CAR-T CELL THERAPY

• Cancer treatment has progressed over 100 years starting with surgery, radiation, chemotherapy, immunotherapy and gene therapy. The availability of adoptive cell immunotherapy allows researchers to genetically reprogram patients’ own immune cells to find and attack cancer cell.
• Over the past decade, therapeutic treatment for multiple types of cancer has proven a success rate, which suggest immunotherapy and adoptive cell therapy valuable knowledge for growth and development. For all discoveries in the medical arena especially in the CAR T cell therapy which is a part of and has made a lot of progress amongst multiple disease as seen in all forms and in the arena of immunology has made a lot of milestones in medical history for immunotherapy have been booming over the last decade. The use of adoptive cell therapies was first discovered in 1988, but the critical improvement occurred in 2002 with the introduction of an immunodepleting preparative regimen given before the adopted transfer, resulting in improved re-population of anti-tumor T cells.
• Chimeric antigen receptor (CAR) T-cell therapy is a type of adoptive cell immunotherapy that has led to remarkable patient outcomes and has the potential to transform cancer treatment.
• Chimeric antigen receptors (CAR) T-cells are a type of adoptive cellular therapy of state-of-the-art technology whose development has been formed by a wide variety of disciplines, including immunology, molecular biology, Cancer biology, anti-body engineering, and virus bacteriology, as well as basic and clinical medicine. CARs genetically modified to redirect and reprogram in order to overcome tolerance in cancer and programed to. These synthetic receptors combine the effector functions of T lymphocytes and the ability of antibodies to recognize pre-defined surface antigens with a high degree of specificity in a non-MHC restricted way.
• The development of CARs was originally considered in clinical research studies for transplantation in B-cell leukemias and lymphomas.
• The first successful CAR therapies were directed against B-cell hematologic malignancies, targeting the CD19marker, which is highly specific to B-cell products. In 2017, the U.S. Food and Drug Administration approved two second generation CD19 directed cell immunotherapies, tisagenlecleucel (Kymriah) and Axicabtagene clioleucel (Yescarta) of produced unprecedented clinical outcomes for relapsed and refectory B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma patients.
• CAR T cells may help guard against recurrence, and they may eradicate all of the cancer cells and remain in the body months after the infusion has been completed. The therapy has resulted in long-term, remissions for some types of blood cancers.
• Additional CAR targets are under investigation for the treatment of other hematological malignancies as well as solid tumors. CAR T cells get produced in the laboratory under specific and careful work instructions.

 

The steps following CAR T Cell Production are below:

• The production of CAR T cells requires several carefully performed steps, and quality control testing is performed throughout the entire protocol as this investigational product for CAR T cells is a live product that it is unknown unwanted mutations occur.
• First, the process involves using leukapheresis to remove blood from the patient’s body, separate the leukocytes, and return the remainder of the blood to the circulation.
• After a sufficient number of leukocytes have been harvested, the leukapheresis product is enriched for T cells. This process involves washing the cells out of the leukapheresis buffer, which contains anticoagulants.
• Enrichment of lymphocytes can be accomplished subsequently through counterflow centrifugal elutriation, which separates T cell subsets at the level of CD4/CD8 composition using specific antibody bead conjugates or markers is an additional step that may performed.
• Lymphocyte-enriched apheresis product is cultured in a sterile bioreactor in the presence of autologous antigen presenting cells (aAPC) and viral vector encoding the CAR; the WAVE biomarker uses a rocking motion for optimal mixing and gas transfer.
• After 9-11 days, the CAR T cell culture would have expanded to about 5L.
• Magnetic bead-based a APCs are removed from the culture. The CAR T-cell culture is concentrated using a cell washer, and then concentrated CAR T-cell product is cryopreserved. aAPC autologous artificial antigen -presenting cell; CAR; chimeric antigen receptor is produced.

Evolution of CAR T-cell Generations: CAR T-cells differ in their intracellular domain portion contributing to their generations.

