Engineering Objectives

Antigen heterogeneity in solid tumors describes the presence of multiple antigens at the tumor site with variable levels of expression. CAR T - cell therapy, reliant on antigen specific action, is therefore rendered largely ineffective when it encounters multiple antigens1. The properties of a solid tumor are maintained by the tumor microenvironment (TME), which comprises a colony of cancer cells. At this site, the cancer cells adhere to the vascular endothelial cells to form a sustainable base for the tumor, enabling metastasis. This adhesion is physically manifested in selectin proteins: E and P - selectin in particular2. The dismantling of this adhesive network can therefore facilitate enhanced T-cell action by negating solid tumor properties.

Figure 1 displays the engineering design flow, starting from the primary objective here described

Figure 1: A representation of the project engineering flow. Legend provided on the top left corner. Created using

Primary Target

  1. Specifications we looked at:

  2. Genetic upregulation (enhancement) of adhesive elements (E and P - Selectin)
  3. Candidates:
    • STAT3
    • NF-κB
    • TNF-α
Why STAT3 over other candidates?
  • ICAM and VCAM Apart from the selectin proteins, STAT3 also regulates the intercellular adhesion molecule (ICAM) - 1 and vascular adhesion molecule (VCAM) - 1 proteins, both crucial to the cell adhesive mechanism in solid tumors3.
  • Epithelial to Mesenchymal Transition (EMT): The EMT process allows a tumor to metastasize. The primary markers of EMT are N-cadherin and vimentin, whereas E-cadherin, an epithelial marker, is downregulated during the same4.
  • STAT3 is found to be a crucial gene in the regulation of EMT, as it increases the expression of EMT markers while down regulating E-cadherin and enhances the subsequent tumor metastasis5. Therefore, STAT3 inhibition, apart from diluting onsite defenses, also neutralizes the spread of cancer to some extent.

Figure 2: Genetic makeup of protein pathways for initiation of Epithelial-Mesenchymal Transition6. Created with BioRender
  • Cell apoptosis:
    STAT3 enhances the apoptosis inhibitors Bcl - x, Mcl - 1 and survivin, thereby supporting cancer survival. STAT3 inhibition therefore assists cancer apoptosis and not simply a reversion to the initial state7.

This unique positioning of STAT3 in the genetic pathways for tumor sustenance thus qualifies it as an excellent target for cancer therapy.

Targeting Agent

  1. Specifications we looked at:
  2. In our research, we focused on evaluating the STAT3 inhibitory activity of specific compounds. We aimed to achieve effective dispersion of these compounds, either through their concentrated self-directed action or by using a carrier to guide them. Tumor penetration was a critical consideration, and we prioritized small molecules for this purpose. It's worth noting that STAT1, another important signaling protein, shares similarities with STAT3 but plays a role in supporting tumor cell apoptosis8 . Consequently, any targeting agent we developed needed to be selective and not interfere with STAT1. To minimize interference from normal cell activity, we opted for compounds that are not natural derivatives. These considerations guided our scientific approach to developing effective and precise therapies. Early on in our project, we spoke to people from the pharmaceutical industry, who also talked about how targeting the STAT3 receptor is being looked at with great interest by companies developing CAR T-cell therapy.

  3. Candidates:
  4. One prominent candidate is identified:


  5. Engineering Advantages: Honokiol
  6. As described later, honokiol provides a viable option of delivery by virtue of its binding activity with CB2 and CB110. Owing to the advantages associated with the use of Cannabinoid receptors (as described ahead), honokiol is found to be the ideal targeting agent.

  7. Testing and Verification
  8. Honokiol treatment (with CAR T - cells): introduction of CAR T - cells alongside honokiol to a tumor patient was studied and modeled mathematically to obtain the requisite data for the following parameters:

    • Tumor cell mass
    • Time taken for action

    To effectively interpret the data, the following conditions were studied:

    • Natural tumor growth
    • Tumor growth after introduction of CAR T - Cells
    • Tumor growth after introduction of drug
    • Tumor growth after introduction of CAR T - cells and drug.

    Kindly refer to the Model page for detailed modeling process and results.

  9. Optimization
  10. To determine the adequate dosage of honokiol, based on the mathematical model, various parameters of the study were varied to obtain optimal results. Upon varying the initial concentration of honokiol, the best results are obtained for 25 µM concentration, with a 70% tumor dispersion observed (upon further increase, the numbers do not change significantly). For detailed results, please refer to the Model page.

