Professor Mrs. Eleftheria Rosmaraki and Associate Professor Mr. Ilias Kazanis

-It is challenging to convert somatic cells into induced Pluripotent Stem Cells (iPSCs) and differentiate them into Natural Killer cells (NK cells) within the competition's timeframe.

On February 12th, we met with Mrs. Eleftheria Rosmaraki, Professor in the Department of Genetics, Cell Biology and Development at the University of Patras, and Mr. Ilias Kazanis, Associate Professor of Developmental Biology in the Department of Biology at the University of Patras. They assisted us in conducting our scientific research by serving as our team's Principal Investigators (PIs). During our initial meeting with our team and the project presentation, they pointed out the time-consuming process of genetic modification of somatic cells into induced Pluripotent Stem Cells (iPSCs), the differentiation of iPSCs into Natural Killer cells (NK cells), and the challenges we would have to overcome. Moreover, they informed us that it is not possible to carry out these genetic modifications within the timeframe of the competition. Additionally, they emphasized the importance of finding innovative aspects in our project, since it is crucial to advance our therapy even in the preclinical stage and highlight the significance of innovation in research. Therefore, we decided to skip the above steps and focus on constructing the CAR receptor on NK cells and ensuring the proper function of CAR-NK cells against pancreatic cancer cell lines.

Molecular Biologist and Researcher Mr. Konstantinos Kabas

- It is necessary to find specialized and experienced scientists for laboratory experiments and for clinical feedback.

On February 18th, we contacted Mr. Konstantinos Kabas, a molecular biologist and researcher affiliated with the Pasteur Institute. During our initial conversation, Mr. Kabas underlined the importance of memory cellular elements, such as NK cells, which contribute to establishing enduring cancer memory within the body. Simultaneously, he emphasized that adherence to a well-structured laboratory protocol for our project necessitates the presence of proficient individuals who can expedite laboratory procedures, thereby optimizing time efficiency. Concerning the selection of the most appropriate antigen featuring superior antibody specificity and effectiveness, Mr. Kabas advised us to implement a comprehensive screening procedure. This procedure entails systematically testing all the selected antigens aligning with the specific types of immune responses under consideration. So, through this meeting, Mr. Kambas emphasized the importance of finding experienced instructors for conducting lab experiments within the timeline and advised us on selecting the appropriate antigen, according to the screening procedure.

Οncologist, Professor Dr. Aristotelis Bamias

-You should focus on adenocarcinoma and be aware of the problem that may occur with the histocompatibility of CAR-NK cell therapy.

On March 3rd , we communicated with Dr. Aristotelis Bamias, an Οncologist and Professor of Therapeutic Pathology-Oncology at the National Kapodistrian University of Athens, and also, Director of the Second Educational Pathology Clinic at the Attica University Hospital. In particular, Mr. Bamias encouraged us to focus our therapy approach on adenocarcinoma of the pancreas (PDAC), as it concerns most cases of pancreatic cancer. On the contrary, he explained that in the other type of pancreatic cancer, which includes neuroendocrine tumors, the healthy function of the pancreas is not significantly affected, and the symptoms remain the same as in adenocarcinoma. In addition, Mr. Bamias discussed some primary symptoms of pancreatic cancer. Regarding jaundice, the tumor appears on the top part of the pancreas, whereas in the case of diabetes, the tumor appears in the lower part of the pancreas. At the same time, he pointed out another main symptom of pancreatic adenocarcinoma: intense back pain due to the retroperitoneal position of the pancreas. He then shared his concern about whether allogeneic CAR-NK cells are compatible with the patient's body, as there is the risk of rejection. He also stressed that we should investigate the application of immunotherapy in the case immunosuppression occurs via T-lymphocytes in the tumor microenvironment. Lastly, Mr. Bamias told us there are no effective immunotherapies for treating pancreatic cancer. Thus, he urged us to continue our research to find a therapeutic approach with satisfactory therapeutic results. Hence, the most crucial thing was that, under his guidance, we redirected our research towards only adenocarcinoma, primarily focusing on its morphology.

Oncologist-Pathologist Dr. Nikolaos Tsoukalas

- There are many challenges in the timely detection of pancreatic cancer stemming from nonspecific symptoms, the restricted range of endorsed therapies and the complexity of applying immunotherapy.

On March 10th, we had the honor of communicating with Dr. Nikolaos Tsoukalas, an esteemed Oncologist-Pathologist currently serving as the Deputy Director of the Oncology Department at Athens 401 GNA Hospital. Dr. Tsoukalas has also held the honorary position of Editor at the Henry Dunant Hospital Center in Athens since 2020. During our meeting with him, we embarked on an insightful discussion regarding the diverse subtypes of pancreatic cancer. These subtypes are notably differentiated by the origin of tumors within the pancreas, encompassing both the exocrine and endocrine functions. The endocrine segment of the pancreas plays a pivotal role in food metabolic processes, whereas the exocrine portion primarily contributes to digestion.

We delved into an analysis of the clinical distinctions between these two subtypes of pancreatic cancer, noting that endocrine tumors tend to exhibit a more gradual progression and manifest fewer metastasis instances. It was also mentioned that glucagon contributes to hypoglycemia, while insulin induces hyperglycemia. On the contrary, tumors originating in the exocrine pancreas present a severe clinical outlook due to their rapid growth, often leading to diagnoses occurring when metastases are already extensive in an advanced stage. Dr. Tsoukalas proceeded to elucidate primary symptoms indicative of pancreatic cancer, encompassing manifestations such as jaundice, diabetes and unexplained weight loss. Another aspect emphasized during our discussion was the noteworthy biological correlation between pancreatic cancer and severe depressive states experienced by patients. At the same time, we talked about another crucial symptom associated with pancreatic cancer: thrombosis, which is frequently underestimated, resulting in late-stage diagnoses and contributing to patient mortality. Further characteristic symptoms indicative of pancreatic cancer adenocarcinoma were also expounded upon, including benign jaundice, alterations in fecal coloration and urinary hyperpigmentation. In addition, Dr. Tsoukalas shared a noteworthy observation regarding the evolving demographic characteristics of pancreatic cancer. Specifically, he highlighted a decreasing average age range for the occurrence of pancreatic cancer, with cases now manifesting in individuals aged 40-45 up to 60-65. A plausible explanation for this is the evolution of scientific understanding and advancements in diagnostic technology that have facilitated earlier disease detection.

