Chimeric Antigen ReceptorIn Gene Therapy

Duy-Khiem Chanh PhamKevin Hugins

Molecular BiologyMarch 13, 2014

Introduction

Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. However, the first FDA-approved trial on humans was not until 1990 on two young females with ADA-SCID, an immune deficiency disorder that leaves them defenseless against infections. Under Dr. William French Anderson and a team of colleagues, the first successful human gene therapy using reconstituted T lymphocytes to treat patients with ADA deficiency produced great results (2). Since then clinical trials involving genetic engineering also show promising results in treating eyesight diseases, Parkinson’s disease, and cancer. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.

Traditionally the battle with cancer has always been fought with chemotherapy, radiation therapy, and even invasive surgeries. These treatments have made an astounding impact as far as being able to destroy some cancerous cells. However, the biggest downfall to chemotherapy or radiation therapy is the non-selective targeting of cells both cancerous and normal host cells. A more innovative approach has now gone through successful clinical trials to combating cancer, specifically leukemias such as chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In fact, Dr. Carl June and his team from a Penn medicine was nationally recognized for being able to treat leukemia patients using their own immune cells. The findings were published in an article, late of 2013, has been feature “in more than 20 top newspapers across the nation” (3). Dr. June and his team started the trial in summer of 2010 with three CLL patients whom were the very first to this procedure. Two of the three patients are now still in remission. The procedure expanded to treat ALL patients and also showed amazing results. Findings from three different trials with adult patients with CLL, pediatric patients with ALL, and adult patients with ALL collectively produced remarkable responses. In adult patients with CLL, 15 out of 32 responded very well to treatment including 7 of them going into complete remission. In 22 pediatric patients with ALL, 19 of them went into complete remission and all 5 patients with ALL also showed complete remission (4).

Furthermore, cancer treatments have never been cheap but one of the major contributors to this cost is multiple treatments that extend over a period of time along with transplants when treatments fail. This is why research is an ongoing process for a more effective one-time approach. With CAR immunotherapy, it seeks to accomplish this and may be the tip of the iceberg for the future. Reported on NBC news, an article by Marilyn Marchionne claims, “lab costs are now $25,000, without a profit margin” for gene therapy. The Leukemia and Lymphoma Society has also donated 15 million dollars to research groups testing out specifically the CAR approach and expanding it (5). This paper will aim to explain the details of CAR immunotherapy, the structure of the receptors, the mechanism in which it works, the vector used for delivery, and the possible side effects.

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Receptor Structure and Function

With the introduction and advancement of cancer therapies using T-cells modified with chimeric antigen receptors (CAR), researchers are having success in overcoming two inherent problems that the immune system has when responding to cancer cells. The cells of the immune system are continually on the hunt for cells that are presenting non-self antigen on the cell surface. Cancer cells are essentially self cells in which cell division has gone awry. When cells of the immune system come into contact with these cancer cells they recognize the antigens on the cell surface as being self and the leukocyte continues on searching for other invaders.

The body has a second major process in recognizing infections using major histocompatibility complex or MHC. One of the functions of MHC’s is to alert immune system cells that a self-cell has been infected internally. The infected cell upregulates the processes involved with MHC. It digests some of the foreign protein, attaches it to an MHC and expresses the antigen protein and MHC on the cell surface. When T Cells encounter these MHC molecules they are activated and an immune response is initiated. (11) However, many types of cancer have the ability to prevent the upregulation of MHC so the immune system does not recognize the pathogenic cells. Again, the immune system is prevented from recognizing a problem with the proliferating cancer cells and is unable to mount a response.

