Overview

Recent advancements in cancer treatment are emerging due to immunotherapy, ranking as fourth fundamental pillar of cancer therapy following surgery, radiation, and chemotherapy. Our vision is to approach an innovative strategy for the immunotherapy of pancreatic cancer, by following the recent developments in the field of synthetic biology and bringing scientific discoveries even closer to the existing available treatments.

Natural Killer (NK) cells that are armed with a Chimeric Antigen Receptor (CAR) can contribute when dealing with solid tumors, like pancreatic cancer. Mesothelin, which is highly expressed by pancreatic tumor cells, is our main target. Herein, we will analyze the whole path of implementing our idea, considering both the part of synthetic biology and the administration of the therapy.

Implementing a well-defined project involving CAR-NK cells, a cutting-edge technology in cancer immunotherapy, necessitates a meticulously orchestrated process. This journey entails defining project objectives, assembling a multidisciplinary team, and formulating a comprehensive project plan. To propel this endeavor, securing diverse funding sources, ensuring regulatory compliance, and establishing state-of-the-art laboratories are vital steps. The project then unfolds through critical phases, from CAR design and preclinical studies to clinical trials and regulatory approvals. Continuous research, post-market surveillance, and community education further bolster its success. Throughout this intricate process, unwavering commitment to scientific excellence and quality assurance remains paramount. Throughout the entire process, encompassing treatment selection, manufacturing, delivery, and administration, there is a vital need for ongoing communication, coordination, and collaboration among pharmacists and the healthcare professionals. This interplay is of paramount significance. We consider ourselves lucky because we are located in Patras (Greece) where The Institute of Cell Therapy (ICT) was established as a part of the University Research Centre of the University of Patras, so we are able to seek help and cooperation easily.

Where to begin?

“Our team aimed to develop a readily available therapy, which could be easily accessed, circumventing the time-consuming procedures in hospitals that might impede the timely delivery of treatment, potentially further complexing the patient's health condition”

Typically, the leukocytes that are used in immunotherapy can be isolated directly from patients' blood, through leukapheresis. Leukapheresis, is a medical procedure conducted by healthcare providers which aims to separate white blood cells from red blood cells. It is primarily employed in cases of lymphocyte-related disorders such as leukemia, with the goal of reducing the quantity of uncontrollably multiplied lymphocytes in the bloodstream. In these cases, leukapheresis is a supplementary procedure and not a treatment option. In immunotherapy, leukapheresis is used to isolate a specific immune cell that is usually intended for autologous purposes, meaning that the donor and recipient are the same person.

Our primary goal is to the issue of allogenicity (using cells from a donor for the treatment of a patient), aiming to develop a readily available product and thereby reducing the need for repeated patient-specific cell derivation. Although leukapheresis supports autologous use, procedures that have been conducted so far, for generating NK cell products, employ immune-magnetic techniques to remove T and B cells selecting CD56+ cells with great precision. Achieving high purity of these products is of utmost importance, due to the potential for residual T-cells causing GVHDs (Graft-Versus-Host Diseases) and passenger B lymphocytes leading to complications. Even though this could indicate allogeneic use, limitations remain.

Exceptionally high purity remains an issue, especially when considering the sufficient amount of NKs that has to be isolated. In trials performed to date, the required number of NK cells varies between 5 x 106 to 5 x 107 cells (CD3-, CD56+) per kilogram. Considering the small values of NK cells found in the blood (5 - 10%), in order to achieve this required amount, it is necessary to enhance the isolated NK cells from the blood, with leukapheresis products from additional donors, thus achieving larger volumes. The process required for high-precision isolation, impacts the yield of NK cells and can lead to reduced effectiveness or even cell death. Obtaining a significant quantity of NK cells from a single leukapheresis procedure becomes difficult due to the lower yield rate, especially when considering the restricted availability of donor-derived leukapheresis products.[1]

Another method of NK recruiting is starting from peripheral blood as a source. This technique involves the isolation and expansion of NK cells derived from peripheral blood mononuclear cells (PBMCs). The expansion process typically employs feeder cells like K562 (mb.IL21s) which is a cell line gene modified to express membrane bound interleukin-21 (IL-21) aiming for an aproximately 300 fold expansion of NK cells after day 28. Even though this method could be a promising alternative with increased available amounts of NK cells, conflicts relating to purity still remain and K562 (mb.IL21s) cell line can only be obtained under a strict Material Transfer Agreement (MTA).[2] The resulting proliferated NK cells retain a low purity of only 60-70%, which makes them incompatible for allogeneic use.

For this purpose, we utilized induced Pluripotent Stem Cells (iPSCs) as a source of NK cells. Pluripotent Stem Cells can be reprogrammed to Natural Killer cells in the lab, bypassing any conflict regarding purity and required quantity. In this way, constructed CAR-NK cells can be utilized as an “off-the-shelf" product that will be appropriately stored making it widely and readily available and ideal for allogeneic use.[3]

Who will it address?

