CARs+in+the+Treatment+of+Acute+Lymphoblastic+Leukemia

Introduction
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Acute lymphoblastic Leukemia (ALL) is a cancer of the white blood cells in the human body. There are two types of ALL, each one affecting one of the two types of lymphoblasts: B-cell ALL and T-cell ALL. Both lymphoblasts types are critical to maintaining a healthy immune system, the B-cells are involved in adaptive immunity whereas T-cells are involved in cell-mediated immunity, which is independent of antibodies. For this project, we will mostly be focusing on the treatment of B-ALL, which account for about 80% of ALLs. [A statement of purpose here]

ALL: Past and Present
Before going into detail on our treatment of interest for ALL, we would first like to discuss the history and the present state of the disease. First off, due to the "liquid" nature of leukemia by its presence in the blood, ALL can metastasize to many other organs; however, it often invades the liver, spleen, and lymph nodes (American Cancer Society 2013). ALL is most common in children ages two to five, but some other risk factors of ALL include exposure to high levels of radiation, carcinogenic chemicals, or genetic predispositions such as Down Syndrome (WebMD).

In the past, there were two main therapies for leukemia. In 1786, Thomas Fowler, a British inventor, developed a general treatment for a plethora of diseases which he coined "Fowler's Solution". The active ingredient of the solution was a small dose of arsenic trioxide. About 50 years later, physicians began using the arsenic containing medicine specifically as an anti-leukemia drug. It was proven effective to a degree; however, it fell out of favor when radiation therapy was discovered. However, due to a significant amount of radiologists ironically contracting leukemia, radiation therapy also began to lose support in the medical community. The crude nature of the therapies at the time and the liquid nature of ALL made it an always lethal illness until 1942. (Antman 2001)

ALL is a significant disease in the history of cancer research because it is one of the first cancers for which an effective chemotherapy was created, leading for the first time ever in human to cancer remission. Sidney Farber, a pathologist in Boston, investigated the possibility of folic acid curing pediatric ALL the same way it helped cure other blood disorders in the past (Mukherjee 2010). Farber then proceeded to inject synthetic folic acid into leukemic children. Farber's hypothesis was horribly wrong, the folate he gave to these children made them drastically worse by accelerating the children's cancer, doubling the white blood cell count in one leukemic child (Mukherjee 2010). This tragic investigation allowed Farber to make the rational conclusion that an anti-folate would cause the opposite effect seen in his first experiment. As history would show, Sidney Farber, the "father of chemotherapy" was correct and soon thereafter he standardized the use of aminopterin for leukemia therapy, which would soon be supplanted by the easier to produce drug, methotrexate (Mukherjee 2010). Farber's research lead to the eventual curing of most childhood ALL, around 90% in children under 15, and is considered "the greatest success story in the history of cancer" (American Society of Hematology). Unfortunately though, the story does not have a happy ending for all. Like most illnesses that are mostly prevalent in children, ALL is much more aggressive in adult and has a much worse prognosis. The treatment response of patients is a function of age (Larson et al 1995). Currently, with the standard treatment of high dosage chemotherapeutics as methotrexate and vincristine, the 5-year survival of adult ALL patients is at 33% (Figure 1), a far cry from the 90% seen in children (Thomas et al 2004). The sharp disparity in treatment response between children and adults has led to a desperate call for new cancer therapies for what many assume is one of the "success stories" in cancer. This leads us into our discussion on an exciting new targeted therapy, chimeric antigen receptors (CARs), designed to treat these B-cell ALL patients that have shown a poor response to the standard chemotherapy and radiation treatment.