The conversation subsequently shifted towards exploring environmental factors that elevate the risk of developing pancreatic cancer. Such factors encompass dietary choices, heightened stress levels, excessive alcohol consumption and tobacco use. Additionally, inflammation of the pancreas, such as pancreatitis, was identified as a contributing factor. Notably, in the case of the endocrine function of the pancreas, certain genetically inherited syndromes (MEN1, MEN2) were noted to heighten the likelihood of neuroendocrine tumors (NETs).

Dr. Tsoukalas proceeded to provide valuable insights into the existing treatments for pancreatic cancer. First, he mentioned surgery as a therapeutic option, although dealing with metastases can complicate the process. Another treatment approach could be adjuvant therapy, such as chemotherapy or radiation. He also discussed utilizing specialized drugs tailored for Neuroendocrine Tumors (NETs) designed to target somatostatin. Lastly, he mentioned that medications aiming to reduce angiogenesis may be applied, noting the challenges associated with drug delivery due to pancreatic fibrosis and inflammation.

Subsequently, Dr. Tsoukalas shed light on the application of immunotherapy in the context of pancreatic cancer, elucidating its limitations and potential avenues for improvement. Immunotherapy is administered to patients with pancreatic cancer who exhibit specific mutations (MSI-H), demonstrating moderate effectiveness in such cases. An essential consideration in enhancing immunotherapy revolves around evaluating the tumor's immunogenicity. Furthermore, we must pay attention to facilitating medication access within the pancreatic microenvironment. In essence, reduced immunogenicity and the unique microenvironment of pancreatic tumors present significant challenges for the effectiveness of immunotherapy. Enzymes such as hyaluronidase and anticoagulants like heparins render immunotherapies more efficacious, while reducing the likelihood of thrombosis and enhancing patients' prospects for survival. Thus, through this discussion, we have validated the research we had conducted regarding the background of pancreatic cancer, its microenvironment and in general the application of immunotherapy in cancer treatment.

Surgeon, Professor Dr. Antonis Vezakis

-Pancreatic cancer has a very poor prognosis and thus, most of the pancreatic tumors are inoperable.

On March 14th, we had the pleasure of meeting with Dr. Antonis Vezakis, a Surgeon and Professor of Surgery at the National Kapodistrian University of Athens, who elaborated on scientifically-based information on pancreatic cancer. The meeting took place at the ARETEION Hospital of Athens. Initially, Mr. Vezakis talked about the general description of the disease. In particular, he assured us about the disease's poor prognosis, which limits the five-year survival rate to about 12% in all cases. This is because the symptoms of the disease are non-specific and thus, it is difficult to diagnose at an early stage. It is, also, very common for most of the patients to be diagnosed with metastases, because pancreatic cancer is found in an advanced stage and has already affected the neighboring organs. For this reason, approximately 80% of pancreatic tumors are inoperable. Also, Mr. Vezakis informed us that the average age of pancreatic adenocarcinoma occurrence is 70 years old, and when a patient is diagnosed with pancreatic cancer, the surgeon must determine the stage of the disease through MRI or CT. Furthermore, we talked with him about the surgical interventions for pancreatic cancer and their challenges. More specifically, there are three types of interventions, which are whipple surgery (the tumor is located in the head of the pancreas), partial/peripheral pancreatoduodenectomy (the tumor is found in the body or tail of the pancreas) and total pancreatectomy (depending on the location of the tumor). So, through this discussion, we understood that our therapeutic approach is innovative, unconventional and it could be used as a solution for all those cases where surgery can not be applied.

Professor of Radiation Oncology and Radiobiology Dr.Dimitrios Kardamakis

- here are limitations in radiotherapy in pancreatic cancer treatment and the research for new therapeutic approaches is a necessity.

On March 22th, we had the opportunity to communicate with Dr. Dimitrios Kardamakis, Professor of Radiation Oncology and Radiobiology at the Medical School of the University of Patras, with whom we discussed the practical aspects of radiotherapy in cancer patients, as well as the limitations, application, and results in the case of pancreatic adenocarcinoma. Initially, we discussed how radiotherapy serves as one of the most common treatment modalities in oncology. Thus, we were able to comprehend the goals of radiotherapy in the case of oncology patients. Ionizing radiation emitted by radiotherapy devices (linear accelerators) induces cell apoptosis, both indirectly (via hydrolysis and the production of free radicals) and directly (on cellular structures such as DNA and mitochondria). Mr. Kardamakis talked about radiotherapy's objectives: tumor eradication, tumor regression, and symptom relief (palliative action). Moreover, he highlighted that 60% of oncology patients can undergo radiotherapy during the development of the disease and that radiotherapy can be applied during all stages of the disease. However, some restrictions exist on applying radiotherapy in certain oncology patients. Such critical contraindications include patients with myelosuppression (low levels of hemoglobin, white blood cells, and platelets) and patients with severe autoimmune diseases, such as scleroderma. Subsequently, Mr. Kardamakis reported that radiotherapy is usually performed as a complementary treatment in combination with surgery. Also, it can be applied as a neoadjuvant therapy (e.g. rectal cancer) or adjuvant therapy (e.g. breast cancer). In Radiation Therapy Centers with appropriate technological equipment, radiotherapy can also be used intraoperatively (e.g. pancreatic cancer). Mr. Kardamakis then explained that we use the term "radiosensitivity" to characterize patients' sensitivity to radiotherapy. Although there are tumors we describe as radioresistant, the correct term is that there is no response to radiotherapy. So, through this meeting, we realized that the applications of the existing radiotherapy of pancreatic cancer are limited and there is a necessity to innovate new approaches.

Pathologist-Oncologist Dr. Michalis Liondos

-The main drawbacks of existing CAR-T cell immunotherapies are the intense side effects, such as cytokine release syndrome, the challenge of CAR-T cell therapies in effectively targeting the dense tumor of the pancreas and the “immunologically cold” pancreatic tumors.

