English Transcript Cell Therapy Primer_edited version.pdf

Proprietary. Do Not Share. Transcript: Cell Therapy Primer Section 1: Cell Therapy Primer Welcome Welcome to the Biotech Primer class Cell Therapy Primer. This course will consist of four sections: Cell Therapy Defined, Chimeric Antigen Receptor, CAR-T’s Commercial Landscape, and Next Generation Ce...

Proprietary. Do Not Share. Transcript: Cell Therapy Primer Section 1: Cell Therapy Primer Welcome Welcome to the Biotech Primer class Cell Therapy Primer. This course will consist of four sections: Cell Therapy Defined, Chimeric Antigen Receptor, CAR-T’s Commercial Landscape, and Next Generation Cell Therapy. Section one will provide an overview of the first cell therapies and compare two types of cells commercially available today for cell therapy development. Section 1: Cell Therapy Primer Objective At the end of this section, you should be able to: • Define the term cell therapy. • Explore varieties of cells in the human body. • Identify the types of cells considered for cell therapy. • Differentiate between autologous and allogeneic cells. • Finally, list the types of cell therapy. Blood Transfusions Cell therapy has been around for over a century in the form of blood transfusions. Blood Transfusions Explained Blood transfusions deliver red blood cells to the patient who has lost blood. Red blood cells are crucial as they have a component called hemoglobin which carries oxygen and allows oxygen delivery to the body after a blood loss. But only some people's blood is compatible with others as the presence or absence of specific antigens and multiple factors determine the four blood groups. 1 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Blood Transfusions Discovery Since Karl Landsteiner discovered blood types in 1901, blood transfusions, as a form of medical therapy, have made surgeries and various therapeutics possible. Bone Marrow Transplants Since then, further advancements have been made in medical transfusions in the form of bone marrow transplants. Bone marrow transplants harvest cells from the bone marrow of healthy donors and insert them into patients who do not produce healthy blood cells. Scientists observed that not all bone marrow transplants were successful. In the 1960s, surface proteins called Human Leukocyte Antigen (HLA) were identified as being used by the immune system to differentiate between the self and foreign cells. Each person has different HLA markers, half inherited from their mother and half from their father. It was found that a minimum of six HLAs should match for a successful bone marrow transplant. Blood transfusions and bone marrow transplants are types of cell therapy. Cell Therapy Defined Cell therapy is a treatment that uses donor cells. The donor cell can be from the patient's body or a different person to help treat a disease or condition. These cells are collected, purified, increased in number, and then reinfused in the patients, as in the case of blood transfusion or bone marrow transplant. Today, we have the technology to manipulate these harvested cells using gene therapy and then reinject the manipulated cells in the patient. The idea is to treat a disease at the cellular level. The goal of cell therapy is to provide functional cells, cells that perform the function they are meant to accomplish. Cell Origin All cells start as pluripotent stem cells. Pluripotent stem cells perform multiple functions until they become specialized or differentiated. This specialization occurs when the cell received signals or cues and becomes specialized or differentiated. Once a cell is differentiated, it mostly maintains that identity and function. Cell differentiation happens in the embryonic stages of 2 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. development. Although all cells have the same genetic information in their DNA, the differentiated cells have restricted access to different parts of the DNA to express that specific protein. Thus, the cells become different from each other in size, shape, and function. Cell Therapy Utilizes Different Cell Types Now that we know cells differentiate from each other to perform specific functions, it becomes crucial to identify which cell type is needed to cure a particular disease or condition. Let’s look at some examples. 1. Patients who have lost too much blood need their red blood cells replenished. 2. Immunocompromised patients with a low white blood cell count need their white blood cells replenished. 