Immunotherapy with Chimeric Antigen Receptor Cells (CAR-Cells)

Immunotherapy is a type of cancer treatment that mobilizes the body's immune system to fight cancer cells. Unlike conventional treatments such as chemotherapy and radiotherapy, which attack both cancer and healthy cells, immunotherapy specifically targets cancer cells by stimulating the immune system to recognize and attack them. This treatment approach involves either boosting the body's natural immune response or providing artificial immune cells. that destroy cancer cells Immunotherapy represents a rapidly growing field in cancer research, with ongoing efforts to develop new and more effective treatments that can harness the power of the immune system.[5]

Intensive research in the field of immunotherapy led to the construction of CAR (Chimeric Antigen Receptor) T-cells. These are T-cells that have been genetically engineered to express a chimeric antigen receptor. This receptor carries a single-chain variable fragment (scFv), which binds specifically to an antigen that cancer cells express on their surface. Once CAR-T cells attach to cancer cells, they trigger the immune system to attack and destroy them. This treatment has shown promising results in combating certain types of hematological cancers, including leukemia and lymphoma, receiving approval for its clinical application from the FDA. Research is focused on expanding the use of CAR therapy to other types of cancer and improving its effectiveness and safety. Τhe clinical production of CAR-T cells is summarized in the following figure.

(1) Peripheral blood is obtained from the patient (autologous mode) or the peripheral blood of a healthy donor can be obtained, induced pluripotent stem cells (iPSCs) or cord blood can be also used (allogeneic mode).

(2) The first stage of T-cell isolation involves the process of leukapheresis.

(3) They are then separated and purified from other leukocytes using anti-CD3/CD28 coated beads. This process is followed by cell activation.

(4) The genetic material encoding the chimeric receptors is then introduced into the T-cells by known methods (such as mRNA transfection), viral vectors (e.g. lentivirus) or transposons.

(5) The engineered CAR-expressing T-cells are then expanded in a bioreactor. The patient is receiving chemotherapy to lower the white blood cell count. After 48–96 hours, the CAR T-cells are re-infused into the patient, followed by close monitoring for a few days to watch for any side effects.

CAR-cells are classified as “a living drug” as they are living cells that multiply in the patient's body and provide long term cancer memory. In recent years research has focused on the use of CAR-NK cells, although no therapy utilizing them has yet been approved.[6, 7]

Chimeric Antigen Receptor Structure & Generations

Let’s get to know the Chimeric Antigen Receptor. The basic architecture of CARs includes an extracellular binding domain followed by a hinge region, a transmembrane domain, and intracellular costimulatory motifs. The extracellular region comprises a single-chain variable fragment (scFv) consisting of a variable light (VL) and a variable heavy (VH) portion, which binds a specific antigen. The intracellular domain is responsible for immune cell activation. This receptor binds specifically to an antigen that cancer cells express on their surface.[8]

Based on the costimulatory patterns, CARs are classified into five generations:

1st generation: Includes only Immunoreceptor tyrosine-based activation motifs (ITAM).

2nd generation: Includes an additional costimulatory motif.

3rd generation: It has two additional costimulatory motifs added.

4th generation: It was designed based on the 2nd generation but coupled to cytokine expression under transcriptional control of NFAT transcription factor.

5th generation: I was also designed based on the 2nd generation with additional intracellular components such as cytokine receptors domains that activate the JAK and STAT3/5 signaling pathways enhancing immune cell proliferation and stability.

CAR-NK VS CAR-T cells

Natural Killers cells are an essential ingredient of natural immunity. They can bind to cancer cells and release lytic granules containing substances such as granzymes and perforin. Perforin is a protein that forms pores in the membrane of target cells allowing granzymes, which are special enzymes, to enter and cause apoptosis. CAR-NK cells have a lot of advantages over CAR-T cells as they offer better safety for the patient, significantly limiting various unwanted effects of immunotherapy such as cytokine release or neurotoxicity syndrome. In addition, they have a greater ability to penetrate solid tumors and a greater potential of allogeneic use. Their production is easier while they have more mechanisms to activate their cytotoxic activity.[9-12]