On April 5th, we had the privilege of establishing contact with Dr. Michalis Liondos, a distinguished Pathologist-Oncologist currently serving as an Assistant Curator at the Oncology Unit of the Therapeutic Clinic at Alexandra Hospital in Athens. At the outset of our discussion, Dr. Liondos illuminated the substantial efficacy of immunotherapies involving CAR-T cells in treating specific hematological cancers. Notably, these therapies have exhibited remarkable success in addressing conditions such as Acute Lymphoblastic Leukemia and various lymphomas, including Hodgkin lymphoma, securing approval from the World Health Organization (WHO). Subsequently, Dr. Liondos analyzed the treatment of pancreatic cancer through existing immunotherapies employing CAR-T cells. He expounded upon the challenges associated with these therapies, primarily stemming from their non-specific targeting when applied to the formidable solid tumor, which is a characteristic of pancreatic cancer. Furthermore, he mentioned the issue of heightened toxicity attributed to cytokine release in the context of immunotherapy with CAR-T cells for pancreatic cancer.Specific antigens, notably the KRAS antigen and PD-1, were brought into focus in the realm of immunotherapies for pancreatic cancer. Moreover, he underscored the defining characteristics of pancreatic tumors that render them "immunologically cold" signifying their resistance to immunotherapeutic interventions. These attributes encompass the tumor's diminished immunogenicity and the pronounced immunosuppressive environment inherent within the tumor microenvironment. Concluding our discourse, regarding the economic considerations of immunotherapy involving CAR-NK cells, Dr. Liondos aptly reminded us that if this therapeutic approach proves efficacious and ultimately extends the life expectancy of patients battling pancreatic cancer, the benefits would outweigh the associated treatment costs. In summary, we have gathered significant information regarding why our approach with CAR-NK cells is more reliable than the existing one with CAR-T cells, specifically identifying the shortcomings of CAR-T treatment.

Psychiatrist Dr. Stergios Kaprinis

-There is a biological connection between pancreatic cancer and depression, so it is important to pay attention to the way of approaching patients so as not to weigh down on their mental health.

Our team embarked on a meaningful endeavor to gain more insight into the mental health difficulties experienced by patients battling pancreatic cancer and focus on understanding their perspective. To accomplish this, on April 9th, we attended an online meeting with Dr. Stergios Kaprinis, a renowned psychiatrist and Associate Professor of Psychiatry at the Aristotle University of Thessaloniki (AUTH), affiliated with the 2nd Psychiatric Clinic of AUTH.

During this informative meeting, we explored the complex mental health profile of individuals diagnosed with pancreatic cancer. Dr. Kaprinis explained that initially most patients appear to have a general sense of denial, as a self protective mechanism. Thus, depressive symptoms are a common occurrence among them. As time passes, they tend to recognize the unavoidable need for adaptation to their new life circumstances, thereby embarking on a journey of acceptance of this reality. However, most patients tend to suffer from different forms of depression or even from psychotic episodes.

Dr. Kaprinis emphasized the unique nature of depression caused by pancreatic cancer, due to its potential to culminate in the development of a paraneoplastic syndrome. Along with that, patients may experience visual illusions associated with organic disorders, which may not be treated through medications. Nonetheless, the broader understanding of the biological interplay between depression and pancreatic cancer remains somewhat limited. Furthermore, Dr. Kaprinis explained the pivotal role of personalized treatment planning in alleviating the mental health distress endured by patients. He emphasized that fostering the concept of self-improvement is crucial in enhancing adaptability and instilling mental health resilience in individuals struggling with this disease. Ultimately, Dr. Kaprinis underlined the vital contribution of psychotropic medications in the management of psychotic episodes, as well as the integral role of invaluable support generated by family members in dealing with patients’ depression. All in all, through realizing the biological link of pancreatic cancer with depression, we were able to empathize with cancer patients and understand the mental health difficulties they have to face, in order to approach them with respect, without triggering negative emotions.

Medical Director Mr. Nikos Nikas

-Pay attention to the clinical benefits of cancer therapy as well as to the financial burden pancreatic cancer puts on the public health system, in order to develop a cost-effective therapeutic product

On April 12th, our team, aiming to integrate our project into the Pharmaceutical Industry, met with Mr. Nikos Nikas, the Medical Director of Boehringer Ingelheim Pharmaceutical Company in Greece. Mr. Nikas explained the importance of finding new therapeutic approaches for pancreatic cancer, emphasizing the lack of effective immunotherapies for this specific type of cancer. He then elaborated on the high cost of cellular immunotherapies and how it should be reduced to make such therapeutic products accessible to more patients. Therefore, we discussed the importance of cost-effectiveness in our proposed treatment without compromising its effect on improving patients' quality of life. In other words, Mr. Nikas helped us better understand that the proposed treatment's clinical benefits determine its cost. He also assisted us in figuring out how reducing hospitalization and mortality rates through efficient therapies, overall benefits the economy of the public health system, thereby achieving cost-effectiveness. In this way, we were able to define our goals better in regards to developing our product and introducing it to the market strategically, aiming to reduce its cost and financial impact on the public health system.

The “ALMA ZOIS” Association of Women with Breast Cancer in Achaia, Greece

-We should focus on understanding the patients’ experience when receiving cancer therapy and aim to develop a therapeutic product which does not put an additional burden to their mental health, by limiting negative side effects and painful administration

The "ALMA ZOIS" Association of Women with Breast Cancer in Achaia, Greece, organized the 2nd Conference on Psychosocial Support in Oncology on May 26th-27th. The Conference focused on studying the mental health of cancer patients as well as the ways in which their families and social environment can provide better emotional support. Also, it shed light on the psychosocial dimension of cancer at all stages of the disease, not only for patients but for their families, too. Therefore, it emphasized the significance of psychosocial oncology as a crucial pillar in the therapeutic management of cancer. Through this Conference, we had an excellent opportunity to engage in discussions with local politicians, regarding how society and the government can effectively contribute to the mental well-being of cancer patients. Furthermore, we had the chance to connect with individuals who have experienced cancer themselves and better understand their needs. All in all, we came closer to the patients’ perspective when receiving cancer therapy and the effect of this disease on the mental health of them and their families, thereby we aim to limit the negative side effects of our therapeutic product and avoid painful delivery methods in order to contribute to the patients’ mental well being.