Principle of CAR Therapy
As mentioned in the introduction, T-cells are involved in the cell-mediated immunity, which is established by the presence of antigen receptors on the surface of T-cells. These receptors can be modified in order to recognize and attack the body's own cells, a method generally called immunotherapy. Scientists are able to synthesize these artificial immune cells by altering the DNA of the T-cells within patients. They accomplish this through the use of viral vectors, which are viruses that have been stripped of their ability to cause illness, but retain the ability to integrate their DNA into the DNA of the host cell. By inserting DNA that codes for a specific receptor into the viral vector and mixing these vectors with T-cells taken from a patient, the scientists are able to create T-cells that artificially express a receptor that will target anything that they wish. This receptor is called a single chain variable fragment, or scFv, and it is linked to stimulatory molecules residing inside the T-cell. When the receptor binds to its complementary molecule, the T-cell becomes activated and fully functional which allows it to proliferate and attack the cancer cells. (Curran et al 2012)

In the case of ALL, the receptor that is targeted is CD19, which is almost exclusively expressed on the surface of B cells. The scientists isolate the DNA that codes for a CD19 receptor, called anti-CD19, and use viral vectors to integrate this piece of DNA into the T-cells of their patient. The T-cells will then begin to transcribe and produce the CD19 receptor. Once expressed, the T-cell is able to seek out any cell that has CD19 on its surface; in this case it is the B cells in the patient. The T-cells have co-stimulatory domains on the inside of the cell which are attached to the anti-CD19 receptor via a trans-membrane domain. The co-stimulatory domains in for the anti-CD19 modified T-cell are called CD28 and CD3 zeta, as shown in Figure 2. These are the domains that allow the cell to become fully functional when it is triggered by a CD19 protein on another cell. These domains trigger the T-cell to proliferate into both cytotoxic T-cells and memory T-cells. (Curran, K. J et al 2012) As seen in figure 3, the process of using this technology as a therapy is quite complex. In order to actually use CAR T-cells, doctors first take blood from the patient. Scientists then mix this blood with magnetic beads that are coated in an anti-CD28 and anti-CD3, which selects for T-cells. Using a large magnet, the scientists are able to isolate the T-cells which are bound to these magnetic beads. The T-cells are then mixed with viral vectors containing the DNA for anti-CD19; this DNA is then integrated into the DNA of the T-cells and begins to be expressed. The population of T-cells is incubated until their numbers reach into the hundred millions because the therapeutic dose of CAR T cells is 30 million cells per kilogram of body weight. These cells are then washed once more to ensure that the population is purely T-cells. The pure sample can be frozen for up to 3 months if the patient is receiving neo-adjuvant chemotherapy at the time. Prior to infusion of T-cells; however, physicians administer cyclophosphamide, a chemotherapeutic agent, to lower the number of leukocytes present in the patient. This is necessary so that the T-cell concentration is higher in the patient and also reduces the chances of having an adverse immune reaction to the therapy. It is also necessary to note that since the CAR T therapy against CD19 eliminates all B cells, not just the leukemic ones, physicians must administered immunoglobulins to support the fragile immune system of the patient. (Hollyman, D. et al 2009)

CAR Kinetics
Many of the initial studies done with chimeric antigen receptors were conducted in patients with chronic lymphoblastic leukemia (CLL) rather than ALL (Hollyman et al 2009; Kalos et al 2011; Porter et al 2009; and Kochenderfer et al 2012). These preliminary studies showed the proof of principal of CAR therapy by showing that the modified T-cells could specifically target the leukemic B cells. From this point, researchers extrapolated the idea of CAR therapy to other blood based cancer such as ALL, which we are studying. The main focus of CAR therapy has been in the blood cancers because the therapy is more effective in liquid. In solid tumors, the modified T-cells would only be able to target the outer layer of cells, but to be effective it would need to target the entirety of the tumor. Below, we discuss the results of the initial studies of CAR therapy on CLL. In Figure 4, the specific elimination of B cells is shown. The T cells, which are modified to target the CD19 surface protein, increase drastically at about 3 weeks after infusion (Kochenderfer et al 2012). At the same time, a drastic drop in B cells is shown. This shows that the modified T cells do, in fact, have a correlated effect on the B cell concentration circulating in the body. CAR therapy achieved its goal by completely eliminating the B cells in the body, which is required before adjuvant therapy can take place.

