Title: Trends in Biotechnology
1Trends in Biotechnology
2- Brief Outline
- Gene Therapy
- Clinical Trials
- Recent Gene Therapy Success
- New Approaches to Gene Therapy
- Virotherapy
- Stem Cells
- Vaccines
- Tissue Engineering
- Xenotransplantation
- Drug Delivery
3 4- Gene Therapy
- Genetic Disorders
- Gene Target Selection
- Gene Delivery Methods
- Viral vectors
- Nonviral Delivery Methods
- Gene Therapy Examples
5- Clinical Trials
- Recent Gene Therapy Success
- New Approaches to Gene Therapy
- Spliceosome Mediated RNA Trans-splicing
- Triplex-Helix-Forming Oligonucleotide Therapy
- Antisense Therapy
- Ribozyme Therapy
6- Virotherapy
- Stem Cells
- Therapeutic Cloning and Embryonic Stem Cells
- Vaccines
7- Tissue Engineering
- Xenotransplantation
- Drug Delivery
- Biosensors
- Biotech Revolution Nanotechnology
8- Learning Objectives
-
- Know how genetic diseases are generated and how
genes are candidates for gene therapy. - List and define the methods used to deliver genes
into cells. - Know the difference between ex vivo and in vivo
gene therapy. - List diseases that have been the subject of gene
therapy research, the causes of those diseases,
and how gene therapy attempts to treat them. - Know the types of clinical trials and the steps
of the clinical trial process required for gene
therapy.
9- List and define new approaches to gene therapy,
such as spliceosome-mediated RNA trans-splicing,
triplex-helixforming oligonucleotide therapy,
antisense therapy, and ribozyme therapy. - Be familiar with the potential of stems cells,
the types of stem cells used in research, and
their applications. What are the issues
surrounding the use of stem cells? - Know the potential applications of biotechnology
regarding vaccines, tissue engineering,
xenotransplantation, drug delivery, and
biosensors.
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12- A. Introduction to Gene Therapy.
- Treat genetic diseases by putting normal genes
into cells to correct a disorder. This is better
than treating the disease with a drug that might
not be a cure. - Treat acquired and genetic diseases, eg cystic
fibrosis and AIDS. - Two types of gene therapy
- Somaticusing only the bodys cells to correct a
disorder. - Germ linepermanently modifying a gene in the
reproductive cells.
13- Genetic Disorders.
- Medical problems caused by a mutation in one or
more genes on the chromosomes. The person can be
born with the mutation or develop it. - Can be grouped into four categories
14- Can be grouped into four categories
- Single-gene
- Multigene disorders
- Mitochondrial disorders.
- Chromosome abnormalities.
15- Single-gene changesa mutation in one gene can
result in a change in the protein product or
possibly the elimination of the protein entirely.
Sickle cell anemia is an example.
16- Multigene disordersalso called multifactor
disorders, result from mutations in more than
one gene, and sometimes along with environmental
influences. Examples of these include heart
disease, diabetes, and cancer.
17- Mitochondrial disordersaffecting many organ
systems, these diseases are caused by mutations
in mitochondrial DNA.
18- Chromosome abnormalitiescomplete chromosomes or
large regions of a chromosome are missing,
duplicated, or modified in some way. Down
syndrome is an example.
19- Gene Target Selection.
- Usually, only a single gene causing a genetic
disorder can be treated (candidates for treatment)
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23- Gene Target Selection.
- The normal and mutated genes must be identified
and well-studied. - The disease caused by the mutation must be
well-understood. - An approved protocol involving a gene delivery
method must be available. - The potential toxic effects of the gene or gene
delivery vehicle must be examined, as well as
whether the therapy produces an immune response.
24- Gene Target Selection.
- The gene must be delivered to the correct cells.
- If the therapy method calls for it, the gene must
be integrated into the host chromosome so the
gene is not destroyed in host cells. - The gene must be turned on (transcription) at the
right time, and is also regulated properly.
25Gene Major Delivery Methods for the insertion of
genes into cells. Ex vivo gene therapy In vivo
gene therapy
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27- Gene Delivery Methods.
- Ex vivo gene therapy
- Cells are removed from the body.
- The gene of interest is inserted into them.
- The cells are cultured for reproduction.
- The cells are returned to the body.
- An advantage over in vivo gene therapy is that
rejection does not occur if the persons own
cells are used. - The transplantation of the cells is the biggest
technical problem.