“Οur team aims to create a more inclusive treatment for pancreatic cancer”

Even though pancreatic cancer more often affects older age groups, younger age groups are not excluded. Depending on patients' overall health profile and consulting health specialist indications, our therapy can be administered to patients from age 18 and older. It appears that the level of mesothelin expression is not dependent on the patient's pathological stage. A study conducted by (Le et al., 2020) found that mesothelin expression in tissue samples does not exhibit a significant correlation with clinicopathological data. Therefore, there is potential to consider MSLN-related immunotherapy for all PDAC patients who show positive MSLN expression, regardless of factors like the patient's pathological stage, degree of differentiation, or lymph node metastasis. This invariance in mesothelin expression regardless of disease stage increases the number of patients who can receive the treatment.[4] This is further confirmed by a study conducted by (Weidmann et al., 2021). Immunohistochemistry on tissue microarray from 599 pancreatic cancer tumor samples was performed and no correlation between cancer aggressiveness and mesothelin expression was found. Mesothelin expression was elevated in 90% of Pancreatic Adenocarcinoma (PDAC) cases, making it an ideal target. Given the poor prognosis associated with pancreatic cancer, the availability of a therapy applicable at any stage assumes paramount significance. In conclusion, the detection of heightened mesothelin expression in a patient's cancer tissue through biopsies may serve as a determinant for considering the administration of CAR-NK therapy. For this purpose, Immunohistochemistry assay could be the preferred technique, in order to detect mesothelin levels and forward MSLN CAR-NK therapy.[5]

The sufficient amount of CAR-NK cells needed for the initial dose may vary but this will be determined in the early stages of clinical trials. Optimal exact dosage which will be administered to a patient should be determined by health care providers and be amplified to the patient's special needs. Immunotherapy with Chimeric Antigen Receptors can be divided into administration phases with increasing dosage. Therapy can last over 3 weeks, with the doctor supervising the progress of the treatment and patients' stage of health.[6]

Distribution

“Product distribution, presents challenges that must be addressed for prompt delivery and administration of the therapy”

All aspects from the moment our cell immunotherapy product leaves our production facilities to final administration should be considered in order for the treatment to be delivered safely, effectively and methodically. Distributed CAR-NK cells can be provided in either a bag or syringe for infusion and are delivered in a dry-shipper containing vapor-phase nitrogen, maintaining a temperature of around -160°C. Monitoring the sequential stages of manufacturing pharmaceutical products as needed through our website will enable the advance scheduling of reception dates and times. This, in turn, facilitates the mobilization of required personnel for reception without any disruption to the cold chain.

Upon receipt, a conformity check will be conducted using reception documents (including all documents, certificates of analysis and release, temperature logs etc). This check involves:

• Examining the cryo-shipper for any visible damage or leaks.
• Opening the metal cassette to thoroughly inspect the frozen cell product.

Ensuring the completeness and accuracy of information printed on the CAR-NK cell label, including patient identity and drug identity. Proper labeling is essential to uphold the Chain of Identity and Chain of Custody throughout the manufacturing process, all the way to the administration of the medicinal product to the intended recipient. Moreover, it should be clearly indicated in the receiving documentation whether there is a backup bag present at the manufacturer's site. If any issues or nonconformities are identified during the reception process, this information becomes highly valuable for pharmacists and hematologists when deciding on the treatment plan. Having a backup bag on hand allows for prompt administration, ensuring that the defective CAR-NK cells can be replaced, and treatment can proceed within 48 hours if necessary.

Storage and administration

“The storage and handling of the product must be done by specialized personnel keeping a detailed record of the procedures”

The formalized storage procedure entails the utilization of vapor-phase nitrogen tanks, with hospitals housing cell processing facilities having the option of employing dedicated nitrogen storage tanks. Regarding nitrogen storage duration, it approximates six months. The concern is averting potential burn injuries and hypoxia incidents during the manipulation of CAR-ΝΚ cells within and outside the cryogenic containers. To mitigate such risks, it is imperative to conduct these operations within well-ventilated facilities, where access is restricted solely to personnel who have undergone comprehensive training and recurrent retraining.

Initiation of the thawing process commences upon approval from the hematologist. The thawing procedures are carried out by the pharmaceutical team on the day of administration, with the focus on minimizing the duration of the process. This necessitates coordinated planning in conjunction with the Haematology Department. The thawing process takes place within the hospital premises or, if outsourced, within the cell processing facility. It involves the double-wrapping of the CAR-ΝΚ cell bag in a protective plastic covering within a sterile environment. Thawing is accomplished in a dedicated 37°C ± 2°C water bath until all ice crystals in the bag have completely dissolved. Depending on local protocols, a dry thaw method may also be considered. As a recommended precaution, it is advised to employ a dual-layered plastic bag for thawing to safeguard the CAR-ΝΚ cell bag. Post-thawing, CAR-ΝΚ cells remain stable at room temperature for a duration ranging from approximately 30 to 90 minutes.