The different ways of NK-mediated tumor killing and immune system regulation are shown in the figure above.(A) NK cells are capable of enhancing the antigen presentation to T-cells by killing the immature dendritic cells while promoting the IFN-g and TNF-a mediated maturation of them.(B) NK cells can specifically recognize the cells that lack the expression of self-MHC class I molecules (Missing-self).(C) They have receptors that are able to recognize antibodies that bind to cancer cells exhibiting antibody-dependent cytotoxicity (ADCC)(D) They use the Fas/FasL pathway as the binding of FasL to Fas results in delivering a “death signal” to the target cell that undergoes apoptosis shortly.(E) Cytokine pathway can also exert anti-tumor potential. (F) NK cell receptors NKG2D are capable of recognizing the “induced-self” ligands that are expressed at a very high rate in response to the activation of tumor-associated pathways.(G) Checkpoint blockade may inhibit NK cell suppression by preventing the interaction of NK cell inhibitory receptors with their ligands.(H) As a result of adoptive NK cells transfer, the mismatch between donor and recipient, inhibitory KIRs, NK cells eliminate the allogeneic tumor cells that lack self-MHC.(I) CAR-NK cells designed specifically to target overexpressed tumor antigens.(J) Bispecific molecules are also being utilized to specifically eliminate tumor cells as these special molecules bind to activating NK cell receptors on one arm and tumor antigens on the other.(K) NK cells can enhance or diminish macrophage and T-cell activities via IFN-g and IL-10 production.[13]

iPSCs as an unlimited source of NK cells

Stem cells are one subpopulation of not differentiated, self-renewing cells found in most adult tissues and are responsible for the replacement of cells which have lost the ability of cell division and so they play a crucial role in the maintenance of most tissues and organs.

Recent studies in the field of molecular biology and genetics have led to the discovery of a method of transforming differentiated somatic cells into pluripotent stem cells (PSCs). The transformation of somatic cells into pluripotent stem cells was first published by Kazutoshi Takahashi and Shinya Yamanaka in 2006.[14] The researchers showed that mouse fibroblast gene expression could be reprogrammed so that to give rise to cells that resemble embryonic stem cells. These cells were calledinduced pluripotent stem cells (iPSCs). The reprogramming required the action of only four transcription factors introduced with retroviral infection. The infected cells were able to differentiate into all cell types.

In subsequent studies it was shown that human cells can be reprogrammed by a similar mechanism. Reprogramming fibroblasts into pluripotent stem cells can now be achieved not only with the four transcription factors they had researchers initially used. Four major transcription factors (Oct4, Sox2, Nanog and Klf4) play a central role in this mechanism of gene expression and are responsible for maintaining pluripotency forming a self-regulating positive feedback loop while suppressing genes that induce cell differentiation. This self-regulating circuit helped the researchers to overcome some difficulties such as for example the fact that some of the initial transcription factors used (e.g. c-Myc) act as oncogenes. Once the process is complete the phenomenon of self-regulation it henceforth maintains multipotency without the need for continuous exogenous expression of the factors that initially caused the reprogramming.

Induced pluripotent stem cells hold immense promise in the field of CAR-NK (Chimeric Antigen Receptor Natural Killer) therapy. By harnessing the regenerative potential of iPSCs, it becomes possible to generate a virtually unlimited supply of genetically stable and customizable NK cells. These iPSC-derived CAR-NK cells can be precisely engineered to target specific cancer antigens, offering a potent and adaptable tool in the fight against various malignancies. Additionally, iPSC-based CAR-NK therapy reduces the reliance on donor-derived cells, potentially mitigating issues related to graft-versus-host disease and donor availability. This innovative approach represents a significant advancement in the development of safer, more effective, and personalized immunotherapies for cancer treatment.