Overall Treatment Design Requirements-

To achieve targeted tumor action in cancer therapy, it is of utmost importance to carefully design the delivery system for the treatment agent. Our approach is using receptor carriers, which can efficiently transport small molecules to tumor cells via specific molecular interactions. By employing receptor carriers, our small molecule drug can be effectively masked from normal cells, thereby minimizing off-target effects and reducing undesirable side effects associated with conventional cancer treatments.

Delivery Mechanism

In order to deliver a small molecule, a carrier must be developed.


  • Receptor: to bind to the small molecule
  • Vector:for transport to the tumor site
  • Anchorage:to link receptor to vector

Figure 3: The receptor based carrier system for our drug. Created using BioRender.


  • Specifications we looked at:
  • Receptors serve as critical components, as choosing a receptor which has specific binding sites which facilitate the binding of small molecules like honokiol is important. While honokiol is not naturally synthesized within the human body, it originates from external sources (plants). Achieving controlled release of these compounds often relies on the availability and strategic use of suitable receptor antagonists, which must have a higher binding affinity to the receptor in the case of a competitive inhibitor, or must be binding to allosteric site such that the conformation of the receptor changes such that the drug gets released at the target site.

  • Candidates:
  • Cannabinoid receptors 1 and 2 (CB1 and CB2) are determined to be ideal candidates through extensive literature review. .

  • Engineering Advantages:CB1
  • CB1 serves as the perfect receptor for our drug, as we had performed a virtual screen of sorts of potential receptors for our candidate drug. The binding scores are illustrated in the Docking Simulations section on our Model page and are also illustrated below.

  • Testing and Verification
    • Binding - CB1 binding with honokiol is studied in AutoDock, a software tool for binding studies. Figure 4 illustrates the binding simulation in PyMOL, a protein editing and visualization tool.
  • Optimization
  • Parameters:

    1. Binding Strength
    2. The free energy of binding describes the energy lost in the process of binding between a receptor and ligand. The greater the amount of energy lost, the more stable the binding. Highest binding strength was found to be -8.72 kcal/mol, followed by -8.54 kcal/mol.

    3. Binding Site
    4. The region of binding for the ligand is another crucial parameter for the binding. Honokiol was found to bind in an opening in CB1, thus forming a secure binding. The region of binding is highlighted in Figure 6.

    Figure 4: 3D-model of honokiol (magenta) binding to truncated CB1 protein. Created on PyMOL.
    1. Engineering Advantages: CB1 - modified
      1. Removal of redundant domains allows for more directed binding.
      2. Reduction of alternative binding sites for other biological agents, which may interfere with the system.
      3. Increase in the probability of competitive inhibition/release of honokiol by AM - 6538, due to restriction to a smaller space.
    2. Modification
    3. The excess domain in the CB1 sequence was removed using the delete feature on PyMOL. The domain was identified as the separate floating lobe opposite the free end (as against the binding end) of the primary group. The resultant is a coherent cylindrical structure functioning as the receptor, which is easier to engineer and fuse with other components.

    4. Testing and Verification
    5. Extraction (in vivo):

      The modified CB1 construct was obtained from GenScript in cloning plasmid pUC57 with the restriction site specified against the type II restriction enzyme EcoRV. The plasmid pET28a is determined to be suitable for transfer and expression of CB1. Both plasmids are digested with EcoRV. For both plasmids, 1µl of sample was digested against 1µl

      EcoRV in a 30µl reaction set for 4 hours. However, no bands were obtained for pET28a, while pUC57 was undigested. The amount of plasmid was then increased 8µl to increase the probability of digestion. This time, pET28a was digested and a proper band obtained, however, pUC57 was still undigested.

      The procedure was then repeated, with the reaction set for 8 hours. Negative results were obtained. The reaction was again repeated, now set for 12 hours, but to no avail. In consultation with the company, the restriction targets were modified to EcoR1 and HindIII. 1 µl each of both enzymes was used to digest 8 µl pET28a and 1ul pUC57. Both constructs were successfully digested. For detailed procedure, kindly refer to the Experiments page.

      Figure 5: Restriction Digestion with EcoR1 and HindIII; gel electrophoresis results shown. pUC57 (digested) shown in lane1. Lane 5 shows positive control. Upper band in lane1 (red) is empty pUC57, at 2710bp. Lower band in lane1 is CB1, at 1083bp.