3. Patients with platelet dysfunction or low platelet counts, due to Dengue fever or hemophilia need their platelets replenished. Each of these blood cells has different proteins that allow them to perform a particular role. For example, hemoglobin helps red blood cells carry oxygen. Antibodies, created by white blood cells, fight off invading pathogens. And platelets express factors that help blood clotting. Therefore, the key to cell therapy is identifying, purifying, and delivering the specific cell type that would benefit a patient with a particular disease. The Cell Source As we stated in the previous slide, the key to successful cell therapy is to identify, purify, and deliver the cell needed by a patient. There are two sources to obtain these cells―autologous cells and allogeneic cells. Autologous cells are cells derived from the patients themselves. And allogeneic cells are cells harvested from healthy donors. The cell source matters as they have inherent advantages and disadvantages. 3 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Autologous Cells Advantages The autologous cells originate from the patient. As a result, this cell type has advantages. Autologous cells have low immunogenicity, meaning the body's immune system will not reject the cells. These cells are persistent and have a longer life span than allogeneic cells. The source of the cell is abundant as the patient can donate samples as needed. And the final significant advantage is the regulatory aspect. If the patient’s cells are only purified and redelivered to that patient (no gene is added), the cells are seen as “minimally manipulated” by the FDA, and the time taken to get approval is far less. Autologous Cell Disadvantages Though autologous cells look very promising for cell therapy, there are some significant disadvantages to autologous cells. The logistics of performing cell therapy from an individual’s cells take time and effort. Cell harvesting can be complicated if the patient is old or sick, which is usually the situation. As we age, cell division slows down, and the quality of the cells is compromised as more mutations accumulate. Moreover, sick patients may not be well enough to go through a biopsy to harvest cell samples. Since these treatments are custom-made for a patient, the variability in starting material requires more research, testing, and validation to ensure cells produce the same desired result. What worked for one patient may not work for the next. Finally, the cost of autologous cell therapy is so expensive that it may be out of reach for most individuals. Considering the challenges associated with autologous cell therapy, an alternative cell source of allogenic cells was pursued. Allogeneic Cells Advantages Allogeneic cells are collected from young, healthy donors and are manipulated to treat patients. Allogeneic cells can be pre-processed for a particular cell type. This is accomplished through standardization, meaning every patient gets a similar and more reliable drug product because 4 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. controls and release criteria such as functionality, number of cells, or quality of proteins produced are standardized. Finally, allogeneic cells can be manufactured centrally and distributed broadly, potentially decreasing production costs due to economies of scale. The vision of the biopharma industry is to eventually have off-the-shelf allogeneic cell therapy banks for specific disease therapy, like today’s blood banks. Allogeneic Cells Disadvantages Though allogeneic cells may be the future of cell therapy, these cells also have associated disadvantages that need to be considered. A patient's immune system will recognize allogeneic cells as foreign and induce an immunological response, sometimes resulting in a cytokine storm or Graft Versus Host Disease or GVHD that can potentially shut down organs and be fatal. There is also a risk of alloimmunization when the allogenic cell therapy delivered a second time precipitates a severe immunological response. It becomes impossible to use allogeneic cell therapy again. The allogeneic cells can be cleared from the body quickly before they have completed their job. The risk of transmitting infectious diseases through allogeneic cell therapy is high so the donors must be screened for contagious diseases before they are eligible to donate samples. Finally, the most significant hurdle is getting through the regulatory phase. Allogeneic cell therapy is highly regulated and requires clinical trials for approval. Section 1: Cell Therapy Primer Summary In this section, we learned: • Cell therapy replaces damaged or non-functioning cells with healthy cells. • The two types of cell sources used in cell therapy are autologous and allogeneic cells. 