28- Possible in vivo engineering
29- Gene Delivery Methods.
- In vivo gene therapy
- The gene is inserted directly into cells within
the body. - Vectors such as viruses are used to target the
DNA to specific cells. - Technical difficulties
- The transferred gene is unstable and the product
produced temporarily. - The methods are not as controlled as ex vivo gene
therapy because cells are not removed from the
body.
30Fig. 10.1 The different methods used to transfer
DNA into cells.
31Viral Vectors (Figure 10.1). Retrovirus. Adenoviru
s. Adeno-Associated Virus.
32- Viral Vectors
- Retrovirus.
- RNA viruses that only infect dividing human
cells. - DNA of interest can only be up to 8 kb in size.
- Site of integration into host chromosomes occurs
randomly. - Viral insertion could inactivate genes or elicit
an immune response.
33- Viral Vectors
- Adenovirus.
- Can infect dividing and non-dividing cells.
- Can engineer proteins on the virus surface to
target specific cells. - DNA of interest can be up to 7.4 kb and the
protein will be highly expressed. - Does not integrate into the genome, so there is
low risk of mutation.
34- Viral Vectors
- Adenovirus.
- Possible problems
- Temporary protein production because no
integration occurs. - The virus may replicate in host cells, killing
them. - Gene products may cause the cell to divide
abnormally. - The virus of gene products may cause an immune
response.
35- Viral Vectors
- Adeno-Associated Virus.
- Infect dividing and non-dividing cells.
- Need a helper virus to infect host cells.
- DNA of interest can be up to 5 kb.
- 95 of the time DNA integrates into a region in
chromosome 19, reducing the chance of genes being
activated or inactivated by the insertion of DNA.
36- Nonviral Delivery Methods.
- Other methods include
- electroporation,
- microinjection,
- biolistics (the gene gun), and
- liposomes (membrane-bound spheres that contain
the DNA).
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39- Gene Therapy Examples.
- First Gene Therapy.
- treatment of severe combined immune deficiency
(SCID), (defective adenosine deaminase gene
causes the immune system to not function
correctly). - 4-year-old girl received T-lymphocytes with the
correct ADA gene.
40- The therapy had to be repeated - enzyme would be
produced for only a few months. - gt 50 of the T cells in the girl had the
corrected gene, in 2003 her body still actively
produces ADA. - A second trial occurred on an 11-year-old girl,
but her immune system developed a reaction
against the virus, and only 0.1 to 1.0 of her
cells produced ADA.
41- Lung DiseaseCystic Fibrosis.
- Caused by a defective ion transport molecule
called the CF transmembrane conductance
regulator (CFTR) in the plasma membrane of a
cell (Figure 10.2). - Affects airways, the intestines, and the pancreas
- People with CF have increased mucus production,
bacterial infections in the lungs, and altered
epithelial cell transport.
42Fig. 10.2 A drawing of the CFTR protein inserted
into the cell membrane.
43- For therapy to work, only a small amount of
molecules need to be produced per cell. It has
been successful in cell culture and in CF mice by
liposomes. - Spraying adenoviruses with the CFTR gene into the
nose - only temporary treatment, and does not
treat organs eg pancreas. - Another alternative is spraying a DNA-liposome
aerosol into the nose.
44- Liver Disease.
- Transfected hepatocytes (liver cells)
- Not very successful
- Only about 10 of the cells go into the liver.
- In 1992, a 29-year-old patient was treated for
familial hypercholesterolemia (FH) - Causes cholesterol to build up in arteries,
leading to heart disease.
45- Caused by a defective low-density lipoprotein
receptor (LDLR) gene. - 250 grams of the patients liver was cultured.
- A retrovirus was used to infect about 25 of the
cells. - The cells put back into the liver.
- Tests confirmed that the cells were expressing
the LDLR gene, and medication contributed to
lowering cholesterol levels.
46- Clinical Trials.
- After the therapy has been tested on animals
(called preclinical trials), it is then tried
on humans. - There are several types of clinical trials
- There are several phases to clinical trials
(Figure 10.3)
47- There are several types of clinical trials
- Diagnostic trialidentifies better tests for
diagnosing diseases. - Treatment trialtests new therapies or drugs.
- Prevention triallooks for new ways to prevent
disease in people who have never had the disease. - Quality of life triallooks to improve the
well-being and quality of life of chronically ill
patients. - Screening trialfinds ways to detect specific
diseases or health conditions.
