatics and structural vaccinology approaches." Molecular BioSystems 13.4 (2017): 699-713.

  9. Parvizpour, Sepideh et al. “In silico design of a triple-negative breast cancer vaccine by targeting cancer testis antigens.” BioImpacts : BI vol. 9,1 (2019): 45-56. doi:10.15171/bi.2019.06

  10. Amorim, K., Rampazo, E., Antonialli, R. et al. The presence of T cell epitopes is important for induction of antibody responses against antigens directed to DEC205+ dendritic cells. Sci Rep 6, 39250 (2016). https://doi.org/10.1038/srep39250

  11. Dar, Hamza Arshad, et al. "Immunoinformatics-Aided Design and Evaluation of a Potential Multi-Epitope Vaccine against Klebsiella Pneumoniae." Vaccines 7.3 (2019): 88.

  12. Tang, Mi, et al. "Antitumor efficacy of the Runx2‑dendritic cell vaccine in triple‑negative breast cancer in vitro." Oncology Letters 16.3 (2018): 2813-2822.

  13. DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost. (2020, October 25). PubMed Central(PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102224/

  14. DiAndreth, B. (2019, January 1). PERSIST: A programmable RNA regulation platform using CRISPR endoRNases. BioRxiv. https://www.biorxiv.org/content/10.1101/2019.12.15.867150v1

  15. Ossevoort, M. (2003, October 20). Creation of immune ‘stealth’ genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1.GeneTherapy. https://www.nature.com/articles/3302098?error=cookies_not_supported&code=bb5e8128-3eea-4a9f-aa12-6ffffd0ab55a

  16. Multiepitope-Based Subunit Vaccine Design and Evaluation against Respiratory Syncytial Virus Using Reverse Vaccinology Approach. (2020, June 1). PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350008/

  17. Castiglione, F., & Bernaschi, M. (2004, April 30). C-ImmSim∗ : Playing with the immune response. Retrieved October 26, 2020, from https://www.math.ucsd.edu/~helton/MTNSHISTORY/CONTENTS/2004LEUVEN/CDROM/papers/316.pdf

  18. C. Denkert, C., P. Narang, M., J. Budczies, M., S. Luen, B., RM. Samstein, C., Aaron M.. Goodman, S., . . . S. Terry, P. (1970, January 01). Immunotherapy in Triple-Negative Breast Cancer: Present and Future. Retrieved October 26, 2020, from https://link.springer.com/article/10.1007/s12609-019-00345-z

  19. KUROKI1⇓, M., MIYAMOTO2, S., MORISAKI3, T., YOTSUMOTO1, F., SHIRASU1, N., & And, Y. (2012, June 01). MASAHIDE KUROKI. Retrieved October 26, 2020, from http://ar.iiarjournals.org/content/32/6/2229.full

  20. Liao, S., & Oldham, R. (2018, January 22). Immunotherapy of cancer is a part of biotherapy. Retrieved October 26, 2020, from https://jcmtjournal.com/article/view/2364

  21. Biotherapy/Immunotherapy. (2019, July 09). Retrieved October 26, 2020, from https://www.wmcc.org/biotherapy/

  22. Immunotherapy for Breast Cancer. (n.d.). Retrieved October 2020,from https://www.cancerresearch.org/immunotherapy/cancer-types/breast-cancer

  23. https://www.healthline.com/health-news/value-and-cost-of-immunotherapy#The-potential-of-immunotherapy-

  24. Sanmamed1, M., & Chen1, L. (n.d.). A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization. Retrieved October 26, 2020, from https://www.cell.com/cell/pdf/S0092-8674(18)31247-9.pdf

  25. García-Aranda, M., & Redondo, M. (2019, November 20). Immunotherapy: A Challenge of Breast Cancer Treatment. Retrieved October 26, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966503/

  26. Jomaa, M. K., & Nagy, A. A. (n.d.). Survival outcomes in Egyptian patients with triple-negative breast cancer: Single institute experience. Retrieved October 26, 2020, from https://ascopubs.org/doi/abs/10.1200/jco.2015.33.28_suppl.158

  27. Emens, L., & Middleton, G. (2015, May). The interplay of immunotherapy and chemotherapy: Harnessing potential synergies. Retrieved October 26, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012642/

  28. García-Aranda, M., & Redondo, M. (2019, November 20). Immunotherapy: A Challenge of Breast Cancer Treatment. Retrieved October 26, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966503/

  29. DeLeo, A., & Whiteside, T. (2008, September). Development of multi-epitope vaccines targeting wild-type sequence p53 peptides. Retrieved October 26, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721363/

  30. Kirman, J., Quinn, K., & Seder, R. (2019, July 08). Immunological memory. Retrieved October 26, 2020, from https://onlinelibrary.wiley.com/doi/full/10.1111/imcb.12280

  31. C. Denkert, C., P. Narang, M., J. Budczies, M., S. Luen, B., RM. Samstein, C., Aaron M.. Goodman, S., . . . S. Terry, P. (1970, January 01). Immunotherapy in Triple-Negative Breast Cancer: Present and Future. Retrieved October 26, 2020, from https://link.springer.com/article/10.1007/s12609-019-00345-z

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