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reflective essay PP

Submitted by jdantonio on Thu, 05/04/2017 - 22:45

Our first project which we began at the beginning of the semester and have continued through to the end is the weekly journal entry. I think when I was first given the assignment I thought this would largely be busy work, something to check off at the end of each week. However as the semester went on I think I continually got more out of it. The reading of papers to create journals entries and just the act of writing made me a better scientific writer. Seeing many examples of  scientific literature allowed me to compare and contrast styles and improve my own writing by synthesising the best parts for my journal entries. It also helped to get me to do projects in other classes in a more reasonable time frame. In the past I would typically do projects at the last second but spreading them out over several days as journal entries made me go at a slower rate and I feel helped to improve their quality. I think in the future I will spread out my work over a larger period of time which will help me improve my writing quality and be prepared for adverse events. I will also try to write more in general as I definitely feel that I have improved my writing skills simply by the act of writing regularly.  

 

reflective essay jornal entry

Submitted by jdantonio on Thu, 05/04/2017 - 21:19

Our first project which we began at the beginning of the semester and have continued through to the end is the weekly journal entry. I think when I was first given the assignment I thought this would largely be busy work, something to check off at the end of each week. However as the semester went on I think I continually got more from it. The reading of papers to create journals entries and just the act of writing made me a better scientific writer seeing many examples of  scientific literature allowed me to compare and contrast styles and improve my own writing by synthesizing the best parts in my journal entries. It also helped to get me to do projects in other classes in a more reasonable time frame. In the past I would typically do projects at the last second but spreading them out with journal entries made me go at a slower rate and I feel helped to improve their quality. I think in the future I will spread out my work over a larger period of time which will help me improve quality and be prepared for adverse events. I will also try to write more as I definitely feel that I have improved my writing skills simply by the act of writing regularly.  

cancer gentics project 3 excerpt 6

Submitted by jdantonio on Thu, 05/04/2017 - 20:33

Prior to the infusion of our selected and modified T-cell lines we will perform a  myeloablation, the destruction of immune cells in the body by high dose full body radiation treatment and the infusion of hematopoietic stem cells (Wrzesinski et al 2010). This pre treatment of patients has been shown to greatly improve the efficacy of ACT treatments in vivo(Perica et al 2015).  This works by reducing the presence of suppressing regulatory lymphocytes that inhibit cytotoxic T-cell activity(Wrzesinski et al 2010). We would infuse both our adaptive cell lines and our chimeric cell lines at the same time following the myeloablation and would concurrently administer IL-2 by and IV (Kochenderfer et al 2012). After the administration of the treatment we would monitor the patient and be on alert for cytokine toxicity a potential side effect of T-cell adaptive therapies caused by T-cell stimulated release of inflammatory cytokines. This can lead to fever and inflammation in the patient and can in very severe cases cause patient death (Barrett et al 2013). For dosage of the treatment, T-cell numbers are a limiting factor in the treatment. So we would infuse all the T-cells we are able to generate in a reasonable amount of time (maybe a month or two of growth), this amount would vary depending on multiple factors.

 

Barrett DM, Singh N, Porter DL, Grupp SA, and June CH. 2013. Chimeric Antigen Receptor

Therapy for Cancer. Annual Review 65: 333-347.

<http://annualreviews.org/doi/full/10.1146/annurev-med-060512-150254>. Accessed

2017 May 1.



Kochenderfer JN, Dudley ME, Feldman SA,Wilson HW, Spaner DE, Maric I, Stetler-Stevenson

M, Phan GQ, Hughes MS, Sherry MR, Yang JC, Kammula US, Devillier L, Carpenter R,

Nathan DAN, Morgan RA, Laurencot C,and Rosenberg SA. 2012. B-cell depletion and

remissions of malignancy along with cytokine-associated toxicity in a clinical trial of

anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 119(12):2709-20.

<http://www.bloodjournal.org/content/bloodjournal/119/12/2709.full.pdf>. Accessed

2017 May 1.

 

Perica K, Varela JC, Oelke M, and Schneck J. 2015. Adoptive T Cell Immunotherapy for Cancer.

Rambam Medical Journal 6(1): e0004. National Center of Biotechnology

Information[NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327320/>.

Accessed 2017 April 29.