48- There are several phases to clinical trials
(Figure 10.3) - Phase I Trialstest on a small number (twenty to
thirty) of human volunteers to find dosage
limits, how to deliver it, and to find toxicity
and safety. - Phase II Trialsfind effectiveness and get more
toxicity and safety information with a larger
group of people (100300). If the therapy is
effective and safe, the researchers begin phase
III trials.
49- Phase III Trials many more people tested
(10005000). Information obtained from phase I
and II clinical trials is used, and the
therapeutic role of the drug is studied. - An application to the FDA is made for approval.
- Phase IV Trials after approval, if any
questions remain regarding safety and efficacy,
as well as the use of the treatment, they are
studied in these trials before the drug is
allowed to be released.
50Fig. 10.3 Drug discovery and approvals. (a) The
process of biotech drug discovery.
51Fig. 10.3 (b) New biotechnology drug and vaccine
approvals.
52- Recent Gene Therapy Success.
- No one has completely been cured by gene therapy,
but small successes mean that gene therapy could
be used for diseases that are difficult to treat.
53- Recent Gene Therapy Success.
- The first commercially licensed gene therapy
- In October 2003 in China, under the name
Gendicine, after 5 years of clinical trials. - Treats head and neck squamous cell carcinoma.
- An adenovirus vector containing the p53 tumor
suppressor gene can be used along with
chemotherapy and radiation therapy to increase
effectiveness. - Only reported side effect is a low-grade fever.
54- New Approaches to Gene Therapy
- Spliceosome Mediated RNA Trans-splicing
- Triplex-Helix-Forming Oligonucleotide Therapy
- Antisense Therapy
- Ribozyme Therapy
55- New Approaches to Gene Therapy.
- Why new approaches are needed
- Sometimes correcting the defective gene is not
effective. - For example, a mutated gene can prevent a normal
protein from functioning properly. This is called
a dominant negative gene and cannot be
corrected by simply inserting the correct gene.
56Fig. 10.4 The effect of a dominant negative
mutation on a normal protein.
57- New Approaches to Gene Therapy.
- Spliceosome Mediated RNA Trans-splicing (SMaRT)
- Instead of correcting the gene, the region of
mRNA that is affected is repaired - An RNA strand that pairs with the intron (by base
pairing) next to the mutated region (exon) of the
mRNA is introduced into cells. The RNA is
genetically modified to contain the correct exon
and a small region that binds to the neighboring
intron.
58- New Approaches to Gene Therapy.
- Spliceosome Mediated RNA Trans-splicing (SMaRT)
- Instead of correcting the gene, the region of
mRNA that is affected is repaired - When the RNA strand binds to the intron, the
duplex (section of double-stranded RNA) causes
the spliceosome to cut and remove the intron and
the defective exon from the mRNA. - The exons are joined, with the corrected exon
ligated into the mRNA, thereby generating a
functional, mature mRNA and protein.
59Fig. 10.5 A new approach to gene therapy using
spliceosome mediated RNA transplicing. (a) An
intron is excised from a normal transcript using
a spliceosome and exons 1 and 2 are ligated.
60Fig. 10.5 (b) In this example, a mutated exons 2
of the mRNA is repaired using SMART.
61- New Approaches to Gene Therapy.
- Triplex-Helix-Forming Oligonucleotide Therapy.
- triplex-forming nucleotides bind to target DNA
regions, and block transcription into mRNA. - The single-stranded string of nucleotides, about
fifteen to twenty-one bases in length, binds to
the groove between the double strands of DNA
where the mutated gene is used. - The triple helix that forms blocks transcription.
- A correct gene can be introduced into cells.
62Fig. 10.6 The prevention of transcription of mRNA
with a mutation using a triplex-helix-forming
oligonucleotide.
63- New Approaches to Gene Therapy.
- Antisense Therapy.
- Targets the mRNA of a mutated gene so that it
cannot be translated into protein. - Is complimentary to the mRNA that is used to code
for the protein, called the sense mRNA.
64Fig. 10.7 A mutated mRNA cannot be translated
using antisense technology.
65- New Approaches to Gene Therapy.
- Antisense Therapy.
- The steps of the method are
- New, antisense RNA is introduced into cells
that is complimentary to the mRNA that is used to
code for the protein, called the sense mRNA.
66- New Approaches to Gene Therapy.
