New England Biomedical Engineering Professional
I originally wrote this paper in Fall 2017 for a graduate-level molecular biology class taken as part of my Master’s degree program.
A new genetic therapy has shown promising results as a treatment for leukemia. In short, the new treatment uses modified HIV as a vector to deliver an anti-leukemia transgene. Because HIV is a retrovirus, the transgene is integrated into the host cell genome, and passed down to progeny. Despite the natural (and entirely reasonable) aversion to HIV-induced AIDS, the transgene integration by retroviral vector enables delivery of a long-lasting therapeutic effect. The safety profile of HIV-vector transgene delivery is better than the safety profile of other vectors, making HIV the delivery vehicle of choice (Berkhout).
HIV and leukemia are two diseases that have both been in the public eye for a considerable amount of time. In 1983, the virus that would later be called HIV was discovered as the cause for acquired immunodeficiency syndrome, or AIDS (Barre-Sinoussi). Since then, the virus’s mode of transmission (sexual activity) has caused HIV/AIDS to become a popular disease for research.
HIV targets CD4+ T-cells in the human immune system. In the natural course of an HIV infection, the virus modifies the genome of its host T-cells, causing them to express the HIV gene when fighting infection (Hiscott). The T-cells are weakened. As the viral infection progresses, the body becomes increasingly susceptible to other opportunistic infections. At this point, the patient’s condition is upgraded from “HIV positive” to AIDS.
Modern prophylactic drugs can stave off an HIV infection if administered immediately following exposure. For those who already have a full HIV infection, a full cure is not currently possible. However, a strict regimen of antiretroviral drugs can reduce the viral load within the body to acceptable levels. These drugs target different stages in the viral replication cycle, attempting to halt the cycle before the virus can spread throughout the body. These drugs are categorized by the stage of viral replication that the drug inhibits: entry, reverse transcription, integration, or assembly (“Guidelines”).
Antiretroviral drug regiments allow the immune system to maintain its activity and avoid the opportunistic infections that lead to death. With such treatments, it is possible to avoid most of the problems caused by HIV infection. However, missing doses of therapeutics can allow the virus to develop a drug resistance, enabling resurgence and making future treatment more difficult.
Leukemia is a category of blood cancers that originate in the bone marrow and cause the production of abnormal or defective white blood cells. The causes of leukemia are varied, and include tobacco use, radiation exposure, viral agents, and carcinogen exposure (Radivoyevitch). The symptoms of leukemia are similarly variable. They commonly include easy bruising, pale skin, and fever in children (Clark). As the disease suppresses the immune system, patients become susceptible to infections and may suffer pneumonia or other opportunistic infections.
Established treatments for leukemia depend on the specific subtype of leukemia, the condition of the patient, and the stage of the disease. Hairy-cell leukemia requires almost no intervention, with occasional regimens of one week or six months being sufficient to keep the disease in check (Robak). At the other extreme, chronic lymphocytic leukemia (CLL) is not currently curable without an allogenic or autologous bone marrow transplant. Chemotherapy for CLL serves only to stall disease progression and suppress its effects. In between the two extremes, acute lymphoblastic leukemia (ALL) requires several stages of treatment, starting with rapid killing of tumor cells and putting the bone marrow into remission, and ending with continuous maintenance therapy to prevent relapse. Acute myeloid leukemia (AML) follows a similar treatment regimen, but may require additional treatment for the central nervous system (CNS). However, the median age for AML diagnosis is 67 years, and so treatment may be complicated by the (comparative) age and frailty of the patient (Brown).
HIV and leukemia share a relationship through the Human T-Lymphotropic Virus (HTLV) family. The two predominant forms of HIV, currently known as HIV-1 and HIV-2, are members of the HTLV family. Other viruses in this family also affect human T-cells, but rather than destroy the host cells, HTLV immortalizes them, inducing cancer. In particular, HTLV-1 is thought to be responsible for Adult T-Cell Leukemia (ATL), a rare form of leukemia. ATL causes hypercalcemia, skin and bone lesions, and osteolysis. Treatment for ATL has not been firmly established, but both antiretroviral and chemotherapeutic agents have been investigated (Taylor).
Additionally, HIV infection renders the patient susceptible to three forms of lymphoma, a related cancer of the lymphocytes, a type of white blood cell found in both the blood and lymph systems. Specifically, weak immune systems are a known risk factor for non-Hodgkin lymphoma, and this includes immune systems weakened by HIV infection (Tran). In fact, lymphoma rates among HIV-postive individuals are 60-200 higher than that of the general population (Grogg). The weakened immune system enables the onset of opportunistic lymphotropic viral infections, such as human herpesvirus 8 and Epstein-Barr virus. Furthermore, the products of HIV gene expression, such as Tat protein, are inherently oncogenic in nature. Tat inactivates the tumor suppressor gene RBL2, which produces Rb2/p130, which is involved with the G0/G1 transition of the cell replication cycle (Grogg). Deactivating this regulator allows the cell to divide uncontrollably, leading to lymphoma.