5 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. • The autologous cells originate from the patient. Allogeneic cells are collected from young, healthy donors and used to treat patients. • Types of cell therapies include blood transfusions, bone marrow transplants, and autologous and allogeneic cell therapy. 6 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Section 2: Chimeric Antigen Receptor Introduction Section two reviews the immune system’s role and how cancer evades it. We end the section by explaining how Chimeric Antigen Receptors (CARs) are used in cell therapy to detect and destroy malignant cells. Section 2: Chimeric Antigen Receptor Objectives At the end of this section, you should be able to: • Describe the types of lymphocytes, their functions, and their mechanism of action. • List the reasons cancer can evade our immune system. • Explain the role of the chimeric antigen receptor. • State the parts of the chimeric antigen receptor. Lymphocytes Let’s begin this section by quickly reviewing the roles of the immune system cells. Lymphocytes are cells that elicit an immune response. There are two main types of lymphocytes: T-cells and B-cells. T-Cells T-cells originate from stem cells in the bone marrow but migrate out of the bone marrow and into the thymus gland, where they mature into Cytotoxic T-cells and Helper T-cells. Both types of T-cells have unique receptors, aptly named T-cell receptors or TCR. Activated Cytotoxic T-Cell Cytotoxic T-cells secrete cytotoxins, a poison, to kill cancerous cells. The Cytotoxic T-cell strategy is two-fold. The first is to release cytotoxic granules that punch a hole in the diseased cell to make a path for granzyme to enter and denature its proteins resulting in the cell’s death. The second is to secrete cytokines that activate and attract macrophages to phagocytize the cancer cells. 7 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Activated Helper T-Cells Helper T-cells help coordinate the overall immune response by secreting signaling proteins called cytokines to activate and direct other immune cells, such as Cytotoxic T-cells, B-cells, and macrophages, to do their jobs. Helper T-cells cannot kill; they recruit other immune cells to do this. Activated B-Cells Activated B-cells produce antibodies that seek out and bind to the malignant cell’s antigens. The antibodies signal for other immune cells― macrophages, neutrophils, and dendritic cells―to destroy and clear the cancer cells. Major Histocompatibility Complex (MHC) A major Histocompatibility Complex is commonly known as the MHC complex or molecule. The MHC presents an antigen in its pocket. That antigen is known as the Human Leukocyte Antigen (HLA). There are two types of MHC molecules, MHC Class I and MHC Class II. MHC Class I span the membrane of almost every cell in a human, while MHC Class II is restricted to the cells of the immune system. MHC molecules are important components of the immune system. The T-cell recognizes the foreign antigen attached to the MHC molecule and binds to it, stimulating an immune response. In uninfected healthy cells, the MHC molecule presents antigens from its cell (self-antigens) to which T-cells do not normally react. Tumor-Specific Antigens and Tumor-Associated Antigens Immunologists distinguish broadly between two types of tumor antigens: tumor-specific antigens, which are found only on cancer cells and not on their normal counterparts, and tumorassociated antigens, which are located on both normal cells and cancer cells but are abnormally expressed on cancer cells. In both cases, these antigens have been shown to evoke an immune response, although not necessarily one strong enough to eliminate the tumor. 8 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Cancer: Malignant and Benign Cancer is a term used for a broad spectrum of diseases, all originating from uncontrolled cellular growth due to genetic mutation. Cancer can be broadly classified as malignant (which invades other body cells and turns them into cancerous metastasis) or benign (where the cells do not metastasize). Cancer: Liquid and Solid Tumors Though cancers can be grouped in many ways, we will consider liquid and solid tumors for this course. Liquid or fluid cancer are mobile cells, notably the blood cancers like leukemia and lymphoma. Solid or tumor cancers are growths in organs or tissues that form lumps and can spread to other body parts if they metastasize. To date, all FDA-approved CAR-T cell therapies are for blood cancers, specifically, cancer found in B-cells. The Immune Challenge Several mechanisms have been identified that allow cancer to avoid recognition and destruction by the immune system. The surfaces of cancer cells may lose antigens recognizable by the immune system. Cancer cells