The first publication regarding CAR was in 1989 by Gross et al. The process involved placing a modified receptor and signaling complex on a T cell. The goal of this process was to modify a patient’s own T-Cells so they could recognize and attack cancer cells while undergoing normal expansion in vivo in order to provide an expanding and persistent immune response. (8)

A native T cell has 2 primary components, the T cell receptor (TCR), which is designed to interact with MHC, and the signaling portion (CD3) which activates the T cell after an antigen is bound. In the first attempts at modifying T cells to attack cancer cells researchers tried to modify the antigen recognition site on the T cell. It was discovered that while this worked well in vitro there was poor response in vivo. This was because as the modified T cells circulated through the lymphatic system, when they reached the thymus they were selected out because the antigen recognition portion was very similar to self-antigens. Gross got past this obstacle by synthesizing an entirely new receptor that could very specifically target malignant cells while avoiding negative selection processes in the thymus. (7) The first step utilizes antibodies which are produced in B-Cells. As is shown in the figure to the right, an antibody is made up of four basic units, two heavy chains and two light chains. At the top of the “Y” the heavy and light chains both contain what is known as the variable region. It is there that the antigen binding sites for the antibody is located. Antigen binding sites have very high specificity for specific antigens. Researchers are able to engineer this binding site specifically for antigens presented on cancer cells in an individual patient. These antibodies can then be clonally expanded in a lab either in mice or isolated tumor cells. After sufficient quantities of the antibodies are made, the variable portions of the heavy and light chains are removed from the constant subunits of the antibody. This product is termed a single chain variable fragment, or scFv. These scFv’s will eventually be incorporated into viable T-Cells.

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Figure 1http://www.biology.arizona.edu/immunology/

tutorials/antibody/graphics/antibody.gif

Like antibodies, T-Cells also have receptors containing antigen binding sites. But, the binding site simply recognizing and attaching to an antigen is just the first part of T-Cell activation. In close

proximity to the receptor is a signal transduction complex that is responsible for providing the T-Cell an intracellular signal signifying that it has found something that is a threat that needs to be responded to. This signaling complex is referred to as CD3. CD refers to “cluster of differentiation”. The T-Cell CD3 has two subunits that have been given the designation of the Greek letter zeta. The body of a ζ unit penetrates deep into the T-Cell and signals it that the pathogen it has attached to needs to be destroyed. These ζ subunits are a critical component of the chimeric antigen receptor. An scFv alone would bind to the cancer cell but without a signal transduction unit no response would occur.

After obtaining CD3 ζ subunits the CAR can be assembled. The light and heavy variable chains obtained from the antibodies are attached toeach other using a flexible linker. Flexibility is critical so that antigen binding sites are able to conform to targeted antigens on the surface of the cancer cells. A ζ subunit is then

attached to the scFv with a spacer that must contain a hydrophobic region that can be inserted into the membrane of the T-Cell which is being modified.

Typically a viral vector is used to introduce the engineered receptor into T-Cells that have been harvested from the patient. The T-Cell population is then expanded before being introduced to the patient. Currently this process takes approximately two weeks from start until the cells are ready to be infused into the patient.

After expansion the chimeric T-Cells are introduced into the patient. The modified T-Cells with the chimeric antigen receptors function like native T-Cells. When they locate and bind to the target antigen on cancer cells the T-Cells release perforins and granzymes which lyse the cell membrane and break down the cancerous cell bringing about its death. Additionally, since the modified T-Cells circulate through the body in the blood and lymph, if the cancer has metastasized, the CAR T-Cells can locate and destroy the renegade cells in other parts of the patient’s body long before their physician has discovered

its proliferation. Because the modification of the T-Cells was accomplished through a virus, researchers hypothesize that persistent immunity will occur as a result of periodic reactivation of the virus. (9)

The engineered structure of chimeric antigen receptors has evolved and improved over time with in vitro and in vivo studies. As shown in the figure, signaling enhancements have evolved as researchers seek to create a more robust response from the CAR T-Cells. These changes have helped increase the effectiveness of the T-Cells. With better signaling mechanisms the ability to kill a cancer cell has increased significantly and

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Figure 2http://course1.winona.edu/kbates/