First Generation CAR:

• Single chain antibody structure from the CD3z-chain or FceRlg links the ITAM at transmembrane region to hold it in place.
• Primary transmitter of signals from endogenous T cell receptor.
• Unable to produce enough interleukin-2 (IL-2) on its own and exogenous IL-2 is given to CARs so cytokines would be signaled due to tumor death.
• Recent studies show that the deletion phosphorylation on ITAM A and C in the CD3z-chain signaling moiety could decrease the apoptosis signal, beneficial for the continuous expression of transgenes so that the expression for transgene can continue.
• CAR T cells with CD3z-chain were more effective at activating T cells and killing tumor cells although they had lower expression levels in vitro and is responsible for cell-to-cell downstream communication.
• The transmembrane domain of the first-generation cells consists of a dimer of homologous and heterologous in combinations of CD3, CD8, CD28, which can meditate optimal cellular activation via the dimerization of CARs and the functional interaction of this receptor with the endogenous TCR.
• Most first generation cells did not achieve the desired outcomes because of inadequate proliferation, a short lifespan in vivo and in sufficient secreted cytokines.

Second Generation CAR:

• Added intracellular signaling domains from various costimulatory proteins receptors to the cytoplasmic tail of the CARs to provide additional signals to the T cell, such as CD28 and CD134 or 4-1BB which improves proliferation, cytotoxicity, and sustained response, and prolong the life of CAR T-cell in vivo
• CD28-meditated co-stimulation is very important in the regulation of proliferation and survival for lymphocytes and plays a key role for the establishment of memory cells and effector cells.
• CD134 can sustain proliferation and strengthen IL-2 production.
• CD137 can maintain the response signal of T cells, which plays a key role in the survival of T cells and the memory of CD8+ T cells.
• The CD28 z CAR T-cell can cause constitutively stimulation, proliferation, and growth. However, the 4-1BB z CAR T-cell can induce early exhaustion which made the limit antitumor efficiency.

Third Generation:

• Combining multiple signaling domains as expectation of more antitumor efficacy, and to acquire further enhanced activation signals, proliferation, production of cytokines and effective function.
• Provided a better fraction of CAR Ts that function as memory phenotype for preventing tumor relapses, but a significant therapeutic effect of emerging uncontrolled activity accompanied with more antitumor efficacy caused life-threatening lysis activity as the most critical adverse effect or toxicity including clinically significant release of pro-inflammatory cytokines, pulmonary toxicity, multi-organ failure, and eventual death.

Fourth Generation:

• Designed to shape the tumor environment by the inducible release of transgenic immune modifiers, such as adding IL-12 to the base of the second-generation constructs, and are known as T Cell Redirected for Universal cytokine-mediated killing (TRUCKs).
• TRUCKs augment T cell activation, activate and attract innate immune cells to eliminate antigen-negative cancer cells in the targeted lesion.

Possible Side Effects of CAR T-Cell Therapy

Cytokine-Release Syndrome (CRS)

• Frequently associated with CAR T-cell therapy. Cytokines are chemical messengers that help the T cell carry out their functions. Cytokines are produced when the CAR T cells multiply in the body and kill the cancer cells. (common after receiving the 4th generation).
• Severe CRS requires intensive treatment. Although most symptoms are reversible, the potential life-threatening risk or CAR T cell therapy should not be underestimated.
  • Mild symptoms include nausea, fatigue, headache, chills, and fever.
  • Serious symptoms include low blood pressure, tachycardia, capillary leakage, cardiac arrest, cardiac arrhythmia, failure, hemophagocytic lymphohistiocytosis, hypoxia, renal insufficiency, poor long oxygenation, Multiple organ failure.
• Macrophage Activation Syndrome (MAS)Closely associated with sever CRS and is a condition caused by the excessive activation and multiplication of T cells and macrophages. It is generally seen in patients with chronic autoimmune and rheumatic disease. This syndrome is similar to CRS and can be mitigated by the infusion of the monoclonal antibody tocilizumab. Corticosteroids and anti-cytokine therapy can be considered as treatment options.Neurological toxicities

  The frequency, severity and nature of neurological effects appear different between CAR T products. Common symptoms include language impairment (aphasia), confusion, delirium, involuntary muscle twitching, hallucination, or unresponsiveness.