 

Wrzesinski C, Paulos CM, Kaiser A, Muranski P, Palmer DC, Gattinoni L, Yu Z, Rosenberg SA,

and Restifo NP. 2010. Increased intensity lymphodepletion enhances tumor treatment

efficacy of adoptively transferred tumor-specific T cells. Journal of Immunotherapy

33(1): 1-7. National Center of Biotechnology Information[NCBI].

cancer gentics project 3 excerpt 5

Submitted by jdantonio on Thu, 05/04/2017 - 18:59

We will augment the endogenous TIL lines by the creation of additional cell lines that have genetically engineered chimeric antigen receptors (CAR). These chimeric receptor will be designed to bind to one of the remaining 10 most prevalent neoantigens that we identified as previously described. We will create ten cell lines, each being given one of chimeric receptors, creating ten cell lines which each bind to a different neoantigen. We will introduce a gene which encodes for a genetically engineered T-cell receptor by a viral vector in vitro. The CTR will be  created by the fusion of the Vh and VL gene sequence segments of a monoclonal antibody that binds to our neoantigen, a hinge-Ch2-Ch3 IgG1 , a transmembrane CD4 domain, and zeta chain of the CD3 cytoplasmic domain (Park et al 2007). This creates a chimeric TCR that recognises one of our discovered neoantigen and can activate T-cell activation (Park et al 2007). The monoclonal antibodies that bind to our neoantigens will be created by using a generalized method of immunization of rodent models with our neoantigens, the rodents will create antibodies against the neoantigen and they will be harvested and screened in a manner similar to the adoptive cells for bind specificity to the neoantigen (Gerdes et al 1983). These antibody are then used to make the gene fragment that is placed in the chimeric vector. We would perform this procedure for the 10 most common neoantigens for our tumor that were not found to have endogenous TIL’s during the adoptive cell therapy. The chimeric gene would be delivered to the cells in vitro by an IVT (in vitro transcription) RNA vector which is one of the standard delivery systems for CARs (Zhao et al 2010). Once the viral vector has been incorporated into the T-Cells we would then modify them further by using CRISPR to knockout the PDL-1 and CTLA-4 receptors on the cells(Albain et al 2015).

 

Ablain J, Durand EM, Yang S,  Zhou Y,  Zon LI. 2015. A CRISPR/Cas9 Vector System for

Tissue-Specific Gene Disruption in Zebrafish. Developmental Cell 32(6): 756–764.

Sciencedirect.<http://www.sciencedirect.com/science/article/pii/S1534580715000751>.

Accessed 2017 May 1.

Gerdes J, Schwab U, Lemke H, Stein H. 1983. Production of a mouse monoclonal antibody

reactive with a human nuclear antigen associated with cell proliferation. International

Journal of Cancer 31(1): 13-20. Wiley Online Library. <http://onlinelibrary.wiley.com/doi/10.1002/ijc.2910310104/full>. Accessed 2017 May 1.

Park JR, DiGiusto DL, Slovak M, Wright C, Naranjo A, Wagner J, Meechoovet HB, Bautista C, Chang WC, Ostberg JR, Jensen MC. 2007. Adoptive Transfer of Chimeric Antigen Receptor Re-directed Cytolytic T Lymphocyte Clones in Patients with Neuroblastoma. Molecular Therapy 15(4):  825–833. Science Direct. <http://www.sciencedirect.com/science/article/pii/S152500161631351X>. Accessed 2017 May 1.

 

Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J,

Levine BL, Albelda SM, June CH. 2010. Multiple injections of electroporated autologous

T cells expressing a chimeric antigen receptor mediate regression of human disseminated

tumor. Cancer Research

70(22):9053-906.<https://www.ncbi.nlm.nih.gov/pubmed/20926399>. Accessed 2017

May 1.