Keratinocytes as a promising somatic cell source of iPSCs

Numerous groups have explored various somatic cell sources to identify the promising sources for reprogramming into iPSCs with different reprogramming factor combinations. To optimize the potential for future biomedical applications, it is highly desirable to create genetically stable induced pluripotent stem cells (iPSCs) from a somatic cell source that meets specific criteria. These criteria include: (i) the cell source should be abundant in tissues, (ii) it should be easily accessible, allowing for minimally invasive isolation, (iii) it should be straightforward to culture and expand rapidly to obtain a sufficient cell count for reprogramming in the shortest possible time, (iv) it should primarily lack critical somatic mutations and chromosomal abnormalities, (v) it should be amenable to efficient and swift reprogramming, and (vi) it should be capable of reprogramming cells from individuals of varying ages and health conditions.[15] The basic characteristics of the most common somatic cell sources are summarized in the following

Taking these into account as well as the existing relevant literature, we propose the use of keratinocytes for the production of iPSCs and their use in cellular therapy. Keratinocytes account for most of the cells in the epidermis of the human skin. Various types of human body hair are suitable for generating iPSCs. In addition to scalp hair, facial hair types such as beard, eyebrow, and nasal hair can also be utilized. Apart from their presence in the dermis, keratinocytes are also found in hair follicles, which are embedded in the dermis and not visible on the skin's surface. These follicles have two root sheaths, the inner (IRS) and outer (ORS) root sheaths, with hair follicle stem cells located in the ORS bulge, giving rise to keratinocytes and other cell types. Mature keratinocytes play a crucial role in producing keratin for hair growth. The human hair cycle includes distinct stages discernible by the shape of the hair bulb, with anagen hair bulbs, characterized by darkly pigmented, hockey stick-shaped bulbs with distinct root sheaths, representing the growth phase. Telogen hair has no or an insufficient root sheath, features a club-shaped bulb, and is less pigmented than anagen bulbs, indicating a resting hair follicle stage. Catagen hair bulbs represent a very short transition phase between the previous stages, with a narrowing bulb and hair atrophy.

To initiate efficient reprogramming, a sufficient number of cells, in this case, keratinocytes, are required. The crucial part is to pluck the hair with an intact root, including the intact ORS. The ORS is identifiable as a white covering around the root. Once plucked, hair can be stored in a standard DMEM medium for several days at room temperature, allowing for easy shipment from various parts of the world without concerns about the ability of keratinocytes to proliferate. This method offers convenience for all parties involved, as donors do not need to undergo clinical and surgical investigations or visit a hospital. Scientists can obtain the hair with minimal effort from the person plucking it.

In order to culture keratinocytes effectively, specialized low-calcium media formulations are accessible to prevent premature senescence. This presents an advantage because, firstly, most other cell types do not proliferate successfully in low-calcium environments. Secondly, after initiating the reprogramming process, a new medium with normal calcium levels is introduced. This causes uninfected keratinocytes to either reduce or cease their proliferation. Only infected cells continue to divide, a prerequisite for reprogramming into stem cells.[16] Once the culture handling procedure is established a single outgrowing hair root provides a sufficient quantity of keratinocytes for a successful reprogramming process. The huge advantage of this isolation process is the non-invasive procedure, which takes only a few minutes. Keratinocytes undergo reprogramming at a notably faster pace, typically within a span of 1 to 2 weeks. This stands in stark contrast to the lengthier period of 3 to 4 weeks required for fibroblasts. Furthermore, fibroblasts exhibit substantially lower expression levels of key genes such as c-Myc and Klf4 when compared to other cell types. The pre-existing elevated baseline levels of these genes in keratinocytes, as observed, might contribute to a more efficient reprogramming process.[17] A typical reprogramming procedure is as follows:

(1) Hair that has been plucked is cultured in flasks until keratinocytes start to grow out from it. These keratinocytes are then transferred to a six-well plate.

(2) Next, the keratinocytes are exposed to infection with a lentivirus that carries the four reprogramming factors: Oct4, Sox2, Klf5, and c-Myc.

(3) Following infection, the keratinocytes that have been infected are moved to a culture plate along with feeder cells, such as rat embryonic fibroblasts, and placed in a reprogramming medium.

(4) Over the course of two to three weeks, colonies of stem cells emerge, and the uninfected keratinocytes do not continue to proliferate in the reprogramming medium.