Associate Professor Mr. Ilias Kazanis and Specialist Senior Scientist Mrs. Florentia Papastefanaki

-Your project should be adjusted on the bioethical regulations of iPSCs for the allogeneic therapy with CAR-NK cells.

In the context of this year's project's holistic approach, on May 20th, we initiated communication with Mr. Ilias Kazanis, one of the Principal Investigators within our team and with Mrs. Florentia Papastefanaki, who holds the position of Specialist Senior Scientist within the Laboratory of Cellular and Molecular Neurobiology in the Department of Biochemistry at the Hellenic Pasteur Institute. During this interaction, we delved into a comprehensive discussion concerning the bioethical implications and issues associated with our project, with a particular focus on the utilization of iPSCs. More specifically, we discussed the Informed Consent, the Privacy and Confidentiality, the Risk-Benefit Analysis, the Safety and Efficacy, the Equity and Access and the Regulations that may vary in different countries.

Regarding our concern about the compatibility of allogeneic CAR-NK cells, Mrs. Papastefanaki proposed to obtain somatic cells (which would subsequently be reprogrammed into iPSCs) from different donors to provide a variety of CAR-NK cells. Additionally, as mentioned, the compatibility issue can be addressed through genetic modification of iPSCs to make them more suitable for the patient.

Emphasis was placed on the importance of obtaining detailed consent from both the donor and the recipient, as they should have a precise understanding of the procedures involved in cell collection, modification, and administration, as well as the purpose of this donation. Simultaneously, the privacy and confidentiality of the cell donation process were underscored to ensure the anonymity of the personal data of both the donors and the recipients. Mrs. Papastefanaki also noted that before a cell donation, the method's safety and effectiveness should be confirmed. The equal access to treatment for all socioeconomic groups was, also, referred to as an ethical concern.

Finally, the discussion revolved around the different legislation regarding cell donation in each country and how it should be anticipated for the execution of such a procedure. Thus, through this discussion, we were informed about all the ethical issues related to our project, from the initial steps to the final ones.

Pharmacist, Head of the Department of Biological Product Evaluation at the Hellenic Medicines Agency Mrs. Angeliki Roboti

-The therapeutic product you are developing belongs in the Advanced Therapy Medicinal Products category and it is integral to ensure its biosafety in every stage of research, by defining the research objectives, the comparability, as well as the end points and criteria of future clinical trials, in order for it to ultimately be approved through central authorization by EMA.

Since we wanted to ensure the biosafety of our product throughout every step of its development, we contacted on the 20th of June with Mrs. Angeliki Roboti, Pharmacist and Head of the Department of Biological Product Evaluation at the Hellenic Medicines Agency (EOF).

During this meeting, Mrs. Roboti emphasized the importance of biosafety of the product. Initially, she pointed out the "therapeutic gap" in advanced pancreatic cancer therapies and the need of developing an innovative treatment for this type of cancer. Moreover, she assured us that our final product falls into the Advanced Therapy Medicinal Products (ATMP) category and thus, it must follow the European preclinical and clinical regulation guidelines referring to this specific category. She then explained that Advanced Therapy Medicinal Products have to be approved through central authorization by the European Medicines Agency (EMA) and provided us with the guidelines that include the clinical trial requirements.

Mrs. Roboti elaborated on the safety assessments biological products have to go through, to evaluate whether their composition is safe for their intended use. All research and development facilities on a preclinical and clinical level must be GMP (Good Manufacturing Practice) certified. Specifically, she gave us valuable insight into the process of biosafety approval of biological products by the Hellenic Medicines Association (EOF), with the aim of proceeding to clinical trials. For this process, it is necessary to provide the Department of Clinical Trials of EOF with analytical in vitro preclinical lab protocols and proven safety results with a recommended therapeutic dose for the proposed treatment. Also, it is essential to precisely choose the population of cancer patients for the clinical trial and define the inclusion criteria of the trial. Furthermore, it is crucial to determine the research objective, the end points of the trial, which are the parameters that define biosafety, as well as the prespecified criteria of the trial and comparability. In this context, Mrs. Roboti highlighted several common evaluation endpoints that should be taken into consideration when testing product biosafety, such as cytotoxicity, irritation, sensitization, systemic toxicity, pyrogenicity, hemocompatibility, implantation, genotoxicity, carcinogenicity and reproductive developmental toxicity.

Furthermore, she explained that the lentivirus we chose as the vector for the transduction of NK cells is the appropriate choice of vector for our project in regards to biosafety, as it presents the best toxicity profile among other vectors. At the same time, lentivirus has the advantage of not integrating into the cell’s DNA. Therefore, through our discussion with Mrs. Roboti, we gained invaluable perspective on the biosafety regulations our therapeutic product must follow during preclinical trials as well as during future clinical trials and we further confirmed that lentivirus is a suitable vector for our project, in terms of biosafety.

Doctorate Student, Research Assistant Ms. Dionysia Kefala

-You should follow these instructions for the Chimeric Antigen Receptor construction, NK cell transduction and NK cell expansion, but also conduct proof-of-concept cytotoxicity studies.

On June 22th, we met Ms. Dionysia Kefala, a Doctorate Student/Research Assistant at the Institute of Cell Therapy at the University of Patras and has extensive laboratory experience differentiating T-cells from induced Pluripotent Stem Cells (iPSCs) and constructing Chimeric Antigen Receptors (CARs). Initially, she addressed the topic of pancreatic cancer, underscoring the significance of the selection of this disease model for our research endeavors. She accentuated the challenging nature of this medical condition, characterized by an unfavorable prognosis and notably limited survival rates following metastatic diagnosis.

Subsequently, she commended our project as both exciting and pioneering due to our utilization of CAR-NK cells instead of CAR-T cells. She also noted that the FDA is in the process of evaluating numerous CAR-NK therapies, suggesting a forthcoming wave of approvals in this regard. She emphasized the importance of selecting an antigen specific to pancreatic tumors, one that is not expressed in normal tissues. She inquired about the specificity of our CAR cells, whether they target one or two antigens, and sought details on the transduction method and our choice of viral vector.