cancer gentics project 3 excerpt 4

Submitted by jdantonio on Thu, 05/04/2017 - 17:47

For the selection of our T-cells that possess endogenous neoantigen receptors we first harvest T-cells by taking blood samples from our patient. The Cells would then be separated from the plasma and the neoantigen cytotoxic T-cells would be selected out by using autologous DC cells that have been loaded with the protein epitome of the neoantigens which we have identified (Yee et al 2002). We will be following the standard procedure for adoptive T-cell generation as described in Yee et al 1999(Yee et al 1999). In short the T-cells are separated and cloned, creating identical clone lines, then screened by testing them for their ability to bind to antigen loaded dendritic cells which are placed in a set of loaded wells for testing. The population of T-cells that are found to bind to the neoantigen are then put through a chromium lystic test. where in the TILs are put on a plate with harvested tumor cells that present the target neoantigen and have been loaded with chromium, the lysing of these cells causes the release of chromium and can be used to test whether the antigen binding T-cells are cytotoxic (Yee et al 2002).  The antigen binding cytotoxic  T-cells will then be raised in culture containing IL-2 and anti CD3-antibodies which activated the growth and proliferation of T-cells, this will be done in 14 day cycles.(Yee et al 2002) We will perform theses screening tests to find a population of cells that can bind to the neoantigens we experimentally derived, we would hope to find cell lines cable of binding to about 10 of the 20 most common of our neoantigens.We will also give preference to those T-cells capable of binding neoantigens caused by driver mutations, mutations that are critical to cancer progression(Stratton et al 2009). With this we will have 10,or more hopefully, T-cell lines that can bind to our tumor and initiate cell death. The use of multiple adoptive cell lines will help to prevent the cancer from become resistant to our treatment, as one single mutation, or even a few, would not be enough for the tumor to evade treatment. Finally we will further the effectiveness by knocking out the T-cell suppression CTLA-4 receptor and the T-cell death initiating PDL-1 receptor by CRISPR knockout as described in detail later in the project (Albain et al 2015).

 

Ablain J, Durand EM, Yang S,  Zhou Y,  Zon LI. 2015. A CRISPR/Cas9 Vector System for

Tissue-Specific Gene Disruption in Zebrafish. Developmental Cell 32(6): 756–764.

Sciencedirect.<http://www.sciencedirect.com/science/article/pii/S1534580715000751>.

Accessed 2017 May 1.

 

Stratton MR,Campbell PJ, Futreal PA. 2009. The cancer genome. Nature 458: 719-724.

<http://www.nature.com/nature/journal/v458/n7239/full/nature07943.html>. Accessed

2017 may 1.

 

Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, and Greenberg PD. 2002.Adoptive

T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients

with metastatic melanoma: In vivo persistence, migration, and antitumor effect of

transferred T cells. Proceedings of the National Academy of Sciences 99(25):

16168–16173. <http://www.pnas.org/content/99/25/16168.full.pdf> Accessed 2017 April

30



Yee C,  Savage PA,  Lee PP, Davis MM, and Greenberg PD. 1999.Isolation of High Avidity

Melanoma-Reactive CTL from Heterogeneous Populations Using Peptide-MHC

Tetramers. The Journal of Immunology 162 (4) 2227-2234. <http://

www.jimmunol.org/content/162/4/2227?ijkey=5e73920a74aaa8e458988ce6c5acc1d1f2f9

ef72&keytype2=tf_ipsecsha>. Accessed 2017 April 30.

cancer gentics project 3 excerpt 3

Submitted by jdantonio on Thu, 05/04/2017 - 16:00

T-cells play a key role as a part of the immune system. They identify pathogenic entities by recognition of non-self antigens on the damaged/infected/foreign cells (Neumann et al 2002). T-cells are able to destroy cells that possess an antigen that they are cable of binding to by three main pathways.(neumann et al. 2002). The first method of lysis for cytotoxic T-cells in the release of cytotoxic granules such as perforin, which break down the target cell's membrane causing death(Neumann et al. 2002). The second is by the activation of the RAS receptor pathway, T-cells can have the ligand for this receptor (CD95L) which activates the apoptosis pathway in the target cell (Cox and Der 2003). The third method that cytotoxic T-cells use to kill their target cells is the release of cytokines such as TNF-α, which can induce apoptotic pathways within the cell (Neumann et al 2002). Our T-cell will use a combination of these pathways to effectively kill tumor cells that present neoantigens that the T-cells bind to.

 

Cox AD and CJ Der. 2002. The dark side of Ras: regulation of apoptosis. Oncogene 22:

8999–9006.<http://www.nature.com/onc/journal/v22/n56/full/1207111a.html>. Accessed

 

Neumann H, Medana IM, Bauer J, Lassmann H. 2003. Cytotoxic T lymphocytes in autoimmune

and degenerative CNS diseases. Trends in Neuroscience 25(6): 313-319.