(5) Once these stem cell colonies reach a certain size, they are manually picked, and it may be possible to transition to a feeder-free system for the cultivation of human iPSCs.

iPSCs differentiation to NK cells

In 2019 Kaufman et al., introduced an improved method for iPSCs differentiation, outlining an enhanced approach to generate NK cells, achieving greater efficiency and reduced time compared to earlier methods, that mentioned before. Τhe new approach is based on iPSCs that are already adapted to a feeder-free environment and utilizes Rho-associated protein kinase inhibitor (ROCKi), resulting in the formation of embryoid bodies (EBs). Both new and old methods employ spinning EBs, with the old method taking 12 days and the new one taking 8 days, to produce hematopoietic progenitor cells (CD34+ cells). These EBs are then directly transferred into conditions conducive to NK cell differentiation. Mature and fully functional NK cells develop within a period of approximately 4 weeks. Successful differentiation is tested through the expression of specific receptors, such as CD56, CD94, CD16 or NKG2D by flow cytometry. Expansion of iPSCs derived NK is performed using artificial antigen-presenting cells (aAPCs), such as membrane-bound IL-21 expressing (mbIL-21) K562 cells, that are first irradiated. Cytokines like IL-2, IL-12, IL-15, IL-18, IL-21, and type I interferons are crucial elements in the development, stimulation, and persistence of NK cells.[18]

Why Mesothelin?

Mesothelin is a 40 kDa glycoprotein that is primarily found in the mesothelial cells lining the pleura (the thin membrane surrounding the lungs and chest cavity), peritoneum (the lining of the abdominal cavity), and pericardium (the sac around the heart). As mesothelin undergoes maturation, a 36-amino acid signal sequence is removed, resulting in the deletion of the 599-621 sequence at the C-terminus. GPI is then attached to this region, enabling mesothelin to remain anchored to the membrane. A segment of the pre-pro MSLN undergoes proteolytic cleavage by furin and is subsequently released. The fragment that becomes soluble after cleavage is known as the Megakaryocyte Potentiating Factor (MPF).[19]

Mesothelin has gained attention in medical research and cancer treatment because it is overexpressed in several types of cancers, particularly in mesothelioma, a rare and aggressive cancer that affects the mesothelial lining of the lungs, abdomen, or heart. High levels of mesothelin are also found in some cases of ovarian cancer, pancreatic cancer, and lung adenocarcinoma. The overexpression of mesothelin in cancer cells has led to the development of diagnostic tests and targeted therapies that aim to detect and treat cancers associated with elevated mesothelin levels. For example, mesothelin-targeted therapies, such as antibody-drug conjugates (ADCs) and immunotherapies, are being explored in clinical trials as potential treatments for mesothelioma and other mesothelin-expressing cancers.

The Mesothelin Challenge

Anchored Mesothelin undergoes a process known asshedding , which is instigated by proteolytic enzymes capable of recognizing various points within the amino acid sequence. At least five of these proteolytic enzymes have been identified as responsible for cleaving at distinct sites, resulting in the retention of only a portion of the mesothelin on the cell membrane.

Nonetheless, the process of shedding poses a challenge when endeavoring to target tumor cells expressing mesothelin. In comparison to healthy tissues, the tumor microenvironment harbors an elevated concentration of diverse proteases, which may, in certain instances, exhibit variation depending on the specific cancer cell line. Thus, the construction of a Chimeric Antigen Receptor, encompassing the single-chain variable fragment (scFv) responsible for mesothelin recognition and precise targeting, necessitates a focus on the persistent fragment that remains tethered to the cell membrane, subsequently inducing apoptosis in cancer cells.[20]

What did we think?

After extensive literature research we thought of combining the work of two research groups. The first group in 2018 compared nine Chimeric Antigen Receptor constructs on NK-92 cells (cell line derived from peripheral blood mononuclear cells from a 50-year-old, White male with rapidly progressive non-Hodgkin's lymphoma) and iPSCs-derived NK cells. Researchers identified a CAR containing the transmembrane domain of NKG2D, the 2B4 costimulatory domain, and the CD3ζ signaling domain to mediate the strongest antigen-specific NK cells signaling among all of the constructs and conferred NK-92 cells the greatest cytotoxicity against ovarian cancer cells.[21]

Τhis research group used the SS1 scFv to construct the chimeric antigen receptor, which is unable to target mesothelin after its partial degradation by proteolytic enzymes. Here comes the work of the second research group on which the project is based, who succeeded and in 2022 published a scFv sequence that binds to the region of mesothelin that remains in the membrane even after its proteolytic cleavage. This research group used the special 15B6 scFv to make CAR-T cells and tested the cytotoxic activity in different cancer cell lines.[22]

So we decided to combine these works by producing CAR-NK cells derived from Induced Pluripotent Stem Cells (iPSCs) by lentiviral transduction. These cells will have a chimeric receptor with a robust structure that will more efficiently target mesothelin of pancreatic cancer cells, representing a potent approach for fighting pancreatic cancer, a malignancy characterized by high mesothelin expression. Our CAR (15B6 scFv-NKG2D-2B4-CD3ζ) belongs to the second generation and is presented below.