- Antisense Therapy.
- The steps of the method are
- The antisense RNA binds to the sense mRNA strands
synthesized by the cell during transcription. - The duplex RNA is blocked from translation into a
protein, eliminating or dramatically reducing the
production of a mutated protein.
67- New Approaches to Gene Therapy.
- Antisense Therapy.
- May be used to treat diseases where there is a
loss of control over gene regulation or if a gene
is overexpressed. - The drug Genasense targets the mRNA for a protein
called bcl-2. The protein is involved in the
resistance of cancer cells to chemotherapy.
68Unfortunately
- Genta's stock took a nosedive yesterday on news
that its lead drug Genasense, a Phase III
candidate for melanoma, did not show a
statistically significant benefit for its
co-primary endpoint of progression-free
survival.Read more Genta tanks on Genasense
data - FierceBiotech http//www.fiercebiotech.com/
story/genta-tanks-genasense-data/2009-10-29ixzz1x
OC6H3X5
69- New Approaches to Gene Therapy.
- Ribozyme Therapy.
- mRNAs that act as enzymes that can cut mRNA made
by the cell. - They exist naturally in cells and have roles in
mRNA splicing (it is a part of the spliceosome)
and the extension of the polypeptide during
translation.
70- New Approaches to Gene Therapy.
- Ribozyme Therapy.
- The steps of this method are
- RNA is engineered to function as a ribozyme and
to bind to the target mRNA. - The ribozyme is introduced into cells.
- The ribozyme binds to the target mRNA encoded by
the mutated gene. - The target mRNA is cut, keeping it from being
translated into protein.
71Fig. 10.8 Ribozymes are catalytic RNA that act as
enzymes to cut mRNA.
72Structure of hammerhead ribozyme
73- Virotherapy.
- Viruses can recognize and bind to a specific
receptor on the host cell surface. Each virus can
attach to a different receptor, so viruses have
cell specificity. - Viruses are engineered to infect and kill tumor
cells, leaving normal cells intact.
74- Three different methods exist for virotherapy
- Infect, reproduce, and kill tumor cells. Viruses
from the killed cells will then infect other
cancer cells. - Engineered viruses that have a tumor-specific
promoter linked to an essential virus gene. The
virus infects both normal and cancer cells, the
gene turns on only in cancer cells and the virus
kills the cancer cells. - Engineered viruses that make tumor cells more
susceptible to chemotherapy.
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76- OncoVex attacks tumors in two ways. It features a
modified cold sore virus that replicates inside
solid tumors, causing cancer cells to die.
Secondly, the drug prompts the immune system to
take out cancer cells. In May 2009, BioVex
released good Phase II data on the drug in
melanoma patients. 13 patients had significant
responses to the treatment, nine had signs of the
cancer completely wiped out.Read more OncoVex
- 10 promising late-stage cancer drugs -
FierceBiotech http//www.fiercebiotech.com/special
-reports/10-promising-late-stage-cancer-drugs/onco
vex-10-promising-late-stage-cancer-drugsixzz1xOFS
pdV0
77- Stem Cells - About Stem Cells
- Genetically engineered undifferentiated stem
cells may act as gene therapy agents. - If stem cells containing a correct gene are
inserted into a patient, the cells could divide
and serve as a source of healthy cells to treat
many diseases.
78- Scientists can isolate stem cells from a
5-day-old mass of cells, called a blastocyst,
that develops into an embryo. The cells are
called embryonic stem cells and have the
potential to develop into different cells of the
body.
79- Goal of using stem cells is to treat damaged
tissue by transplanting stem cells into a region
of the body where they divide and differentiate
into healthy tissue.
80Fig. 10.9 Stem cells obtained from bone marrow
can be cultured and induced to differentiate into
blood cells.
81- There are several types of stem cells
- Embryonic.
- Fetal.
- Umbilical cord blood.
- Adult.
82- Types of stem cells
- Embryonicthese cells are considered to be the
most valuable type of stem cell because they can
become almost any type of cell in the body. Can
be obtained from in vitro fertilization (IVF)
procedures.
83Fig. 10.10 Embryonic stem cells are obtained from
a blastocyst or embryo.
84- Types of stem cells
- Fetal - found in fetal brain tissue and are a
natural source of dopamine neurons. Must come
from prematurely terminated human fetuses or
late-stage embryos. A human embryo is considered
a fetus eight weeks after the egg is fertilized.