Release Mechanism

To release the small molecule at the tumor site for effective penetration, a release trigger is required.

  1. Specifications we looked at:
  2. Once the drug is localized at the tumor site by the receptor system, it must be released in order to effectively penetrate the tumor microenvironment and inhibit STAT3. The key features of such a release mechanism are defined by the receptor and the primary drug. Furthermore, a release agent non-native to the human body is crucial so as to avoid accidental or uncontrolled release of the drug. We also talked to multiple experts about one of the major problems with chemotherapy - which is unwarranted side effects. To combat this, we thought of the following components:

    1. Antagonist: an antagonistic small molecule to competitively bind to the receptor and release the drug.
    2. Antagonist delivery:
      1. Time displaced action: the antagonist may be freely injected a specified time period after the introduction of the main vector, allowing the T-cell vectors to be localized at the tumor site.
      2. Nano-particle: A guided small molecule carrier with release at the tumor site A guided small molecule carrier with release at the tumor site
  3. Candidates:
    1. Antagonist: enzymatic
    2. Antagonist: AM - 6538 (small molecule)
    3. Antagonist Delivery:
      1. Timing mode of introduction
      2. Nano-particle
  4. Engineering Advantages
    1. AM - 6538
    2. AM - 6538 is a known cannabinoid antagonist with high affinity for CB1 receptors11. A structural analog of rimonabant, the small molecule is an ideal model for a competitive release agent against honokiol.

    3. Timing mode of delivery
    4. In order to ensure effective delivery and release of the drug, a time gap must be put between the administration of the primary drug and the release agent. This period allows the primary drug carrying vectors to accumulate at the tumor site and localize. When the release agent is introduced, it now only releases the primary drug at the tumor site, thus preventing out of site release. Furthermore, the drug release is almost simultaneous due to the prior accumulation, allowing for better concentration of the dosage.

  5. Testing and Verification
  6. Binding studies between CB1 and AM - 6538 were performed on Autodock and Molecular Dynamic Simulations to determine efficiency of binding. Kindly refer to the Docking section of our Model page for detailed results.


    1. Binding efficiency
    2. Binding sites


  • Binding energy of the antagonist must be higher than honokiol to CB1. Best scores for binding energy obtained from Autodock are -14.6 kcal/mol and -14.27 kcal/mol , found to be much better than honokiol at -8.72 kcal/mol and -8.54 kcal/mol.
  • Binding sites for high scores are found to be coincidental for honokiol and AM - 6538. Figure 6 demonstrates the binding regions for honokiol and AM - 6538.
  • Binding energy of the antagonist with a receptor-drug complex must be significantly high. Binding energy scores of -11.36 kcal/moland -10.35 kcal/mol are obtained on Autodock, indicating highly favorable competitive binding.

Figure 6: Comparative images of CB1 binding with 1. Honokiol and 2. AM - 6538. Only Specific binding regions are shown. Created on PyMOL.


  • Specifications we looked at:
  • A specific vector is required to deliver the drug precisely to the tumor site, while carrying an extracellular element (honokiol) on it. It should also express and sustain the receptor associated with the drug. Furthermore, the vector should localize at the tumor site to allow effective drug release.

  • Candidates:
  • T - cells from the patient.

  • Egineering Advantages: T - cells
  • T - cells provide natural specificity to the delivery mechanism as to the location of the tumor. This helps avoid out of site release while also reducing the time required for drug delivery. Furthermore, T - cells naturally localize at the tumor site, thus functioning as an anchor for drug release. T - cell accompaniment also ensures that immunological action initiates parallel to tumor dispersion, thereby increasing the potency of the treatment.


  1. Specifications we looked at:
  2. With the drug, receptor and vector determined, a final component is required to adhere them all together. As the small molecule is not naturally occurring and cannot be expressed by a cell, it must be carried by the cell externally. Therefore, the receptor must be expressed on the cell surface. Moreover, the receptor must be anchored to the cell surface. To this end, a fusion protein with cell surface expression is required.

  3. Candidates:
  4. Two prominent surface expression vehicles are identified: Ice Nucleation Protein N (INP - N)and Lipoprotein Outer membrane protein A (Lpp - OmpA)12.

  5. Engineering Advantages: INP - N
  6. The INP protein is a bacterial surface protein generally expressed in Gram negative bacteria. The general InaK protein comprises three major components: a hydrophobic N - domain, the Central repeated Domain (CRD) template and the hydrophilic C - domain. This functions as a well-defined surface expression vehicle13.