  Neurotoxicity has been reversible in most cases and the symptoms have resolved over several days without intervention or apparent long-term effects. However, there can be life-threatening adverse neurological events. The cause of neurotoxicity is the subject of intense investigation by researchers.

  Tumor Lysis Syndrome (TLS)

  Another known side effect of CAR T cell therapy is tumor lysis syndrome (TLS), a group of metabolic complications that can occur due to the breakdown of dying cells, typically at the onset of toxic cancer treatments. However, TLS can be delayed and may occur one month or more after CAR T cell therapy. TLS can cause organ damage and can be life-threatening complication of any treatment that causes breakdown of cancer cells, including CAR T cells. The complication can been managed by standard supportive therapy.

  Anaphylaxis (Life-threatening allergic Reaction)

  There is potential for a patient receiving CAR T-cell therapy to have an overwhelming immune response against the CAR itself, called anaphylaxis, Symptoms associated with anaphylaxis includes hives facial swelling, low blood pressure.

Safety strategies to overcome the toxicity of CAR T-cells

HSV-tk suicide gene

• Herpes Simplex virus thymidine kinase (HSV-tk) is the best characterized suicide gene and widely used in combination with ganciclovir for the treatment of a variety of malignant cancers.
• HSV-tk phosphorylates specific nucleoside analogues, such as GCV, forming toxic GCV-triphosphate compound that competes with triphosphate as a substrate incorporated into DNA via DNA polymerase, dealing to the inhibition of DNA synthesis and subsequent cellular death.
• Important role of the HSV-tk suicide gene is to eliminate GVHD in the allo-stem cell transplants.
• Donor T cells expressing HSV-tk suicide gene were infused into patients with leukemia after a haplo-HSCT to facilitate immune reconstitution and control GVHD.
• Limitations of HSV-tk include: compromised survival of modified T cells, the gene requires activation by a prodrug (like GCV) that remains a crucial pharmacologic agent for the treatment of cytomegalovirus infection; Interfering DNA synthesis to induce T cell death is a gradual progress, it takes more time to eliminate CAR-T cells by HSV-tk.

Caspase 9 (iCasp9) suicide gene

• Inducible safety switch, that contains modified human caspase 9 fused to the human FK506 binding protein (FKBP).
• Chemical inducer of dimerization (CID) (AP1903) forms dimerization and activates the downstream caspase molecules, resulting in apoptosis of cells expressing the fusion protein.

CD20/EGFRt

• Third generation anti-CD20 CAR T-cells contain both CD28 and 4-1BB co stimulatory domains were transduced with iCasp9 suicide gene and truncated CD19 selectable maker.
• T cells infused caused effective cytotoxicity to CD20-positive tumor cells and induced potent cytokines secretion.
• EGFRt is truncated epidermal growth factor receptor.
• Efficiently and specifically eliminated with clinically approved monoclonal antibody rituximab or cetuximab through the complement dependent compound.

EGFRt-rituximab suicide switch has been incorporated into the CAR-T cells to mitigate toxicity

Synthetic Notch Receptor

 

Synthetic Notch (SynNotch)

• Wild -type proteins contain three parts: an extracellular ligand-binding domain, a transmembrane domain, releasing transcriptional regulator through ligand-induced cleavage, and the intracellular effector Domain.
• Intracellular transcription domain could be replaced to activate downstream interesting target genes.
• SynNotch plus the AND-gate T cell was tested in GFP and CAR against CD19. Study showed selective cytotoxicity to ROR1 + tumor cells but not ROR1 CAR T-cells.

Cancer is among the major causes of death all over the globe. Scientists and physicians worked hard in the past few years with the conventional cytotoxic approach for the various neoplastic diseases. However, the effectiveness was limited due to the heterogeneity of cancer cells. A new and improved way following therapeutic approach such as immunotherapy has been developed which increases the quality of the immune system of the cancer patient from ordinary. Immunotherapy works on the concept of antibodies, and immune cells can kill and recognize the cancer cells.