may lose all MHC Class I molecules from their surface, which prevents Cytotoxic T-cells from recognizing them. Some cancer cells produce immunosuppressive chemicals that inhibit T-cells directly. Cancer cells shed some of their antigens that bind to the receptors on Cytotoxic T-cells, plugging the TCR up so that the T-cells cannot bind to and identify the cancer cell for elimination. Cancer cells can outmaneuver an immune response by growing so rapidly and becoming so dense that immune cells cannot come in contact with the cancer cells. Chimeric Antigen Receptor Chimeric Antigen Receptors (CAR) are genetically engineered receptors that can replace the TCR on T-cells. These genetically engineered cells are called CAR-T. The chimeric receptor empowers the immune cell in two ways―freeing them of their MHC dependence and helping them identify the tumor-specific antigens more efficiently. The genius of the Chimeric Antigen 9 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Receptor is that it can be engineered to target specific cancers. CARs recognize tumor-specific antigens specific to cancer cells, induces patients' immune system to target that cancer cell’s antigens, and safely neutralizes the tumor without damaging healthy cells. Chimeric Antigen Receptor Design Let’s end this section by learning the parts of the Chimeric Antigen Receptor or CAR. The Target Element is the chimeric part of the receptor. These artificially made constructs of the variable regions of antibodies provide specificity to the receptor. In the case of cancer, the Target Element would bind to the tumor-specific antigen. The Linker is also called the spacer or hinge region. It is a protein that acts as a flexible connector that can join to any Target Element forming the CAR. The linker is present outside the cell surface. Its length depends on the region where the receptor is present. The Transmembrane Domain is part of the receptor embedded in the cell membrane. It helps dock the receptor in the membrane and interact with the neighboring receptor. The Co-Stimulatory Domain is the internal part of the receptor that, when activated, induces the cell to make cytokines. Lastly, the Signaling Domain is another internal part of the receptor that signals and activates other biochemical processes within the cell, like rapid cell division. Section 2: Chimeric Antigen Receptor Summary In this section, we learned: • B-cells and T-cells are different lymphocytes forming part of the immune system. B-cells produce antibodies that flag cancer cells for destruction. Cytotoxic T-cells kill the malignant cells directly by releasing the cytotoxic granules. Helper T-cells assist other immune cells in mounting an attack. 10 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. • Cancer can evade the immune system by losing antigens, producing immunosuppressive chemicals, using its antigens to plug up Cytotoxic Tcells, and growing so rapidly that the immune cells cannot penetrate the tumor. • Chimeric Antigen Receptor (CAR) is a genetically engineered receptor specific to an antigen on a malignant cell. • CAR-T cells are T-cells genetically modified to express a Chimeric Antigen Receptor (CAR). • The parts of the CAR are Target Element, Linker, Transmembrane Domain, Co-Stimulatory Domain, and Signaling Domain. 11 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Section 3: CAR-T’s Commercial Landscape Introduction CAR-T was first developed by Israeli immunologists Zelig Eshhar and Gideon Gross between 1989-1993. In section three, we discuss the commercial landscape of CAR-T. Section 3: CAR-T’s Commercial Landscape Objectives At the end of this section, you should be able to: • Describe how CAR-T cells are made. • Recognize CAR-T cell therapy products available today. • Discuss the efficacy of CAR-T cell therapy products. • Explain the side effects of CAR-T cell therapy. • List the CAR-T cell therapy efficacy challenges. CAR-T As explained in the last section, a Chimeric Antigen Receptor (CAR) is a genetically engineered receptor. CARs can be placed on various cells, the T-cell being the current industry favorite. CAR-T cell therapy is considered a type of cell-based gene therapy because it involves altering the genes inside T-cells. The FDA categorizes CAR-T as a gene therapy. CAR-T Inputs The cells used for CAR-T are autologous T-cells. This means the T-cells are obtained from the patient and are genetically modified specifically for that patient. The inputs needed to make CAR-T cells include: 12 • The patient’s T-cells. • A pre-identified gene that codes for the receptor to be present on the CAR. • A viral vector. Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Obtaining T-Cells The process begins with the extraction of blood from the patient. The blood sample is processed by a method called leukapheresis which isolates the T-cells of interest. These T-cells need to be activated before proceeding to further steps. T-Cell Activation First, a gene that codes for the desired chimeric antigen receptor is placed into a lentivirus vector. A lentivirus vector is the most used viral vector for cell therapies because it can carry a CAR gene which is 8,000-10,000 bases in size. Then the lentivirus vector is incubated with the patient’s T-cells. The industry favors lentiviral vectors for ex vivo cell therapies for several reasons. Lentivirusral vectors integrate the CAR gene directly into the chromosomes of the T-cell, so the gene replicates with the rest of the genome each time the cell divides. This integration leads to sustained protein expression, even in rapidly dividing cells. This sustained protein expression is the reason CAR-T leads to longlasting health outcomes. Approved CAR-T Products Multiple FDA-approved CAR-T cell therapy products are currently available. These include Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykti. All these CAR-T cell therapy products share some standard features: 13 • They all use autologous T-cells. • They are customized for individual patients. • They target liquid cancer cells. • They are indicated for lymphomas or leukemias. • They specifically recognize and target cancers of the B-cells. • They are manufactured in a similar way. Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Drug Product Design Four approved CAR-T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi) bind the Bcell CD19 antigen. Two (Abecma and Carvykti) bind to the B-Cell Maturation Antigen (BCMA) often found on multiple myeloma cells. CD19 is a tumor-specific antigen on the surface of cancerous B-cells. The CAR-T cells are made with a targeting gene that produces a CAR that binds the CD19 antigen. This enables CAR-T cells to recognize cancerous B-cells with CD19 on the surface, become activated, release cytotoxins, and kill the cancerous B-cells. Similarly, BCMA is a tumor-associated antigen present in high amounts on multiple myeloma cells. In this case, the CAR-T cells are made with a targeting gene that produces a receptor that binds the BCMA antigen, and once bound, the CAR-T cell will release cytotoxins to kill the multiple myeloma cell. Carvykti Development To learn more about the efficacy of CAR-T, we drill down and examine a key clinical trial result from Carvykti. Carvykti was developed by Legend Biotech and its partner, Johnson & Johnson. CAR-T Cells Work! This heavily treated patient group typically has a life expectancy of a year. In contrast, 70% of the patients from the Carvykti pivotal trial were still alive 27 months after a single infusion! That is an unbelievable improvement over the previous standard of care. Carvykti is one specific Bcell therapy, but it's representative of the data coming out against other types of B-cell cancers. Side Effects of CAR-T Though CAR-T cell therapy is very promising, Cytokine release syndrome (CRS) and neurotoxicity are well-known side effects of CAR-T cell therapy. According to the American Society for Blood and Marrow Transplantation (ASBMT), there are four grades of side effect severity for both CRS and neurotoxicity. Low-grade (grade 1) events can be treated with 14 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. supportive care, while for grades 2-4, corticosteroids and other drugs can be used. Let’s take a closer look. Cytokine Release Syndrome When CAR-T cells identify an antigen, they activate and mount an immune response by secreting cytokines that activate other immune cells. Once the target is destroyed, all the cytokines enter the circulation creating a storm of cytokines in the blood. This is what is known as Cytokine Release Syndrome (CRS). CRS is an acute inflammatory immune response where high levels of released cytokines send a deluge of signals to activate the immune system into overdrive. The most common symptoms of CRS are fever, hypotension, and hypoxia. Currently, these stages can be controlled with medications to known side effects. Neurotoxicity Neurologic events can occur along with Cytokine Release Syndrome. In such cases, the nerves of the peripheral nervous system are damaged and cannot properly communicate information to the organs around them. As a result, it causes varying degrees of loss of function. The symptoms may include hallucinations, tremors, and seizures. Although neurotoxicity can be managed and reversed under certain circumstances, it is still a significant concern in sick and