  7. Optimization
  8. The load of the carrier is further optimized to a truncated sequence INPN, developed by iGEM19_CAU_China team. It comprises an inbuilt anchoring domain (N - domain) to allow for surface expression, while an attached linker sequence allows it to fuse to the receptor (CB1). Furthermore, the sequence is very short, making it easy to obtain and engineer.


  1. Chen N, Li X, Chintala NK, Tano ZE, Adusumilli PS. Driving CARs on the uneven road of antigen heterogeneity in solid tumors. Curr Opin Immunol. 2018 Apr;51:103-110. doi: 10.1016/j.coi.2018.03.002. Epub 2018 Mar 16. PMID: 29554494; PMCID: PMC5943172. (Link)
  2. Ferreira IG, Carrascal M, Mineiro AG, Bugalho A, Borralho P, Silva Z, Dall'olio F, Videira PA. Carcinoembryonic antigen is a sialyl Lewis x/a carrier and an E‑selectin ligand in non‑small cell lung cancer. Int J Oncol. 2019 Nov;55(5):1033-1048. doi: 10.3892/ijo.2019.4886. Epub 2019 Sep 26. PMID: 31793656; PMCID: PMC6776192. (Link)
  3. Cho YS, Kim CH, Ha TS, Ahn HY. Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell. Can J Physiol Pharmacol. 2016 Nov;94(11):1220-1226. doi: 10.1139/cjpp-2015-0150. Epub 2015 Nov 18. PMID: 27681094. (Link)
  4. Serrano-Gomez, S.J., Maziveyi, M. & Alahari, S.K. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol Cancer 15, 18 (2016). (Link)
  5. Liu Z, Ma L, Sun Y, Yu W, Wang X. Targeting STAT3 signaling overcomes gefitinib resistance in non-small cell lung cancer. Cell Death Dis. 2021 May 31;12(6):561. doi: 10.1038/s41419-021-03844-z. PMID: 34059647; PMCID: PMC8166856. (Link)
  6. Lamouille, Samy, et al. “Molecular Mechanisms of Epithelial–Mesenchymal Transition.” Nature Reviews Molecular Cell Biology, vol. 15, no. 3, Mar. 2014, pp. 178–96. (Link)
  7. Mulu Geletu, Stephanie Guy, Rozanne Arulanandam, Hélène Feracci & Leda Raptis (2013) Engaged for survival, JAK-STAT, 2:4, DOI: (Link)
  8. Meissl, K., Macho-Maschler, S., Müller, M. and Strobl, B., 2017. The good and the bad faces of STAT1 in solid tumours. Cytokine, 89, pp.12-20. (Link)
  9. Pan J, Lee Y, Zhang Q, Xiong D, Wan TC, Wang Y, You M. Honokiol Decreases Lung Cancer Metastasis through Inhibition of the STAT3 Signaling Pathway. Cancer Prev Res (Phila). 2017 Feb;10(2):133-141. doi: 10.1158/1940-6207.CAPR-16-0129. Epub 2016 Nov 14. PMID: 27849557; PMCID: PMC6005650. (Link)
  10. Lin Y, Li Y, Zeng Y, Tian B, Qu X, Yuan Q and Song Y (2021) Pharmacology, Toxicity, Bioavailability, and Formulation of Magnolol: An Update. Front. Pharmacol. 12:632767. doi: 10.3389/fphar.2021.632767 (Link)
  11. Paronis CA, Chopda GR, Vemuri K, Zakarian AS, Makriyannis A, Bergman J. Long-Lasting In Vivo Effects of the Cannabinoid CB1 Antagonist AM6538. J Pharmacol Exp Ther. 2018 Mar;364(3):485-493. doi: 10.1124/jpet.117.245647. Epub 2018 Jan 8. PMID: 29311110; PMCID: PMC5803641. (Link)
  12. Earhart CF. Use of an Lpp-OmpA fusion vehicle for bacterial surface display. Methods Enzymol. 2000;326:506-16. doi: 10.1016/s0076-6879(00)26072-2. PMID: 11036660. (Link)
  13. Li Q, Yan Q, Chen J, He Y, Wang J, Zhang H, Yu Z, Li L. Molecular Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli. Int J Biol Sci 2012; 8(8):1097-1108. doi:10.7150/ijbs.4524. (Link)