<http://www.cell.com/trends/neurosciences/fulltext/S0166-2236(02)02154-9>. Accessed

2017 May 3.

cancer genetics project 3 PP

Submitted by jdantonio on Fri, 04/28/2017 - 13:12

The first step in our treatment process is the identification of our cancer’s neoantigens. These would vary from patient to patient and would have to be Identified experimentally. Identification of neoantigens begins by first sequencing the genome of the patient's tumors using massive parallel sequencing(MPS). MPS is a system which readily identifies tumor cell mutations by comparison of the genome of the cancer cells to the genome of a somatic cell (Gubin et al 2015).  This method of genome analysis has been shown to be an effective means to identify cancer cell gene mutations (Shiraishi et al 2011).We will use a hybrid exome sequencing technique which allows for the analysis of only genes which encode proteins and allow sequencing on a time scale that is relevant to clinical treatment (Hodges et al 2009). Once the tumor genome has been sequenced and analyzed to identify mutations we will then determine which of the tumors mutations are in oncogenes capable of binding to the MHC protein within the cell (Gubin et al 2015). This will be accomplished by utilizing bioinformatic databases and softwares, specifically the  NetMHCpan algorithm system which identifies a wide range probable MHC binding sequences in Human and nonhuman primates (Nielsen et al 2007).We will then harvest lymphocytes from the patient and test them for neoantigen binding specificity in vitro and select T-cells with tumor suppressing ability that possess the receptor for one of the neoantigens we derive from or cancer cell genome analysis. Following this we would grow these cells in culture to create a large amount of tumor infiltrating lymphocytes (TIL) (Perica et al 2012). These TIL’s will be further modified to improving their binding specificity and resistance to T-cell suppression.

 

Gubin MM, Artyomov MN, Mardis ER,and Schreiber RD. 2015. Tumor neoantigens: building a framework for personalized cancer immunotherapy. The Journal of Clinical Investigation 125(9): 3413–3421. National Center for Biotechnology Information[NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588307/>. Accessed 2017 April 24.

 

Hodges E, Rooks M, Xuan Z, Bhattacharjee A, Gordon DB, Brizuela L, McCombie WR, and Hannon GJ. 2009. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nature Protocol 4(6): 960-974. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990409/>. Accessed 2017 April 24.

 

Nielsen M, Lundegaard C, Blicher T, Lamberth K, Harndahl M, Justesen S, Røder G, Peters B, Sette A, Lund O, Buus S. 2007. NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence. PLOS one 2(8): e796. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949492/>. Accessed 2017 April 24.   

Shiraishi T,Terada N,  Zeng Y, Suyama T, Luo J, Trock B,  Kulkarni P, and Getzenberg RH. 2011. Cancer/Testis antigens as potential predictors of biochemical recurrence of prostate cancer following radical prostatectomy. Journal of Translational Medicine 9: 153. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184272/>. Accessed 2017 April 24.

Cancer genetics project 3 excerpt 2

Submitted by jdantonio on Fri, 04/28/2017 - 12:06

One of the major problems that the immune system faces when fighting cancer is a lack of T-cells that are able to recognize the cancer as a pathogenic entity and begin an immune response to the cancer cells.(Drake et al 2014)We propose that we create adapted T-cells that are capable of recognising and binding to tumor cells via tumor specific neoantigens (Rosenberg 2014). We will accomplish this with a multi step T-cell adaptation which will include selection for T-cells with endogenous neoantigen bind receptors and an addition of a chimeric neoantigen specific receptor via gene transfer to those cells already selected for endogenous receptors. These double neoantigen  receptor possessing T-cells will then be further genetically modified to remove T-cell inactivation pathways thus preventing tumor cell inhibition of T-cell activity. Specifically we will be be down regulating the PD1 receptor which stimulates T-cell death and the CTLA-4  which inhibits T-cell activation.(Rosenburg 2014)This will lead to T-cells that have a very high binding specificity to cancer cells and that are resistant to T-cell suppression mechanisms thus allowing them to activate the cell death pathway in cancer cells and elicit a greater immune response against the cancer cells.

Drake CG, Lipson EJ, and Brahmer JR. 2014. Breathing new life into immunotherapy: review of melanoma, lung and kidney cancer. Nature Review 11: 24-37.  <http://www.nature.com/nrclinonc/journal/v11/n1/full/nrclinonc.2013.208.h....



Rosenberg SA. 2014. Decade in review—cancer immunotherapy: Entering the mainstream of cancer treatment. Nature Review 11: 630-632. <http://www.nature.com/nrclinonc/journal/v11/n11/full/nrclinonc.2014.174.....