Furthermore, she encouraged us to explore various online resources for existing Chimeric Antigen Receptor sequences, which could aid in the design of our plasmid of interest. She also elaborated on the advantages of utilizing serum-free media and recommended the use of feeder cells to achieve optimal expansion of NK cells. Additionally, she provided insights into the utilization of a cytokine cocktail, comprising IL-2, IL-12, IL-15, IL-18, and IL-21, elucidating the unique benefits associated with each cytokine. She recommended the utilization of flow cytometry as a way to monitor and assess the quality and efficacy of the transduction process. She explained that we have the option to select the transduced cell clones through the use of either an antibiotic resistance gene or shorting techniques.

Moreover, she conveyed that the process of reprogramming somatic cells into iPSCs is notably challenging and adhering to the competition's time constraints might pose difficulties. However, she provided an in-depth overview of the laboratory procedures we would undertake if we were to pursue this approach. In conclusion, she urged us to conduct proof-of-concept cytotoxicity studies on pancreatic cancer cells and evaluate the cytokine secretion by NK cells. Recognizing her extensive expertise aligned with the requirements of our project, we proposed that she assume the role of an instructor, an offer she graciously accepted. So, this meeting proved to be particularly significant for the progress of our project, as we not only formed a partnership with an instructor but also gathered essential information about the antigen, the viral vector, the plasmid and other aspects that we should pay attention to during the experimental process.

Ph.D. Student Mr. Arampatzis Asterios

-These specific cell lines should facilitate the transduction and, also, you can consider developing an “off-the-shelf” product.

On June 28th, we initiated contact with Mr. Arampatzis Asterios (M.D., MSc), a Ph.D. student at ETH Zurich (Swiss Federal Institute of Technology), specializing in the intersection of synthetic biology and control theory. Our discussion encompassed both the conceptual framework and the practical implementation aspects of our project. Following a detailed presentation of our project, he underscored the pivotal significance of the innovative component within our project. This component encompasses not only the treatment of solid tumors but also the potential development of an “off-the-shelf” product. Furthermore, he emphasized the importance of conducting experiments, as part of the proof-of-concept, to assess the viability and cytotoxicity of CAR-NK cells within the pancreatic cancer instead of utilizing appropriate pancreatic cancer cell lines.

Subsequently, he offered guidance on the some stages of the research path we should pursue. In particular, he identified specific cell lines, namely HEK 293 and HeLa, known to facilitate transduction, which could be employed for testing purposes. Regarding the transduction process, he advised us to consider either the electroporation method or the lentiviral transduction method as viable options. Subsequently, he advised us on how to express the CAR receptor sequence within the NK cells, suggesting the Transfer method without Integration and the Genomic Integration method. Ultimately, with regard to establishing the proof of concept, he advocated for the thorough repetition of experiments, emphasizing the importance of conducting multiple iterations to ensure their testability and evaluability. In conclusion, Mr. Arampatzis not only encouraged us to pursue our idea of an “off-the-shelf” product but also provided us with valuable insights into transduction and specific cell lines.

Associate Professor of Pathology and Hematology Dr. Georgios Vasilopoulos

-Pancreatic adenocarcinoma (PDAC) has a complex tumor microenvironment and Mesothelin is the right choice of target-antigen since it is overexpressed in PDAC.

On July 6th, we met with Dr. Georgios Vasilopoulos, who is an Associate Professor of Pathology-Hematology at the Hematology Clinic of the Medical School at the University of Thessaly and a Collaborating Researcher at the Institute of Medical Research of the Academy of Athens (IMRAA, Department of Genetics and Gene Therapy).

We initially discussed our project's difficulties and its innovation in our country, as there is currently no research in Greece regarding CAR-NK cells derived from induced Pluripotent Stem Cells (iPSCs) for cancer therapy. Specifically, Mr. Vasilopoulos emphasized the time-consuming process of converting iPSCs into NK cells, which would exceed the time frame of the competition. Consequently, we decided to support this aspect of the project through literature references. In addition, Mr. Vasilopoulos pointed out that the pancreatic adenocarcinoma has significant heterogeneity due to the tumor microenvironment and he assured us that the antigen Mesothelin (MSLN) is the most suitable target for our project. Therefore, Mesothelin is a pretty specific antigen that is overexpressed in pancreatic adenocarcinoma and Mr. Vasilopoulos confirmed that we made the right choice for our target-antigen by choosing MSLN.

Pharmacology Professor Ms. Lefkothea Papadopoulou

-You can use the NK-92 cell line acquired from ATCC through my laboratory and specific reagents to enhance the cytotoxic activity of the cells.

On July 8th, we initiated communication with Ms. Lefkothea Papadopoulou, a distinguished Professor in the Pharmacy Department at the Aristotle University of Thessaloniki. She is renowned for her expertise in cancer immunotherapy, specifically in the utilization of CAR-T cells. She and her research team have presented a technique known as "In vitro-transcribed (IVT)-mRNA-mediated transfection", which serves as a method for introducing the CAR receptor into T-cells. This method could provide a much safer CAR therapy since IVT-mRNA leaves no ultimate genetic residue in recipient cells.

So, we presented our project to Mrs. Lefkothea Papadopoulou, who provided valuable guidance on the specific aspects of our experimental methodology that warrant particular attention and consideration. She also conveyed her substantial expertise in cultivating NK cells and generously offered access to the NK-92 cell line acquired from ATCC through her laboratory. She emphasized the demanding nature of working with these cells and encouraged us to commence laboratory practice promptly. Furthermore, she recommended the utilization of specific reagents to enhance the cytotoxicity of the cells and even extended an offer to share established protocols utilized within her research team.

During our discussion, we delved into the good manufacturing practices employed in developing cell therapy products within clinical contexts, such as using serum-free media and feeder-free methodologies. Ultimately, Mrs. Papadopoulou also recommended that we engage in a dialogue with her Ph.D. student, Mrs. Androulla N. Miliotou, who possesses extensive experimental expertise in the precise area of our research focus. Therefore, the most significant takeaway from this meeting was that we secured the NK-92 cell line, giving us the opportunity to proceed with cytotoxicity tests.