immunosuppressed patients undergoing CAR-T cell therapy. Other Side Effects of CAR-T While CRS and neurotoxicity are short-term side effects that appear by the first week of starting the therapy, there are some long-term side effects associated with the CAR-T cell therapy, such as B-cell aplasia (extremely low B-cell count) resulting in hyperglobulinemia (low serum antibody levels) and cytopenia (low blood cell count). B-cell aplasia may require treatment with monthly immunoglobulin G infusions. Commercial Challenges of CAR-T CAR-T is a new and exciting field of cell therapy but is yet to overcome many challenges. While this therapy is successful, not all patients respond to CAR-T cell therapy. We discussed the side 15 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. effects of CAR-T cell therapy in the previous screen; now let’s look at a few challenges that impact their efficacy. CAR-T Cell Exhaustion Known as CAR-T cell exhaustion, the CAR-T cells get turned off by a negative feedback mechanism due to excessive antigen signaling. When CAR-T cells face constant stimulation or overstimulation and release excessive cytokines, these cytokines activate the inhibitory receptors on the CAR-T cells and shut down their production in the cell. As a result, the CAR-T cells no longer recognize the antigen or release cytokines and eventually stop killing the target cells. In addition, the quality of the T-cells harvested from the patient also matters, as dysfunctional T-cells are only persistent for a short time. CAR-T Cytokine Shut Down The tumor's immediate surroundings, including the blood vessels, neighboring cells, signaling molecules, and the extracellular matrix that hold the tumor cells together, is known as its microenvironment. This microenvironment is immunosuppressive, limiting immune cell proliferation and activity. The CAR-T cells cannot function well in this environment as the tumor cell releases anti-inflammatory signaling molecules that shut down the CAR-T cell's cytokine production mechanism. Even if CAR-T cells are injected into the tumor directly, they lose efficacy quickly and cannot completely wipe out the tumor cells. Section 3: CAR-T’s Commercial Landscape Summary In this section, we learned: • CAR-T cells are made from autologous T-cells that are genetically modified using viral vectors. • There are currently FDA-approved CAR-T cell therapies for use against Bcell cancers. • The CAR-T cell therapy products such as Carvykti have a reasonable success rate. 16 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. • CAR-T cell therapy can have severe side effects like cytokine release syndrome and neurotoxicity. • CAR-T cell exhaustion and tumor microenvironment that shuts down cytokine production pose significant challenges to the success of CAR-T cell therapy. 17 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. Section 4: Next-Generation Cell Therapy Introduction In this last section, we explore the next-generation derivatives of CAR-T cells and investigate exciting new cell line frontiers. Let’s get started. Section 4: Next-Generation Cell Therapy Objectives At the end of this section, you should be able to: • Explore next-generation derivatives for CAR-T cells. • List cell lines, other than T-cells, being explored for cell therapy. Next-Generation Cell Therapies Now that we are aware of the challenges faced by CAR-T cells inside the body, which result in resistance and/or relapse, it is only logical that efforts are made to overcome these challenges. This can be achieved in two ways, to improve the existing CAR-T cell therapy or explore more cell types for their potential use in cell therapy. Attempts To Improve Existing CAR-T Cell Therapy Scientists are trying to improve CAR-T cell therapy. Some of the most promising strategies include: • Increasing localization and specificity. • Dual receptor activation or combinatorial activation. • Suicide switches or STOP CAR. • Gene-edited interleukin CAR-T cells Increasing Localization and Specificity Physical barriers and insufficient expression of chemokine receptors on CAR-T cells affect the homing ability of these cells to target tumor cells. Tumor cells secrete cytokines, and if CAR-T cells are modified to overexpress cytokine receptors along with costimulatory domains, it would 18 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. increase the trafficking of these cells toward the tumor sites and increase the specificity towards the tumor cells. This could be very useful for targeting solid tumors with better precision. Dual Receptor Activation or Combinatorial Activation Also known as Split CAR, this innovation helps target deep-seated tumors. The strategy depends