 

Chimeric Antigen Receptor (CAR) T-cell therapy is a kind of immunotherapy which includes the genetic modification of the autologous T-cells of the patient as a part of the natural immune system to give strength against cancer. A sample of the blood is taken from the patient’s blood by the process of apheresis and T cells (a type of immune cell) are extracted to modify the special structures by binding onto the harmful cancerous cells to kill them. A large number of CAR-T cells are produced and multiplied in the labs. The modified cells are then artificially infused within the bloodstream of the patient at the time of treatment. The cells will later multiply in number within the bloodstream and will recognize and attack the cells having the targeted antigen on their surface. The CAR-T cells can effectively eradicate nearly all the cancer cells and may even remain inside the patient’s body for months after the infusion has been done. CAR-T therapy has shown long-term remissions for different types of blood cancer. CAR-T therapy is officially FDA approved as a standard of care. It is ideal for treating acute lymphocytic leukemia in young adults and kids and with some forms of refractory, aggressive non-Hodgkin lymphoma. It is also recommended for the patients with refractory or relapsed acute lymphoblastic leukemia up to the age of 25. Several trials of CAR-T cell therapy are also in progress for the various forms of blood cancer Stem cell transplants, and chemotherapy is mainly the first preference for the treatment of cancer. If the respective treatments are not showing any effective results or cancer relapses even after treatment, then CAR-T therapy is the preference. CAR-T cell therapy is also considered as the last stage of the cancer treatment for many people. The therapy can only be conducted at a few special cancer centers because sometimes it can cause some side effects and fatal outcomes.

 

The process involves:

1. Collection of T-cells

A special machine is set up to collect the T-cells from the blood. The following process is called leukapheresis. There will be two intravenous (IV) lines in the veins of arms. One intravenous sends the blood from the body to the machine, and the other one returns the blood in your body. During the final process, the patient can do a calm activity such as listening to music, read books, and much more. The process can even take a few hours so the patient can relax as it is not painful as well.

 

2. Changes in T-cell

After the collection of the cells, the cells are taken to the laboratory where the new genes are added to them. The addition of the genes sprouts special proteins on the surface of the cells. The following CARs or Chimeric Antigen Receptors helps the T cells to find and stick on the antigens present on tumor cells. The following process can take a few weeks for the budding of these new cells called CAR-T cells. The time may vary from person to person.

 

3. Low dosage of chemo

When the changes in the T-cells in the laboratory are taking place, the doctors tend to give a low dosage of chemo for a few days to cut back on the immune cells of the body. This process is called lymphodepleting chemotherapy. The low dosage of chemo helps to pave the way for the CAR-T cells to enter the bloodstream and do their job with widespread ease.

 

4. Infusion

In the matter of a few days, the CAR-T cells are deeply frozen are sent to the cancer centre or hospital. The CAR-T cells are targeted to put back in the body by an IV in a vein in the arm. The process resembles blood transfusion. The cells find the tumor cells in the whole body and multiply on their own to find more of the cancer cells present in the body.

 

5. Recovery

It usually takes around 2-3 months to recover from CAR-T cell therapy. The therapy may have various side effects, so it is advised to stay near the treatment center for at least 30 days in order to stay safe. A full-time caretaker is also assigned to the patient to deal with sudden complications and problems. The patients usually feel tired and have less appetite in the recovery process.

 

Side Effects of CAR-T cell therapy

• Neurologic events which may include confusion, encephalopathy (brain malfunction), aphasia (difficulty in speaking and understanding), agitation, drowsiness, altered consciousness, loss of balance.
• Cytokine release syndrome, headache, nausea, vomiting, joint or muscle pain, diarrhea, faster heart rate than usual, low blood pressure, shortness of breath. The following symptoms are usually mild in most of the patients but can be serious and result fatally in exceptional cases.
• Neutropenia (low white blood cell count).
• Anemia (low red blood cell count).

 

Mainly all the following side effects can be cured or managed by medicines without the need for special treatment. The care team will guide through the management of the side effects related to the body of the patient.

 

CAR-T cell therapy is considered as one of the most effective steps to cure cancer. It has over 80% success rate on the patients who were part of the therapy. It was observed that either the patients would either fully cured or a partial response (reduction of the cancer cells in the body). It is usually advised to perform the therapy after a few months for the long-term effects. The data for the effectiveness and time durations are to be collected and observed to bring improvements to the therapy.