Molecular Pharmacologist Ph.D. Ms. Androulla Miliotou

-It is crucial to optimize the transduction method of the NK-92 cell lines and meticulously design the plasmid sequence, whilst paying attention to the functionality of modified NK cells and their cytotoxic activity.

On July 11th, our team had an online meeting with Mrs. Androulla N. Miliotou, Ph.D. Molecular Pharmacologist and Head of the Department of Medical Representatives and Pharmacy Assistants at KES College in Cyprus. Since Ms. Miliotou has extensive experience in the laboratory development of CAR-NK cells, we discussed the optimal transduction method of the NK-92 cell lines. Moreover, she addressed the issues that we should take into consideration during the design of the plasmid, such as sequences that are important to be included in the plasmid, apart from the Mesothelin chimeric antigen receptor gene. We also examined which biological assays to utilize regarding the functionality of the modified NK cells. Furthermore, Ms. Miliotou pointed out that we should proceed with cytotoxicity trials on pancreatic cell lines, where surface antigen Mesothelin is highly expressed. Overall, Ms. Miliotou gave us insightful directions in choosing the best plasmid sequence, the transduction method of NK-92 cell lines and the biological assays of the modified cells, while highlighting once again the importance of performing cytotoxicity tests.

Associate Professor of the Department of Computer Engineering and Informatics of the University of Patras Mr. Konstantinos Tsichlas

-I advise you to use Machine Learning and Bioinformatics algorithms in antibody design.

On July 20th, we communicated with Mr. Konstantinos Tsichlas, an esteemed Associate Professor within the Department of Computer Engineering & Informatics at the University of Patras. Mr. Tsichlas provided invaluable assistance by offering suggestions concerning our modeling approach, which forms the basis of our primary mathematical mode. He also helped with our endeavors in optimized antibody design, an intersection of computer science and cancer biology. His counsel about the precision of neural networks and the application of diffusion-based neural networks was particularly insightful, shedding light on critical concerns. The efficiency of our dry lab simulations was significantly attributed to his recommendations. These were based on the most recent research findings and benefited from his profound expertise in intelligent systems computing and algorithm design. Furthermore, Mr. Tsichlas underscored the potency of neural networks while cautioning that their outcomes should be thoroughly scrutinized through wet and dry lab testing to confirm their biological efficacy. Recognizing our constraints in terms of time and resources was another reason for advising the adoption of this approach, which is a pioneering technique and has demonstrated promising results compared to classical network architectures.

Also, we engaged in a comprehensive analysis of the principles of the central dogma of molecular biology. This discourse served as a foundation for a more profound comprehension of the antibody optimization concept and the framework of our primary dry lab model. Furthermore, he advised us to explore the RCSB PDB site to access pdb files that could prove instrumental in our simulations. His counsel was of particular significance that our PBD files should ideally be derived from experimental determinations via X-ray crystallography instead of relying solely on deep learning sequence predictions, such as those generated by AlphaFold. This distinction was critical in crafting the core simulation involving our CAR receptor and the mesothelin antigen, guiding our selection of the most suitable pdb files for each facet of our simulation.

He also shared a comprehensive understanding of the PDB file format, which encapsulates the three-dimensional crystal structure of macromolecules, along with coordinates illustrating their structure in 3D space. Furthermore, he outlined the expected outcomes of our simulations, emphasizing that we should verify the structural stability of our CAR design over time and measure its affinity in binding to our antigen and the antibody-antigen affinity. In conclusion, Mr. Tsichlas's extensive experience in string algorithmic design and his background in machine learning augmented our algorithmic knowledge. Our discourse encompassed a thorough exploration of bioinformatic algorithms such as FASTA and BLAST, explaining how these tools could facilitate the retrieval of PDB structures and enable predictions of diverse protein structures based on their amino-acid sequences.

Associate Professor of the Biotechnology Department of the School of Applied Biology and Biotechnology, at the Agricultural University of Athens Mr. Dimitrios Vlachakis

-I advise you to perform Molecular Dynamics Simulations and Molecular Docking to figure out the interaction between CAR and Mesothelin antigen.

On July 30th, we conducted a meeting with Dimitrios Vlachakis, an Associate Professor in the Genetics Laboratory of the Biotechnology Department of the School of Applied Biology and Biotechnology, Agricultural University of Athens. Possessing expertise in computational biology and molecular simulations, he advised us to conduct molecular dynamics simulations to explore the interaction between our CAR and the mesothelin antigen, rather than relying solely on a basic molecular docking approach. Also, he elucidated that the docking process is static, providing a singular representation of our protein-ligand complex under specific experimental conditions, including temperature and pressure. In contrast, molecular dynamics simulations offer the capability to assess the stability of our CAR design over time and its binding affinity with our antigen. So, he proposed various molecular dynamics simulation programs, including NAMD, GROMACS, and GROMOS, ultimately recommending GROMACS for our specific needs.

Associate Professor of the Department of Computer Engineering and Informatics at the University of Patras Mr. Hadjidoukas

-I advise you to integrate high-performance computing in your project and achieve fast simulations using parallel computing methods in your modeling workflow.

On August 5th, we contacted Mr. Hadjidoukas, an Associate Professor in the Department of Computer Engineering & Informatics at the University of Patras. His significant impact on our project simulations was noticeable, owing to his research experience enclosing parallel processing, high-performance computing, runtime support for programming models, thread and system libraries, as well as parallel and distributed machine and deep learning.

He was a valuable collaborator during our debugging process, mainly when working on GROMACS and Diffab. Furthermore, his expertise extended to optimizing our simulations by implementing parallel processing via the MPI protocol and thread utilization during runtime on our high-performance supercomputer. This strategic approach allowed us to harness the complete capabilities of our supercomputer's CPU and NVIDIA CUDA® GPU parallel programming model, ideally suited for applications requiring extensive parallelism in matrix computations and advanced data science computing.