on recognizing two different antigens by the CAR-T cells; one antigen that is expressed by every cell within the organ and another antigen that's only expressed in the cancer cells. This would increase the specificity of the CAR-T cells. This way, the CAR-T cells can be delivered to the correct organ, and only the cancer cells within that organ will be attacked by the CAR-T cells. The same strategy can be used for multiple antigen recognition, which would also help overcome the homogeneity issue of tumor cells. Suicide Switches or STOP CAR Switches are embedded within the CAR-T cells. In a massive immune reaction, the CAR-T cells can be turned off. Inducible Caspase 9 (icasp9), Herpes Simplex Virus Thymidine Kinase (HSVTK), and CD20 are a few proteins that induce apoptosis in T-cells and are being explored for use as suicide switches. These switches can be included in the CAR-T cell construct. Gene-edited interleukin CAR-T cells The next generation of CAR-T cell therapy is the addition of interleukin receptors. Interleukin is a type of cytokine used by many immune cells because it can overcome the tumor microenvironment. It is more effective against tumor cells because it is more persistent and remains active in the patient longer. It enables the CAR-T cells to stay active longer to kill tumor cells and is used for anti-tumor therapy. The main construction methods for studying gene-edited interleukin-associated CAR-T include the co-expression of a single interleukin, two interleukins, interleukin combined with other cytokines, interleukin receptor, co-expression of interleukin subunit, and fusion ICR. The construction is similar to that of CAR-T cells. The cytokines produced by these cells now also 19 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. include interleukin. These cells are at the clinical stages of development and show great promise, but they are yet not approved for treatment. Exploring Other Cell Lines for Cell Therapy T-cells are not the only cell type amenable to a CAR-type genetic modification. Scientists are also exploring other cell types to use similar to the CAR-T approach. Progress is also being made with Mesenchymal Stromal Cells (MSCs), Natural Killer Cells (NK cells), Dendritic Cells (DC), and Macrophages (M). All these cell lines are in various stages of development and show promise for treating solid tumors. Mesenchymal Stromal Cells (MSCs) Mesenchymal stromal cells (MSCs) are multipotent stem cells; they can differentiate into a few cell types but not all. They can self-renew and differentiate into different cell types, including muscle, bone, cartilage, and fat. They help induce blood vessel production, modulate the immune response by repressing overactive immune activity, and help repair wounds. These MSCs, due to their pronounced tropism to tumors and lower immunogenicity, have become a promising vehicle for cytokine delivery and drug delivery. They are currently being explored for modification with CAR to generate CAR-MSCs which can recognize and attack cancer cells efficiently. They are being tested to treat various conditions, such as cardiovascular disease, acute respiratory failure, and wound healing for colon and muscular conditions. Natural Killer Cells (NK Cells) The mechanism of action of Natural Killer cells (NK cells) differs from Cytotoxic T-cells as NK cells do not depend on MHC presentation; they use a different signaling pathway. NK cells produce cytotoxic granules that punch a hole in the diseased cell to make a path for perforins or granzyme to enter and denature the target cell’s proteins resulting in the cell’s death. CAR-NK cell lines focus on natural killer cells. Their primary function is to detect early stages of cancer and neutralize them. NK cells can be harvested from healthy individuals as they are not antigen-specific like the T-cells. Thus, allogenic cell lines can detect solid tumors before they 20 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. metastasize. However, many solid tumors avoid detection from natural killer cells by embedding themselves inside normal tissue. CAR-NK cells also use a similar construct as the CAR-T cells. The target element is modified to recognize a particular cancer cell type. They have engineered a co-stimulatory domain to overcome the challenges of the tumor microenvironment, including NK activation, increasing cytotoxicity, and production of interleukin and lytic enzymes. Dendritic Cells (DC) Dendritic cells recognize antigens and activate T-cells. Dendritic cells are being modified by CAR to generate CAR-DCs which may be more efficient in identifying antigens and mounting an immune response. CAR-DC can be used alone to treat