Natural Killer (NK) cells also increase as the T cells increase, indicating an immune response within the body. NK cells are cytotoxic lymphocytes involved in the innate immune response within the body and are recruited to aid the body with the destruction of foreign antigens that enter the blood stream. In the case of ALL, the B cells that express CD19 surface receptor are marked as foreign by the T cells that have been modified to target these cells. This demonstrates proof of principal of the targeted therapy through use of CARs because it is possible to use the immune system that is already present to destroy the cancerous cells (Kochenderfer et al 2012). Figure 5 again shows that the concentration of B cells can be drastically reduced by the infusion of T-cells modified to target the CD19 surface protein. However, in this study the researchers used immunohistochemistry (IHC) to show that the bone marrow in the patient has also been affected. In the above graphic, the areas stained red are those that express the CD19 surface protein. These cells predominate prior to treatment with CARs. After 13 weeks, effectively all of these cells have been eliminated (Kochenderfer et al 2012). This is significant in the treatment of ALL because the stem cells that produce cancerous B cells need to be eliminated just as the already produced B cells do. This is possible to achieve with CAR therapy because B lymphoblasts express the CD19 surface protein as well. With the body free of leukemic B cells, physicians can introduce healthy donor cells and marrow to reestablish the B cell population in the body. Figure 6 shows how effective CAR therapy actually is. Part B is the most interesting part of the graph because it shows how specific immunotherapy can be. As shown, the amount of White Blood Cells is not affected through use of the therapy because normal WBCs do not express the surface protein CD19 that is being targeted on the leukemic B cells (Kalos et al 2011). This allows the body to continue with partial immune response because not all of the cells get eliminated through CAR therapy. In contrast, it is also demonstrated that those cells that do express this protein have drastically lower concentrations as the duration of treatment increases. Through use of specific antigen targeting, it is possible to decrease the number of cells that are expressing the desired surface protein. This is the idea that was then used for cases of ALL. The caveat to this is that it could only be used on cases that also involved the B cells, known as B cell malignancies. This is because the researchers had only identified CD19 as a viable target because it was nearly exclusively expressed on B cells.

Clinical Trials
As mentioned above, CAR therapy trials were first done in chronic lymphoblastic leukemia patients. These CARs used in the therapy of adult B-CLL exhibited the expected kinetics, proved to be relatively safe (a point we will address below) and demonstrated “potent” and “profound” antitumor effects (Kalos et al 2011). When the efficacy of CARs as a future leukemia therapeutic was proven in the Kalos (2011), Porter (2011), and Kochenderfer (2012) clinical trials, a research group in New York (Memorial Sloan Kettering Cancer Center) looked to use CARs in the treatment of adult B-ALL patients. One could say that these CARs were changing direction.

The trial specifically looked at patients that were initially in complete clinical remission but had a return of the disease. These patients with a return in their cancer are known as “relapsed,” in which a patient showed no signs of B-cell hyperplasia and no clinical manifestation of leukemia prior to the return of the illness, or “refractory,” in which patients just show no clinical manifestation of the leukemia but do have notable B-cell hyperplasia while they are in remission. Relapsed and refractory B-ALL patients have an even poorer prognosis and their only hope is an allogeneic hematopoietic stem cell transplant (allo-HSCT, also known as a bone marrow transplant) that gives the patients a blank slate from which to build their blood cell line. However, in order for ALL patients to qualify for an allo-HSCT, they must show that they have no minimal residual disease (MRD-) of their secondary cancer because a bone marrow transplant by itself has shown poor long-term survival (Brentjens et al. 2013). The therapeutic goal of the Brentjens group was to treat the patients with CAR modified T-cells, induce CR, and qualify these patients for an allo-HSCT.