Eppstein-Barr virus (EBV) is present in 40-50% of HIV-associated lymphomas. The loss of EBV-nuclear-antigen-1 specific memory CD4 and CD8 T-cells is associated with progression to malignant lymphoma in AIDS patients (Grogg). In other words, the immune function required to keep potential malignancies at bay has been lost, allowing tumor cells to proliferate unchecked.
Despite the unique and deadly relationship between HIV and leukemia/lymphoma, the former has been explored as an avenue for treating the latter, with some initial success. By modifying HIV to contain an anti-leukemia transgene, the patient’s T-cells can be reprogrammed to fight the cancer. Because HIV is a retrovirus, its genome is integrated into that of the host cell, and the modified genome is passed down to daughter cells. Although HIV/AIDS is a deadly and as-yet-uncurable disease, the virus itself is a highly efficient and effective vector for delivering the anti-leukemia transgene. The transgene is delivered to hematopoietic stem cells obtained from the patient, which then become T-precursor cells. The resulting genetically modified T-cells are replanted in the patient to fight the leukemia (Berkhout).
This solution is elegant in concept, but is not without its risks and drawbacks. The treatment depends on an allogenic hematopoietic stem cells, which may be difficult to obtain from a patient who has been undergoing chemotherapy (Berkhout). The alternative, of course, would be directly injecting live HIV into the patient. However, the spontaneous development of pathogenic strains cannot be precluded, and so safety cannot be guaranteed. Tests with Simian Immunideficiency Virus strains have resulted in the development of AIDS, despite multiple gene deletions from the original viruses. Studies with multiply-deleted HIV-1 strains have shown HIV to be capable of restoring deleted replication functionality, and reproducing with delayed kinetics (Berkhout et al). These “revertant” HIV strains are of unknown pathogenic potential. Because multiply-deleted live HIV so readily reverts to an infectious strain, it cannot be used for therapeutic purposes, as safety cannot be guaranteed. Therefore, to prevent any viable viral material from entering the patient, any use of HIV as a genetic manipulation vector must be done ex vivo.
Similar approaches to leukemia therapy with other vectors have also caused safety issues. A first-generation retroviral therapy had been trialed for X-linked Severe Combined Immunodeficiency Disease (SCID-X1). In that trial, the goal of the retroviral therapy was to restore the missing IL2RG gene to CD34+ precursor cells in ten patients. Nine of the ten were successfully treated, but four of those nine developed T-cell leukemia within five and a half years. Investigation revealed that the transgene had integrated near proto-oncogenes LMO2, BMI1 or CCND2. Additional genetic defects were discovered, including deletion of tumor suppressor gene CDKN2A. Three of the four inadvertently-induced leukemia patients were successfully treated with chemotherapy, killing the tumor cells but allowing them to continue to benefit from the retroviral therapy for their SCID-X1 (Salima). The accidental activation of oncogenes or proto-oncogenes has not yet been observed in retroviral therapy for leukemia.
In trials for leukemia, the anti-leukemia transgene causes the expression of a chimeric antigen receptor (CAR) for antigen CD19, an antigen expressed in both cancerous and healthy B cells. The modified T-cell then not only attacks and destroys cancerous cells, but also proliferates within the body for long-term protection against leukemia. The initial patient for this therapy received a dose of 1.5 x 105 engineered T-cells per kilogram of body weight. Tumor lysis syndrome was apparent at 22 days after treatment. The number of circulating CAR-positive T-cells increased 1000-fold, accounting for 20% of circulating lymphocytes at peak levels. The CAR-positive cells remained at high levels for at least six months following infusion. As the number of CAR-positive T-cells grew, the number of circulating CD19-positive B cells dropped. Remission was ongoing ten months following the initial treatment. (Porter).
The CAR contains several features that enable this anti-leukemia functionality. It has an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The CAR recognizes an antigen – in this case, CD19 on the surface of B cells. The intracellular signaling domain is intended to trigger the immune response. In first-generation CAR, this included the ζ-chain of the T-Cell Receptor protein (TCR) and the γ-chain of an immunoglobulin receptor. These initiated the tyrosine kinase cascade, leading to cell activation against a diseased cell. Perhaps most significantly, the CAR protein activates on CD19 without regard to histological surface markers – it does not have the ability to recognize a cell as belonging to the individual organism (Dai).