His guidance proved instrumental in crafting structured and analytical code, while also giving us access to one of the most high-performing supercomputers within the Department of Computer Engineering and Informatics at the University of Patras. This access significantly expedited our ability to conduct molecular dynamics simulations and optimize antibodies with precision. In a broader sense, he integrated his advanced research knowledge in high-performance parallel computing into our project, thereby facilitating the creation of a swift and resilient dry lab modeling workflow.

Team of Researchers affiliated with the University of Torino

-It is crucial to meticulously design an in-silico CAR model, simulating the real receptor, in order to be able to choose the optimal 3D protein structure prediction.

On September 5th, we engaged in a productive discussion with a team of esteemed researchers affiliated with the University of Torino. These researchers specialize in molecular modeling and analysis and hold a collaborative research partnership with our sponsor, InSyBio.

Our discourse held notable significance as we analyzed our dry lab modeling workflow and particular emphasis was placed on the connection between biology and computation. In this frame, we showed that we have constructed a computational model representing the biological experiments conducted and the molecular models within our project, which are the interactions between the CAR and mesothelin antigen. During our conversation, the researchers introduced us to the concept of in-silico antibody optimization, which further confirmed what we had previously discussed with Professor Konstantinos Tsichlas. Thus, we successfully implemented this concept using Diffab.

Furthermore, the researchers provided guidance emphasizing the necessity for a well-prepared and carefully designed in-silico CAR model. This model should accurately depict a real CAR and include various deep-learning algorithms to facilitate 3D protein structure prediction. The selection of the most suitable prediction is based on the specific requirements of our project. Ultimately, they advised us that the primary software choice that suits our molecular dynamic simulations is GROMACS. So, this call was essential in understanding the connection between biology and computation, the importance of utilizing Diffab and the significance of a thoroughly designed in-silico CAR model.

Dry Lab Advisor Mr. Eleftherios Bochalis

-We should focus together on the GROMACS molecular dynamics simulations and the Mathematical Modeling development.

Discussions and collaborative efforts were undertaken throughout our project with Mr. Eleftherios Bochalis, our Dry Lab Advisor. He played a pivotal role as our GROMACS mentor, facilitating exceptional results in our molecular dynamics simulations. This collaborative endeavor involved a series of Zoom meetings, during which we collectively addressed and resolved challenges that surfaced.

Moreover, Mr. Bochalis engaged in discussions centered on the mathematical model of our project, bridging the divide between the realms of computer science and biology. This endeavor was pivotal in unifying these two disciplines within the framework of our Dry Lab work. From the inception of our project, he introduced us to the concept of Dry Lab, offering a comprehensive explanation of the work that must be conducted within the context of iGEM. His tutoring extended to an in-depth exploration of the PDB file format, which encompasses details about each atom's coordinates in three-dimensional space. He further imparted a clear understanding of the expected outcomes from our simulations, underscoring the necessity of the structural stability of our Nanostructure over time.

Mr. Bochalis proposed various molecular docking programs and web servers, including HDOCK, HADDOCK, ZDOCK, and SWISS-MODEL. Additionally, he presented various molecular dynamics simulation programs, such as NAMD, CHARMM, and GROMACS. Subsequently, we seamlessly integrated GROMACS into our modeling workflow under his guidance. Our extensive discussions enclosed Molecular Dynamics protocols, which typically contain the following steps: Simulation Box formation, Molecule solvation, Energy minimization, Structure Equilibration, and Molecular Dynamics simulation. These directives were meticulously followed accordingly.

Furthermore, it was underscored that the preparation of input files in PDB file format is of paramount importance in the Molecular Dynamics process. Our advisor, also, offered substantial support throughout the debugging process of our GROMACS code, providing insights into various programming approaches to harness the total computational power and optimize simulation times on our supercomputer. Finally, Mr. Bochalis played a pivotal role in guiding us through the membrane construction within our in-silico CAR design, a process facilitated using CHARMM-GUI. This membrane model was integrated into GROMACS to facilitate extensive molecular dynamics simulations. In conclusion, as our dry lab advisor, his role was very significant in various aspects of the dry lab team’s progress, including the use of GROMACS, the Mathematical Modeling development, the membrane construction and the molecular docking.

“Ennovation” yearly University Competition on Entrepreneurship and Innovation by Athens Center for Entrepreneurship and Innovation (ACEin)

-Implementing a thorough market research approach and developing our branding and business knowledge is essential for the improvement of our business plan and introduction of our product to the market.

In an effort to expand our business knowledge and learn more about how to carry out our market research in the context of our business plan, we participated in “Ennovation” which is a yearly University Competition on Entrepreneurship and Innovation with a duration of four months in total. The competition runs by a network of 20+ universities in Greece and Cyprus under the coordination of Athens Center for Entrepreneurship and Innovation (ACEin) of the Athens University of Economics & Business. Through “Ennovation” we gained significant perspective in how to turn our entrepreneurial idea and scientific research results into a sustainable business. Our team applied to “Ennovation” on March 31st 2023 and got promoted to the second phase of the competition, following the evaluation of our project by the judging committee. During the second phase of the competition, we had the pleasure to attend a series of intensive, valuable seminars, as well as workshops organized by the Athens Center for Entrepreneurship and Innovation, that helped us understand how to strategically address the market of our final product and advance our business plan. We also received several coaching and mentoring sessions by experts in the field of Economics and Business, which assisted us in developing the entrepreneurial prospect of our project and its introduction in the business world. The theme of the seminars and workshops we attended, revolved around Market Research, Business Validation, Team Work, Branding and Finance. Therefore, our participation in the “Ennovation” competition gave us invaluable insight on how to carry out our market research for our business plan and address the end customers of our product more effectively.

Economic Analyst specializing in business plan development

-Pay attention to developing a sustainable company and achieving optimal growth rates, while also meticulously figuring out the cost of your product.

During our participation in the MIT "Global Start-up Workshop Conference," we had the opportunity to meet prominent individuals in economics and entrepreneurship. At this networking event, we encountered an economic analyst specializing in developing business plans. He graciously offered his assistance once we reached the final stages of our project. Therefore, we had an online meeting with him on September 10th to discuss our business plan.