Five patients were treated with CART19 cells using the aforementioned methods (Reference: Principles of CAR therapy) and their background and results are summarized in Table 1. All five of these adults were at one point in complete remission (CR) and now had either relapsed or refractory ALL, with the two “refractory” patients (MSK-ALL04 and MSK-ALL05) having the higher B-cell load at the time of the trial. Upon CART19 therapy, all patients were ultimately MRD—. Getting these patients into their second complete remission took anywhere from 8 days (MSK-ALL05) to 59 days (MSK-ALL04). Within four months following the treatments, four patients had qualified for or already gotten an allo-HSCT. It is worth noting that patient MSK-ALL04, one of the two refractory patients, was not eligible for an allo-HSCT and soon thereafter died. This is arguably because of the patients high load of B-cells upon initial T-cell treatment. Although all CAR treated patients thus far have experienced a strong immune response as typical in immunotherapy, MSK-ALL04 experienced significant fever, hypotension and a transient altered mental status that required the doctors to treat this patient with steroids to alleviate the CART19 cells’ immune response. Steroid treatment, as argued by Brentjens et al 2013, is possibly what is to blame for this patient having the poorest response to the treatment. Calming the immune response possibly decreased the efficacy of the treatment, allowing the patient’s relapse within 90 days of showing MRD-. Patient MSK-ALL05, the other refractory patient in the group, also experienced a similar cytokine response with similar symptoms to MSK-ALL04 that required he also be treated with an anti-inflammatory, however, MSK-ALL05 showed the best response of all patients in the group, going into remission within 8 days of treatment, as stated earlier.

The long-term effects of CART19 therapy could not be investigated for longterm efficacy in this trial because all treated patients either had a bone marrow transplant or expired. Although CARs are being touted as a complete replacement for an allo-HSCT, the paper states that it was an ethical matter that they treat these patients with the standard level of care once they determined the safety and efficacy of the treatment in the phase I study. Other trials have been published in the last two years on CART19 therapy in B-ALL that have similar results. The Grupp research group at the University of Pennsylvania, treated two children, a 7-year-old and a 10-year-old with their own anti-CD19 modified CARs and induced remission in both patients (Grupp et al 2013). The 7-year-old patient, Emma Whitehead (pictured below), had a [|moving piece] written about her therapy and life post-cancer in the New York Times around Christmas last year that has served as one of the largest forms of publicity for CAR therapy. Most recently, the Davila group, also out of Memorial Sloan Kettering Cancer Center, treated 16 relapsed B-ALL patients ranging from 18 to 74 and 14 went into complete remission (88%) (Davila et al 2014). Davila’s larger patient population and replicated therapeutic success has served as great support for the advancement in CART19 cells in B-ALL therapy into phase II.

Future Directions
Currently, CAR therapy has piqued the interest of oncologists and cancer researchers, to put it modestly. The therapy has proven efficacious in treating both ALL and CLL. The questions for CARs though remain in its safety and management. As shown in Brentjens et al 2013 (Table 1), it is questionable whether a patient with a relatively high load of target cells can withstand the "cytokine storm" that follows without the use of steroids. Almost all new research is going to reduce the side effects of CARs so that they can be given in more than "last hope" situations. A suggestion mentioned in the literature is for newer generation CARs to be accompanied by a "suicide gene" that will be co-expressed in the T-cells so that their immune response time is shortened, limiting the kinetics of the therapeutic. However, it is still unclear if the elimination of CART cells would mitigate the dramatic cytokine response and how a reduced CART cell population would affect tumor relapse (Maher et al 2014; Cheadle et al 2014; Turtle et al 2014; Davila et al 2014; Sentman 2013; Sadelain et al 2013).

Chemotherapy for cancer was first seen in 1942 when Sidney Farber showed that aminopterin could induce remission in children with acute lymphoblastic leukemia. Now, chimeric antigen receptors are being used in "liquid" cancers to show that immunotherapy for cancer can be an efficacious treatment, as well. And like chemotherapy, CARs are being developed that can target "solid" tumors. There are currently dozens of receptors other than CD19, that are being targeted by CARs in the hopes of treating such cancers as breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, and more (Tables 3 and 4).

Many of these CARs have reached phase I, while some are still to reach human trials. However, it is worth noting that CAR research and development has been solely done in academic institutions for the last decade, completely void of pharmaceutical company aid. The advancement of CARs is thus even more impressive considering the limitations in funding and R&D that are available to giant pharmaceutical companies. Due to the recent results being published and the publicity CARs have received though, Novartis, a 58 billion dollar corporation and the second largest pharmaceutical company in the world, has partnered with the University of Pennsylvania and also purchased a cell processing plant for $53 million, all in the hopes of advancing CAR trials through FDA approval and making CAR therapy accessible to thousands of patients (Palmer 2012).