Second-generation CAR+ cells achieve improved activity by incorporating costimulatory ligands. These signal for the increased cytokine production and T-cell proliferation, resulting in a stronger anti-leukemia immune response. Third-generation CAR+ cells added more costimulatory signals, further increasing cytokine production and tumor suppression in mice. Fourth-generation CAR+cells deposit universal killing cytokines in the tumor, stimulating a natural immune response in addition to that of the engineered CAR+ T-cells. This stronger immune response can kill tumor cells that may not be detected by CAR+ T-cells (Dai).
Just as the therapeutic mechanism of CAR has undergone revision for improvement, so too has the delivery vector. Nonviral gene transfection methods were initially used due to low immunogenicity and low risk of mutagenesis. These included plasmid transfection and mRNA insertion. However, the modifications were not passed down to progeny, and these treatments did not provide a long-term therapeutic effect (Dai).
The highly positive outcome of this trial, with sustained remission of the leukemia over time after just one treatment, points to the replicative nature of the therapy as the cause for its efficacy. In previous attempts, the re-injected T-cells killed just a few tumor cells before dying out (Berkhout). Without the replication and persistence of engineered T-cells, retroviral therapy is largely ineffective and more dangerous than helpful. Additionally, it is speculated that the elimination of normal (and cancerous) B-cells facilitated immunological tolerance of the CAR-positive cells, since no immunological rejection was observed.
However, cytokine release from proliferating T-cells can cause Cytokine Release Syndrome (CRS), a cytokine toxicity effect, in patients undergoing modified T-cell therapy. This is a dangerous but manageable side effect of the cancer treatment. Cytokine toxicity manifests as high fever, hypotension, hypoxia, and can potentially cause organ failure. Several avenues exist to address cytokine toxicity. Cytokine blockers such as tocilizumab have been used to control CRS without degrading T-cell performance. The magnitude of the CRS effect has been correlated to overall tumor burden within the patient. Therefore, conventional chemotherapy before or after T-cell infusion to reduce the tumor load may also help alleviate the toxic effects of cytokines. Chemotherapy or radiotherapy to artificially deplete natural lymphocytes can also increase the engraftment of the engineered lymphocytes, further improving long-term efficacy. Steroids and other anti-inflammatory drugs have also been explored (Dai).
In order to be effective, the CAR-positive engineered T-cells must overcome immunosuppressive conditions within the tumor microenvironment. Tumors are, by their nature, rapidly mutating and not very predictable. Thus, they are likely to express immunosuppressive factors. If therapy itself creates a selective pressure in favor of immunosuppression activity, then the efficacy of the therapy will be diminished. There are a variety of immunosuppressive factors that may be expressed in a tumor, and the the retroviral therapy must be able to overcome them. A dominant-negative receptor for immunosuppressive cytokine TGF-β can invert the signal into an immune-promoting effect. Alternatively, inhibitory immune receptors such as CTLA-4, which functions as a checkpoint for the immune response, can be down-regulated to silence the effect of that signaling pathway. The CAR-positive cells can also be engineered to recognize and attack NKG2D ligands expressed on immunosuppressive cells. A PD1-CD8 chimeric receptor can be used to convert a programmed cell death signal, in the form of PD1, into a CD8-positive T-cell activation signal, increasing the immune response in other, natural, cells (Dai).
Alternatively, CAR-postiive T-cells with CAR-induced IL-12 cytokine secretion enhance tumor suppression by recruiting a secondary wave of immune cells to target the tumor. This secondary wave of immune cells is capable of targeting tumor cells that have stopped expressing the CAR antigen. Without the secondary wave or other co-signals engineered into the T-cells, these tumor cells would escape destruction and enable the cancer to survive.
Resistance to other immunosuppressive factors can be increased with conditioning chemotherapy, such as cyclophosphamine or fludarabine, prior to engineered CAR-positive T-cell transfusion (Dai).
Additional trials have had promising results, but more trials need to be done before retroviral therapy gains regulatory approval. In August 2017, the FDA approved tocilizumab for treatment of CRS, but that is only one element of the whole treatment scheme.
The methodology of modifying autogenic T-cells op recognize and attack leukemic tumor cells has been extended in recent experiments to other immune cells. In particular, Natural Killer (NK) cells have been successfully modified with the CAR transgene. They naturally express a variety of costimulatory receptors that can recognize ligands expressed on the surface of tumor cells (Hu). NK immunotherapy is still in the pre-clinical stage (as of August 2017) but shows good promise. A key advantage of engineered NK cells over engineered T-cells is that CAR-positive NK cells could potentially be used as an “off the shelf” product, eliminating the need for personalized therapy (Rezvani). Engineered T-cell immunotherapy, by contrast, must be done with a sample of the patient’s own T-cells. For patients whose healthy T-cell count has dropped, obtaining a sample may be difficult or not feasible. These patients would benefit from engineered NK-cell immunotherapy.