Immunology/images/figure_05_06.jpg

Figure 3http://upload.wikimedia.org/wikipedia/

commons/4/47/CAR_cartoon.png

Figure 3http://www.hindawi.com/journals/bmri/2010/956304/fig3/

proliferation, resistance, and persistence of the T-Cells have all been enhanced. As additional in vivo trials on humans are approved this mechanism will continue to be fine-tuned. (10)

The Vector System

There are several ways to introduce therapeutic DNA to cells but the most common and still widely use is through viral vectors. Viruses can be rendered non-pathogenic but still be effective in incorporating DNA into host’s cells for protein expression. A few considerations should be taken when choosing a vector for delivery such as dividing or non-dividing target cells, and short or long-term expression. Some of viruses that have been used as vectors include lentivirus, adenovirus, herpes virus, and retrovirus. Although all of these viruses have their advantages and drawbacks, the adenovirus and retrovirus demonstrate to be most useful.

The adenovirus is a non-envelope virus that utilizes double-stranded DNA. It allows up to 5 kb of exogenous DNA to be inserted using recombinant DNA technology and the transmission of these genes to host nucleus without disturbing the chromosome. Viral DNA is taken within the nucleus and exists as “extra” DNA that will be replicated and expressed into proteins. Therefore, it will not likely disrupt important cell functions. Another feature of the adenoviral-vector is the viral DNA will vanish making it less effective for chronic diseases but a more potent system to stimulate an immune response against cancer (6). Incorporating adenovirus into a host cell is first accomplished through certain interactions on the outside membranes of the cell and the virus. The structure of the adenovirus (Figure 1) has a fiber region that binds to the cell receptor. A second stimulatory interaction is through a motif found on the virus and an integrin molecule on the host cell. Both of these interactions are required for the entry of the virus following the endocytic pathway by forming a vesicle surrounded by a clathrin coat (Figure 2).

Figure 4 Figure 5http://www.genetherapynet.com/viral-vectors/adenoviruses.html http://www.genetherapynet.com/viral-vectors/adenoviruses.html

Adenovirus used as vectors have shown to be advantageous because they can infect various human cells with proper receptor, there are at lease 43 different serotypes in humans, and they can cause mild symptoms that usually accompanies a common cold. Furthermore, adenoviruses can be easily employed with therapeutic DNA using recombinant DNA. Once the necessary interactions are establish with host lymphocytes and the virus gains entry it has high gene transfer efficiency with low chance of mutations.

Another viral vector used in modification of immune cells are retroviruses. These viruses are enveloped and use single-stranded RNA in the form of mRNA. Retrovirus infects host cells and replicate using reverse transcription, a process where RNA is transcribe to DNA before going through

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transcription then translation and then expression of genes. This is opposite to conventional processes where DNA is transcribed to RNA and RNA is translated into proteins.

Figure 6 Figure 7http://www.ohsu.edu/xd/about/services/integrity/upload/IBC_Presentation-Choosing-a-Viral-Vector-System.pdf http://www.ohsu.edu/xd/about/services/integrity/upload/IBC_Presentation-Choosing-a-Viral-Vector-System.pdf

The structure of the retrovirus (figure 3) shows the viral RNA genome surrounded by structural proteins (gag) and further encase within an envelope with surface glycoproteins. Retroviruses possess its own enzymes such as integrase, reverse transcriptase, and protease to carry out its unique reverse transcription process in immune cells. A basic mechanism depicting retrovirus introduction (Figure 4) into host immune cells for gene expression shows a process for long-term through integration. Retroviruses can only infect dividing cells, a slight disadvantage compared to adenoviruses.