Cancer genetics project 3 excerpt

Submitted by jdantonio on Thu, 04/27/2017 - 20:00

The first step in our treatment process is the identification of our cancer’s neoantigens, this would vary from patient to patient would have to be experimentally Identified. This is done by first sequencing the genome of the cancer tumors using massive parallel sequencing a system which readily identifies tumor cell mutations by comparison of the genome of the cancer cells to the genome of a somatic cell (Gubin et al 2015).  This method of genome analysis has been shown to be an effective means to identify cancer cell gene mutations (Shiraishi et al 2011).We will use a hybrid exome sequencing technique which allows for the analysis of only genes included in the cells exomes and allows for faster sequencing in a time scale that is relevant to treatment (Hodges et al 2009). Once the tumor genome has been sequenced and analyzed to identify mutations we will then determine which of the tumors mutations would be indicative of an oncogene capable of binding to the MHC protein within the cell (Gubin et al 2015). This will be accomplished by utilizing bioinformatic databases and softwares, specifically the  NetMHCpan algorithm system which identifies a wide range probable MHC binding sequences in Human and nonhuman primates (Nielsen et al 2007). We will then harvest lymphocytes from the patient and test them for neoantigen binding specificity in vitro and select from among those harvest T-cells with tumor suppressing ability that posses the receptor for one of the neoantigens we derive from or cancer cell genome analysis. Following this we would grow these cells in culture to create a large amount of tumor infiltrating lymphocytes (TIL) (Perica et al 2012). These TIL’s will be further modified by the introduction of  chimeric receptor that binds one of the other  neoantigens which we identified.

 

Gubin MM, Artyomov MN, Mardis ER,and Schreiber RD. 2015. Tumor neoantigens: building a framework for personalized cancer immunotherapy. The Journal of Clinical Investigation 125(9): 3413–3421. National Center for Biotechnology Information[NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588307/>. Accessed 2017 April 24.

 

Hodges E, Rooks M, Xuan Z, Bhattacharjee A, Gordon DB, Brizuela L, McCombie WR, and Hannon GJ. 2009. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nature Protocol 4(6): 960-974. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990409/>. Accessed 2017 April 24.

 

Nielsen M, Lundegaard C, Blicher T, Lamberth K, Harndahl M, Justesen S, Røder G, Peters B, Sette A, Lund O, Buus S. 2007. NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence. PLOS one 2(8): e796. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949492/>. Accessed 2017 April 24.   

Shiraishi T,Terada N,  Zeng Y, Suyama T, Luo J, Trock B,  Kulkarni P, and Getzenberg RH. 2011. Cancer/Testis antigens as potential predictors of biochemical recurrence of prostate cancer following radical prostatectomy. Journal of Translational Medicine 9: 153. National Center for Biotechnology Information [NCBI]. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184272/>. Accessed 2017 April 24.

Immunotherapy PP

Submitted by jdantonio on Fri, 04/21/2017 - 10:36

Immunotherapies seek to increase the activity or effectiveness of T-cells and increase T cell cancer targeting specificity (Drake et al 2014).  This can be done by the administering of a vaccine that contains a cancer specific antigen to the body, this in turn activates DC cells that recruit and activate T-cells that recognise the cancer specific antigens (Drake et al 2014). The affects of vaccines to induce more T-cell activity have been found to be effective in some cancer types but not all as the expression of immune checkpoints greatly reduce treatment effectiveness (Drake et al 2014). Antibody suppression of immune checkpoints, so they no longer interfere with endogenous T-cells,  has been shown to be an effective treatment in melanoma cancer in large scale clinical trials. (Drake et al 2014). Another method of immunotherapy is the genetic modification of existing T-cells to target cancer specific antigens, this is done by viral transfection of engineered antigen receptor genes to endogenous T-cells (Drake et al 2014). An alternative to this method is ACT treatment a process by which endogenous T-cells are harvested and selected in vitro for cancer cell binding specificity, then raised in  culture and reintroduced in vivo to the patient (Rosenberg 2014).These methods are necessary in many cancer types as relatively few endogenous T-cells have receptors for cancer specific antigens thus making other treatments like vaccines and immune checkpoint therapy ineffective (Drake et al 2014). While immunotherapies are a promising addition to traditional cancer treatments they face one major obstacle, the lack of Identified cancer specific antigens (Drake et al 2014). To further immunotherapy as a treatment option more cancer specific antigens must be discovered (Drake et al 2014).     

 

Drake CG, Lipson EJ, and Brahmer JR. 2014. Breathing new life into immunotherapy: review of melanoma, lung and kidney cancer. Nature Review 11: 24-37.  <http://www.nature.com/nrclinonc/journal/v11/n1/full/nrclinonc.2013.208.html>.

Rosenberg SA. 2014. Decade in review—cancer immunotherapy: Entering the mainstream of cancer treatment. Nature Review 11: 630-632. <http://www.nature.com/nrclinonc/journal/v11/n11/full/nrclinonc.2014.174.html>.  

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