Although CAR-positive T-cell therapy is only effective against leukemia so far, some advancements have also been made towards applying the method towards solid tumor cancers as well. Obstacles towards solid tumor immunotherapy include identification of target antigens, and trafficking of modified T-cells to the tumor. Because the tumor is solid rather than distributed, activation of the T-cells is not consistent, and successful activation presents a local izedcytotoxicity problem (Yeku).
Glioblastoma presents an immediate obstacle in the transport of T-cells into the brain. A pre-clinical study with repeated intracranial infusions of first-generation CAR-positive T-cells targeting IL-13Rα2 yielded only a temporary antiglioma response in two patients out of the three. An experimental trial with second-generation CAR-positive T-cells prevented local tumor recurrence, but did not control tumor progression at metastatic sites (Yeku).
Pre-clinical trials for neuroblastoma reported a complete remission rate of 27% with first-generation CAR-positive T-cells targeting GD2. However, subsequent treatments failed to control disease progression. Improved performance has been observed with second-generation engineered T-cells, but the transport issue remains problematic (Yeku).
CAR-positive T-cells targeting CD28 and HER2 – two oncogenes associated with breast cancer – have shown positive results in transgenic mice. However, breast tumor cells can develop resistance to therapy by cleaving the extracellular domain of the targeted surface markers. This can be mitigated by using a dual-target CAR system, such that the loss of a single antigen type does not allow the tumor cell to evade destruction. An alternative solution is using CAR-positive T-cells that secrete inflammatory cytokines to elicit a natural immune response (Yeku).
Mesothelioma evades immune activity by creating an immunosuppressive microenvironment. Any engineered T-cell approach to treating mesothelioma will necessarily have to feature some means of subverting that immunosuppressive environment. This can include dominant-negative PD-1 receptors to invert the immunosuppressive signal into an immune-promoting signal. In addition to being expressed in mesothelioma tumors, Mesothelin is also expressed in breast and ovarian cancers, and is also a potential target for engineered CAR (Yeku).
Prostate cancer has more promising initial trials. Prostate stem-cell antigen and PSMA are two antigens that can be used as CAR targets for immunotherapy treatments. Prostate stem-cell antigen is anchored on the cell surface, while PSMA is a transmembrane protein that translocates to the extracellular side of the epithelium during the transformation to malignant prostate adenocarcinoma. Tests in mice with third-generation CAR-positive T-cells showed proliferation of the engineered T-cells, and mouse survival rates were increased. Other tests, with CAR targeting CD28 and OX-40, also showed robust proliferation and cytokine production. These T-cells delayed tumor growth and prolonged survival (Yeku).
Metastatic renal cell carcinoma expresses carboxy-anhydrase-IX (CA-IX). Although it’s a functional target for modified CAR-positive T-cells, CA-IX is also expressed in healthy tissues such as intestinal epithelium, duodenum, and the biliary tract. CA-IX expression can also be induced in other tissues by hypoxic conditions. Two preclinical studies using first-generation engineered T-cells to treat three patients each showed robust cytokine production and cytotoxicity, but also liver enzyme toxicity. Furthermore, all three patients in one of the studies developed antibodies against the CAR fragment used for immunotherapy. There was ultimately no clinical response among the patients (Yeku).
Sarcomas are, as a rule, heterogeneous, and thus it is difficult to select a target for CAR-positive T-cell therapy. HER2 and CD28ζ NKG2D have both been exploited with second-generation engineered T-cells, but these markers can also be found in other tissues. In nude mice, CAR-positive T-cells targeting the IL-11 receptor α-chain were effective against primary sarcomas as well as pulmonary metastatic tumors. Because angiogenesis and vascularization are highly important to sarcomas, additional modifications to the T-cells to suppress angiogenesis may be desired. For example, T-cells that also secrete inhibitors for angiogenic factor VEGF may have more success (Yeku).
Although engineered T-cell immunotherapy has had excellent initial results against leukemia, the problems posed by solid tumors currently prevent the same methodology from being successfully deployed against other cancers. In particular, target factors expressed in healthy tissue can make T-cell immunotherapy difficult.
Retroviral-mediated T-cell immunotherapy is a promising technology, especially for the treatment of leukemia and other blood cancers. As promising as it is for blood cancers, there is still a long ways to go to produce a safe and effective treatment for cancers of solid tumors. The basic methodology – using a retrovirus to transfect T-cells with a custom gene for a custom antigen-recognition protein – is quite elegant. The use of HIV to perform this transfection is not only novel but also downright beautiful. With further innovation, it has significant potential to overcome existing barriers to use. As engineered T-cell technology advances, it will hopefully become easy, reliable, and effective in the fight against cancer.
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