The timeframe for the period between thawing and administration typically falls within a range of 30 to 90 minutes, which varies depending on the specific manufacturer's guidelines. This necessitates meticulous time management, including the transportation of cells from to the designated department in a dedicated container maintained at room temperature. The distribution path from the moment the cells are thawed, to the delivery to the nursing staff responsible for administering injections within the Haematology Department, should be documented. This documentation should include the time of distribution.[7]

Delivery

“We propose three different delivery therapy systems which can be combined depending on the situation”

I) The intravenous delivery of CAR-NK cell therapy presents a promising approach for the treatment of various malignancies. By administering CAR-NK cells directly into the bloodstream, this method allows for wide distribution throughout the body, enabling potential engagement with tumor cells at multiple sites. The non-invasive nature of intravenous delivery minimizes patient discomfort and provides a systemic approach that targets both primary tumors and potential metastases.[8]

II) Α localized and specialized tumor targeting method is endoscopic ultrasound guided fine needle injection which can be used to access and inject directly into tumors in the pancreas. It has been used to deliver chemotherapeutics and has been proposed for use in locoregional delivery of CAR-NK cells. This innovative technique combines the precision of endoscopic ultrasound imaging with the localized delivery of CAR cells directly into the tumor site. By using a fine needle during the EUS procedure, CAR cells are precisely injected into the tumor, allowing for maximum drug concentration within the cancerous tissue while minimizing exposure to healthy surrounding cells. This method offers the advantage of reduced systemic side effects often associated with conventional chemotherapy, as the drug is selectively administered to the tumor area. EUS-guided fine needle injection holds the potential to improve treatment efficacy and patient quality of life by providing a focused therapeutic strategy that targets pancreatic cancer with increased accuracy.[9]

In the context of solid tumors, the likelihood of recurrence depends on the completeness of the surgical margins. While the removal of a tumor through surgery can lead to a patient's cure, any remaining cancerous cells in the surrounding edges, known as margins, can trigger its return. Completely eliminating all residual cells poses a significant challenge for surgeons, mainly due to the difficulty in distinguishing between the tumor and healthy tissue, making it crucial to achieve precise margin clearance. Recurrences after surgical resection of PDAC are unfortunately still common, the rate being as high as 80%, even after R0 surgery where no cancer cells seen microscopically at the primary tumor site.[10]

III) Our potential breakthrough therapy delivery system to address this problem is based on a recent study published in Science Advances developed by researchers from the University of Pennsylvania and offers hope for addressing solid tumor recurrence. The researchers introduced CAR-T cells into a specially designed fibrin gel used for preventing post-surgery bleeding. Applying this gel to the surgical wounds of 20 mice immediately after resecting hard-to-treat tumors led to the prevention of recurrence in 19 of them, all without hindering the healing process. The promising results have prompted

plans for human trials.[11] Based on this work we propose the introduction of CAR-NK cells targeting mesothelin for the treatment of PDAC into an existing product called Tisseel, a fibrin sealant manufactured by Baxter, which surgeons commonly use to control bleeding in challenging surgical sites. It consists of two components: a fibrinogen solution and a thrombin solution. When these two components are mixed, they initiate a process which mimics the body's natural blood clotting mechanism. By carefully adjusting the concentrations of the gel's components we can ensure that CAR-NK cells would not migrate too far from the target area while still reaching the tissue surrounding the tumor. This novel therapeutic approach holds significant potential for improving outcomes in solid tumor surgeries and may offer a promising avenue for future human clinical trials. The CAR-NK cell gel has a lot of potential to be effective, and remarkably, without causing notable side effects, like CAR-T cell gel did.

This novel therapeutic approach holds significant potential for improving outcomes in solid tumor surgeries and may offer a promising avenue for future human clinical trials. The CAR-NK cell gel has a lot of potential to be effective, and remarkably, without causing notable side effects, like CAR-T cell gel did.

Potential as an “off-the-shelf” product & personalized medicine

“Our therapy can be readily available and personalized”

The development of off-the-shelf CAR-NK (Chimeric Antigen Receptor Natural Killer) cell products derived from induced pluripotent stem cells (iPSCs) holds immense promise in revolutionizing cancer immunotherapy. iPSCs have the unique ability to differentiate into various cell types, including NK cells, and can be generated from a variety of sources, including patients themselves. This approach offers the advantage of producing a standardized, scalable, and readily available CAR-NK cell product that can be used for multiple patients without the need for individualized cell harvesting and engineering. By utilizing iPSCs, the challenges associated with donor availability and compatibility can be mitigated. This innovative strategy enables the creation of an o “off-the-shelf” CAR-NK product that can be manufactured in large quantities, stored, and administered to patients as needed. The potential of iPSC-derived CAR-NK off-the-shelf products lies in their ability to provide a convenient and effective therapeutic option, streamlining the treatment process and expanding the accessibility of cutting-edge immunotherapy to a broader patient population.