The gentleman (whose identity we have been asked not to disclose) proved to be an invaluable resource for the entrepreneurial aspect of our project. He underscored the importance of focusing on our product's and the company's sustainability. Also, he provided valuable insights into the cost analysis of our processes, indicating how the product's shelf life influences its pricing. He emphasized the significance of considering the number of personnel operating the machinery and their required expertise. Additionally, he shared information about profit margins, drawing similarities with other industries, and stressed the need to align our strategies accordingly. He also offered an essential perspective on the concept of the break-even point and the requisite growth rate for our company to achieve optimal profitability. Furthermore, he provided crucial information about managing clinical trials and securing initial funding for our venture. At the same time, he shared strategies that could help us in approaching investors and participating in financing programs. This discussion was highly beneficial in determining the feasibility of our company, developing our business plan efficiently to achieve optimal growth rates and profitability, as well as strategically figuring out the cost analysis of our product.

Head of the Department of Biological Product Evaluation at the Hellenic Medicines Agency Mrs. Angeliki Roboti

-Develop a cost-effective cellular immunotherapy by becoming a spin-off company of your University and introducing insurance systems with a payment compensation plan and collaborate with the Committee for Advanced Therapies (CAT) of EMA to better evaluate your therapeutic product.

In order to figure out a way to strategically evaluate our product and effectively develop our business plan, we arranged another call with Mrs. Angeliki Roboti, Pharmacist and Head of the Department of Biological Product Evaluation at the Hellenic Medicines Agency (EOF). Throughout the journey of research and development of our product, she advised us to collaborate with the European Medicines Agency’s (EMA) Committee for Advanced Therapies (CAT) which will provide us with guidelines and expertise that is needed to evaluate advanced therapy medicines like ours. In this way, EMA can provide us with invaluable directions and possible improvements that may be applied to our product, more than once, at any stage of the development process. Furthermore, Mrs. Roboti confirmed that there is a “market gap” regarding advanced cellular immunotherapies like ours and the existing cellular immunotherapies are very expensive and thus, not available to many patients who need them. Therefore, she suggested that we become a spin-off company of the University of Patras in order to reduce our profit and the cost of our therapy. Additionally, she proposed the idea of providing outcome-based reimbursment plans in order for insurance systems to pay for our therapeutic treatment in different doses in predefined time periods after evaluating prespecified criteria such as "relapse free survival" and "disease free srvival". In this way, the cost of our therapy is going to be further reduced. Hence, Mrs. Roboti played an integral part in helping us introduce a cost-effective therapeutic product to a niche market, thus, making therapy accessible to more patients, while indicating factual steps to achieve that and implement these solutions in our business plan.

Computer Engineers from the InSyBio Company

-We propose that in the future your therapeutic approach could be personalized for each patient, through bioinformatics.

On September 20th, we had an online meeting with members of the company InSyBio, specifically computer engineers specializing in bioinformatics and data analysis systems. Their expertise encompasses cutting-edge tools and methodologies tailored for analyzing biological data, with a particular emphasis on extracting valuable insights, such as identifying potential disease biomarkers

During this meeting, we engaged in a discourse regarding the practical application of personalized therapy, leveraging the science of bioinformatics to analyze genetic data as a requirement for patient-specific treatment recommendations. This approach involves pre-screening, a vital step in which the patient's genetic profile is comprehensively evaluated, including the specific genetic mutations present in pancreatic cancer. The objective is to ascertain the viability and efficacy of immunotherapy involving CAR-NK cells for the particular patient in question. Consequently, through pre-screening, we gain insights into the forthcoming effectiveness of our proposed treatment, thereby enhancing the patient's life expectancy and overall quality of life.

Data Protection Officer (DPOaaS) and business consultant in GDPR, CSR, medical tourism and sustainable development Mr. Yannis Kalantzakis

-Figuring out ways to implement General Data Protection Regulations in your product development and distribution is crucial to ensure the protection of health data of the future somatic cell donors and patients who receive your therapy.

Since we were interested in exploring the possibility of receiving somatic cells of healthy donors in the future for our therapeutic product development, we contacted Mr. Kalantzakis in order to learn more about the process of ensuring the protection of health data of potential future donors of somatic cells, as well as patients receiving the therapy. Mr. Yannis Kalantzakis is an entrepreneur of health care services, certified Data Protection Officer (DPOaaS) and business consultant in regards to General Data Protection Regulations (GDPR), medical tourism, Corporate Social Responsibility (CSR) and sustainable development (EU ETS, Ecovadis etc).

Mr. Kalantzakis suggested that following the future development of our spin-off company and the increase of our growth rates and profit, we could consider collaborating with cell donation banks, to get the somatic cells and reprogram them to induced Pluripotent Stem Cells (iPSCs), instead of buying the iPSCs derived NK-cells from another company. Therefore, the process of buying iPSCs derived NK-cells could just be a temporary solution for us, so as to perform our initial preclinical trials. The cell donation bank is going to keep the health data of the donors whose somatic cells we are going to use for our product development, under well defined conditions complied with GDPR, in order to use suitable technical and organizational measures for their protection. Also, Mr. Kalantzakis explained that it is very beneficial for us to collaborate with a cell donation bank if we want to get somatic cells in the future, since in this way, the donors are going to go through medical examinations, in order to ensure that they are healthy. Furthermore, he suggested that it is important to determine the transportation and storing conditions of the donor’s somatic cells to our lab facilities. Most importantly, he pointed out that we should take into consideration the “Substance Of Human Origin (SOHO)” regulation of the European Union which indicates how the safety of health data of the donor and the patients who receive therapy is secured. Moreover, he explained that it is important for us to define whether we are going to use somatic cells from one or more donors for the development of our therapeutic product. However, he highlighted the issue of the increase of the final product’s cost that we would have to tackle in case we collaborate with somatic cell banks. So, we would have to figure out a way to ensure the cost-effectiveness of our product, if we considered starting the development of our therapy from somatic cells. Thus, the contribution of Mr. Kalantzakis in understanding the process and regulations of human cell donation was of utmost significance, since in the future, it is integral to implement GDPR throughout our therapy development and distribution, in case we collaborate with somatic cell banks.