Both viruses ultimately can achieve the same endpoint by introducing therapeutic genes to host for expression. Choosing a proper vector system to use is not easy and is heavily dependent on the circumstances. Adenoviruses utilize a more transient gene expression whereas retroviruses offer long-term expression. Adenoviruses also can infect dividing and non-dividing cells, trigger a high immune response in target cells with great transduction efficiency. Retroviruses is more effective targeting non-dividing cells, can produce a moderate response in target cells with moderate transduction efficiency. Although these vectors are most commonly used, clinical trials are not limited to these two but are continuously trying different vectors.

Conclusion

The recent breakthroughs in the use of chimeric antigen receptor modified T-cells in the treatment of cancer in which all traditional treatments have failed shows great promise for the future. A National Cancer Institute article declared that some Doctors and Researchers see immunotherapy as “the fifth pillar” of cancer treatment. (10) There is still research and expanded human trials needed before modified T-cell treatment for cancer can begin to be used on a wider basis. Most of the success in this arena has been with various types of leukemia. Researchers are having less success with solid tumors such as is seen in lung or brain cancer. With current techniques there is still a risk of serious side effects including death. The most common deleterious side effect experienced by patients is cytokine release syndrome, a condition in which the inflammatory response is excessive and has a significant systemic effect on the patient’s whole body. However, recent progress seems to be developing quickly and researchers are optimistic that soon targeted immunotherapy techniques will dramatically alter the way cancer is treated.

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References

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2) Blaese, R. M., Culver, K. W., Miller, A. D., Carter, C. S., Fleisher, T., Clerici, M., ... Anderson, W. F. (1995). T Lymphocyte-Directed Gene Therapy for ADA$^-$ SCID: Initial Trial Results After 4 Years. Science, 270(5235), 475-480. Retrieved March 13, 2014, from http://www.jstor.org/stable/2889066

3) Abramson Cancer Center. (n.d.). Retrieved March 13, 2014, from https://www.penncancer.org/penn_news.cfm?ID=2125

4) Penn Medicine Team Reports Findings from Research Study of First 59 Adult and Pediatric Leukemia Patients Who Received Investigational, Personalized Cellular Therapy CTL019. (n.d.). Retrieved March 13, 2014, from http://www.uphs.upenn.edu/news/News_Releases/2013/12/ctl019/

5) Marchione, M. (2013, December 8). Gene therapy scores big wins against blood cancers. NBC News. Retrieved March 13, 2014, from http://www.nbcnews.com/health/cancer/gene-therapy-scores-big-wins-against-blood-cancers-f2D11708740

6) Vorburger, S. A., & Hunt, K. K. (2002, February 01). Adenoviral Gene Therapy. The Oncologist, 7(1), 46-59. Retrieved March 13, 2014, from http://theoncologist.alphamedpress.org/content/7/1/46.abstract

7) Turtle, C. (2013). Chimeric antigen receptor modified T cell therapy for B cell malignancies. International Journal of Hematology, 99(2), 132-140. Retrieved March 13, 2014, from http://link.springer.com.ozone.nsc.edu:8080/article/10.1007%2Fs12185-013-1490-x/fulltext.html

8) Chekmasova, A. A., & Brentjens, R. J. (2010). Adoptive T Cell Immunotherapy Strategies for the Treatment of Patients with Ovarian Cancer - Alena A Chekmasova - Discovery Medicine. Discovery Medicine. Retrieved March 13, 2014, from http://www.discoverymedicine.com/Alena-A-Chekmasova/2010/01/22/adoptive-t-cell-immunotherapy-strateg....

9) Lee, D. W., Barrett, D. M., Mackall, C., Orentas, R., & Grupp, S. A. (2012, May 15). The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clinical Cancer Research, 18(10), 2780-2790. Retrieved March 13, 2014, from http://clincancerres.aacrjournals.org/content/18/10/2780.abstract

10) Cancer Research Updates. (n.d.). Retrieved March 12, 2014, from http://www.cancer.gov/cancertopics/research-updates/2013/CAR-T-Cells

11) Parham, P., & Janeway, C. (2009). The immune system. London: Garland Science.

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