Lentiviral Transduction

Lentiviral transduction is a viral vector-based technique that allows for efficient and stable integration of exogenous genetic material into a wide range of host cells. These vectors, which are descended from the retrovirus family, are widely utilized in the field of gene therapy and constitute a commonly used method for gene delivery in mammalian cells. The vector DNA is translated into RNA inside the packaging cells, and viral proteins, which are produced by the helper plasmids, further package the RNA into the virus. The virus is then released into the supernatant, where it can infect the desired cells. In our case formed lentiviral vectors that are released by HEK 293 cells in the supernatant, are then harvested and ready to transduce iPSCs-derived NK cells. Lentiviral particles are produced by transfecting HEK 293 cells using 2nd generation lentiviral plasmids.These plasmids carry the lentiviral genome, which has been modified for safety reasons, retaining only the genes necessary for lentiviral vector synthesis and thereby preventing any replication capability. In the 2nd generation lentiviral plasmid system, the viral genetic material is fragmented into three separate plasmids.

Ι) Packaging plasmid contains essential genes such as gag, pol, tat and rev that are necessary for assembling the vector. Structure-related proteins are encoded by gag, while enzymes necessary for reverse transcription and genome integration are encoded by pol. The export of unspliced or partly spliced viral mRNA from the nucleus to the cytoplasm, which is essential for the creation of viral proteins, depends on Rev. The encoded Rev protein functions by attaching to the Rev Responsive Element (RRE), a structural component of viral RNA. Tat is a regulatory gene that promotes the activation of the 5' LTR (Long Terminal Repeat) region of transfer plasmid initiating transcription, in 2nd generation lentiviral systems. Generally, wild type LTRs are tat dependent.

ΙΙ) Transfer plasmid contains the sequence of MSLN-CAR, that is cloned between LTR regions, that are found at both ends of the plasmid and they are essential for initiating transcription and correspondingly for its termination. Ψ element is also included in transfer plasmid and is responsible for encapsulating the genome into viral particles.

III) Envelope plasmid, generally the stable VSV-G envelope, is necessary to give the viral particle receptor binding and membrane fusion properties. VSV-G-pseudotyped lentiviral vectors are capable of infecting a large variety of host cells, including both proliferating and non-dividing cells. This broad tropism makes them ideal for transducing a wide range of cell types, including primary cells and diverse cell lines.[23, 24]

“Off-the-shelf” product

An "off-the-shelf" product is a product that has been created and is readily available for use. In the context of cell therapy, it refers to a product that is manufactured in advance and can be used to treat many patients without the need for customization or modification. Off-the-shelf therapeutic products provide many advantages over custom cell therapy products. Some of these include:

Availability: Off-the-shelf products are readily available and can be used to treat many patients without the need for adaptation or modification. This means patients can receive treatment more quickly and easily, without having to wait for a customized product to be manufactured.

Cost-effectiveness: Off-the-shelf products are usually less expensive than customized or customized products, as they can be manufactured in large batches and do not require the same level of customization or personalization.

Consistency: Manufactured to a consistent quality and standard, ensuring that every patient receives the same quality of treatment. Highly personalized or customized products, on the other hand, can vary in quality depending on factors such as the patient's health status and the manufacturing process.

Safety: They are often safer than personalized or customized products as they do not require the use of the patient's own cells. This reduces the risk of side effects such as graft-versus-host disease (GVHD) and other immune-related complications.

In the case of CAR-NK cell therapy, for example, an "off-the-shelf" product would be one made using NK cells that have been isolated or produced from some source and genetically modified to express a chimeric antigen receptor ( AR). This product can be stored and used to treat multiple patients without the need to adapt or modify for each individual patient.