The utilization of off-the-shelf CAR-NK (Chimeric Antigen Receptor Natural Killer) cell products aligns seamlessly with the principles of personalized medicine. These products, derived from iPSCs, can be engineered to express CARs targeting specific antigens relevant to individual patients' cancer types. This approach allows for the creation of a diverse library of CAR-NK cell lines, each designed to target different antigens commonly found in various cancers. When a patient is diagnosed, their tumor's antigen profile can be analyzed, and the most suitable off-the-shelf CAR-NK product can be selected and administered, offering a rapid and tailored therapeutic response. This not only circumvents the time-consuming process of patient-specific cell collection and engineering but also ensures that patients receive a CAR-NK product optimized for their unique cancer profile. The combination of off-the-shelf convenience and personalized targeting exemplifies how this approach can seamlessly integrate with the tenets of personalized medicine, providing efficient and targeted treatments for individual patients.[12]

The difficulties so far and how to overcome them

“Our therapy offers the possibility to overcome the difficulties arising from conventional therapies”

Current therapies that utilize Chimeric Antigen Receptors, are mostly combined with T-lymphocytes. However, T-cells indicate limitations:

Unsuitable for allogeneic applications

The successful transfer of CAR-T cells obtained from donors can face challenges, primarily stemming from the risk of alloreactivity attributed to the wide range of TCRs (T-cell receptors) expressed by mature T cells. TCRs, not only recognise antigens presented by MHC-I (Major Histocompatibility Complex) molecules, but also the MHC-I molecules themselves, which are highly polymorphic. When donor T cells, carrying their TCRs, are transferred into the recipient, there is a possibility that these TCRs might perceive the recipient's tissues as foreign, triggering a harmful immune response known as Graft-versus-Host-Disease (GvHD). GvHD occurs when alloreactive donor T cells multiply and infiltrate, resulting in the destruction of host tissues, including those found in the skin, liver, and gut.[13]

Increased toxicity

Therapies that CAR-T cells were utilized, indicated danger for Cytokine Release Syndrome (CRS) and neurotoxicity. More specifically, CAR-T cell therapy is also linked to a substantial incidence of side effects. During the initial phase when CAR-T cells are activated and release cytokines as a natural response to CAR engagement, systemic cytokine toxicities can arise. CRS can arise from cytokines directly generated by the CAR-T cells themselves or from immune cells like macrophages, which might produce cytokines in response to those produced by the infused CAR-T cells.[14]

Immune evasion

Exhausted CAR-T cells exhibit decreased proliferative capacity, impaired anti-tumor activity, and attenuated persistence. CAR-T cells anti-tumour activity mostly depends on CAR activation, which is easily limiting their capacity to invade and kill tumors. This is almost prohibited for solid tumors, such as pancreatic cancer.

Allogenic Use

Since NK cells are not triggered via the MHC pathway and have a lower likelihood of alloreactivity, there's no need for the production of autologous NK cells for CAR NK cell manufacturing. More specifically, the Major Histocompatibility Complex (MHC) interacts with Killer Cell Immunoglobulin-like Receptors (KIR) on natural killer (NK) cells, leading to the inhibition of NK cell activity. This is a really important benefit that NK cells provide, considering that there is no danger of GvHD occurrence, creating an “off-the-shelf' product that can be widely used.

Low toxicity risk

NK cells show limited toxicity compared to T cells. This can be a result of the different cytokines released upon activation. When CAR-T cells are activated, they release inflammatory cytokines like tumor necrosis factor alpha, IL-1β, IL-2, and IL-6, among others. On the other hand, patients undergoing CAR- NK therapy do not experience an elevation in these inflammatory cytokines.[15]

Enhanced target methods

NK cells are armed with multiple targeting methods and a variety of unique receptors that enhance their anti-tumour activity. Most importantly, their ability to activate when MHC molecules are missing, is crucial for recognising cancer cells. Tumor cells have developed cytotoxic T cell escape mechanisms, one of which is based on the concealment of MHC-I molecules on their surface. This poses a challenge for CAR-T therapy, but when it comes to CAR-NK cells, this is not a hindrance since they have the capability to identify cells lacking MHC molecules. Furthermore, they have the ability to identify Fc regions through their CD16 receptors, primarily from Ig antibodies. This process is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). In this way, coated target cells can be identified and killed.