cancer or in combination with CART cell therapy, where they promote the proliferation of CAR-T cells. However, the use of CARDCs is in the very early stages of development. Macrophages Macrophages perform limitless functions, such as engulfing malignant cells in a process called phagocytosis, releasing cytokines, and presenting antigens of the digested diseased cell on its Class II MHC molecules. Tumor-associated macrophages (TAMs) are one type of macrophage. It is the most abundant innate immune cell and constitutes up to 50% of the cell mass within the tumor microenvironment. TAMs play an indispensable role in anticancer therapy because of their profound effect on stopping tumor progression, namely reducing tumor cell proliferation, angiogenesis, metastasis, immune suppression, immune escape, and drug resistance. Due to the promising approach of CAR in targeted cancer therapies and the characteristics of TAM, it is only logical that these cells are being explored to construct CAR-M cells. CAR-M cells are genetically modified macrophages. 21 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. B-Cells B-cells are a type of lymphocytes that produce antibodies. These cells recognize antigens and secrete antibodies that kill the infected cell directly. B-cells also flag infected cells for destruction by other immune cells, such as macrophages or natural killer cells. Hematopoietic Stem Cells (HSCs) In this class, we have emphasized the progress made with a CAR approach to treat various cancers. It should also be noted that considerable advances are being made by genetically engineering a population of cells called Hematopoietic Stem Cells (HSCs). HSCs originate in our bone marrow and develop into our immune and blood cells. Many blood disorders, including sickle cell disease and beta-thalassemia, can be cured potentially through an ex vivo gene therapy treatment of HSCs. The HSC therapeutic process is similar to a CAR-T approach. First, HSCs are either removed from bone marrow or collected from peripheral blood using a process called apheresis. These HSC cells are then isolated and engineered with a lentivirus vector. In the case of sickle cell disease, multiple companies are pursuing the strategy of genetically altering the HSCs so that the red blood cells will increase their protein expression for hemoglobin. Hemoglobin is the gene necessary to circulate oxygen, and it is defective in sickle cell patients. Two separate genetic medicine companies, Bluebird Bio and Vertex, are both engaged in late-stage clinical trials aimed at reaching the large sickle cell patient group with their ex vivo approach. To date, Bluebird Bio has received FDA approval for two separate hematopoietic stem cell gene therapy products. Skysona is indicated to treat a rare disease called CALD (cerebral adrenoleukodystrophy). Zynteglo is indicated for beta-thalassemia. Pluripotent Stem Cells (PSCs) Pluripotent Stem Cells (PSCs) are a type of stem cell that can differentiate into any cell. They are also referred to as Embryonic Stem Cells (ESCs). Due to their ability to differentiate into any 22 Copyright 2023 Biotech Primer Inc. Proprietary. Do Not Share. cell type required, these cells have immense potential in regenerative medicine, where they may be used to generate different cells such as cardiac cells to treat heart diseases, neurons to treat neurological disorders, insulin-secreting beta cells of the pancreas to treat diabetes, and leukocytes to treat different diseases. But PSCs are not present in adults, but they can be generated by a process called reprogramming, which converts adult cells into pluripotent cells. Such cells are called induced Pluripotent Stem Cells (iPSCs). In patients undergoing later stages of treatment, patients do not have enough T-cells to be harvested for making another batch of CAR-T cells. In such a scenario, iPSCs help generate T-cells for CAR-T cell production. Hence, though iPSCs are not directly involved in targeting and killing cancer cells, they do have the potential to supplement cancer treatment and other kinds of cell therapy. Section 4: Next-Generation Cell Therapy Summary In this section, we learned that: • Various attempts are being made to improve the existing CAR-T cells to reduce or eliminate the associated side effects. These strategies include increasing localization and specificity, dual receptor activation or combinatorial activation, suicide switches or STOP CAR, and gene-edited interleukin CAR-T cells. • There are also considerable advances being made by genetically engineering a population of cells called Hematopoietic Stem Cells (HSC) and Pluripotent Stem Cells (PSC). 23 Copyright 2023 Biotech Primer Inc.