Can possibly be used to treat Parkinsons disease.
85- Types of stem cells
- Umbilical cord bloodmultipotent stem cells. Even
though they naturally become blood cells and
immune system cells, they have the potential to
become many different types of cells. Cause less
rejection problems because they have not yet
developed antigens that can be recognized by the
immune system.
86- Types of stem cells
- Adult multipotent adult stem cells that develop
into cells of a specific type of tissue. Believed
to be the least flexible in being able to develop
into any type of tissue. Research today is
focused on trying to get these cells to become
different cell types.
87- Most experiments involve cells that are in
culture and are harvested from bone marrow,
peripheral blood, and umbilical cord blood.
88- Using bone marrow stem cells can treat diseases
such as leukemia because they develop into white
blood cells, by killing all of the abnormal bone
marrow and white blood cells and replacing them
with donor stem cells, which replace the damaged
marrow and cells.
89- Therapeutic Cloning and Embryonic Stem Cells.
- Can obtain stem cells from embryos cloned in a
similar fashion to Dolly, the cloned sheep.
90- Therapeutic Cloning and Embryonic Stem Cells.
- Can obtain stem cells from embryos cloned in a
similar fashion to Dolly, the cloned sheep. - Performed in the following manner
- Nucleus is removed from an egg cell, and donor
cells are extracted from the patient. - Donor cells are cultured so that differentiation
is reversed. - Electrical pulse fuses the donor cell with the
egg cell. - The cell is induced to divide and form a
blastocyst to extract stem cells from. - Stem cells generated this way are not rejected
because they are an exact genetic match to the
donor.
91Fig. 10.11 Therapeutic cloning enables stem cells
to be produced that are genetically identical to
the donor patient.
92- Vaccines.
- New vaccines are being developed using new
vaccine vectors and new delivery approaches,
immunoenhancers, and nucleic acids. - Vaccines are being developed against pneumonia,
malaria, herpes virus, and others. - New vaccines usually contain the antigen and not
the organism. - Small pieces of DNA from a microbe also induce an
immune response, and are being studied to develop
vaccines against malaria and HIV.
93- Tissue Engineering.
- Focuses on the development of substitutes for
damaged tissues and organs. - Combines biology and engineering to develop new
tissue or the implantation of new cells.
94- Tissue Engineering.
- Occurs in the following way
- Tissue-inducing compounds, such as growth
factors, serve as signals to stimulate the growth
and development of new tissues. - The cells reside within a natural or synthetic
extracellular matrix that can be incorporated
into a patients tissue. Scaffolds or networks of
polymers may serve as a substrate for cell growth
and formation into tissues. - Isolated cells can be kept from host tissues to
avoid rejection while they develop. - When implanted in the body, blood vessels grow
and provide nutrients. - Stem cells can potentially be used for tissue
engineering.
95- Tissue Engineering.
- Research has focused on transplanting functional
pancreatic islet cells coated with alginates to
treat diabetes. The cells are implanted into
animals, and are linked to the bloodstream by a
tube that prevents antibodies and white blood
cells from making contact with the implanted
tissue.
96- Tissue Engineering.
- A biohybrid kidney was developed that maintains
kidney function until an injured kidney recovers.
The kidney was made of hollow tubes seeded with
kidney stem cells that divided until they lined
the tubes and became functional kidney cells.
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99Fig. 10.12 Tissue engineering offers hope to
hundreds of thousands of patients who need
functional tissues and organs. (a) Tobular
biodegradable scaffolds to from tubular tissue
such as veins, arteries, and intestines.
100Fig. 10.12 (b) Scanning electron micrograph of a
biodegradable polymer scaffold made of poly
(glycolic acid).
101- Scientists in London created an artificial
windpipe which was then coated in stem cells from
the patient. - The technique does not need a donor, and there is
no risk of the organ being rejected. - A windpipe can be made within days.
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103- 1 Trachea is removed from dead donor patient2 It
is flushed with chemicals to remove all existing
cells3 Donor trachea "scaffold" coated with stem
cells from the patient's hip bone marrow. Cells
from the airway lining added4 Once cells have
grown (after about four days) donor trachea is
inserted into patient's bronchus
104- Xenotransplantation.
- An organ or tissue transplanted from one species
(such as a pig, baboon, or chimp) into another
(such as a human) is a xenotransplant. An organ
or tissue transplanted between two members of the
same species (such as two humans) is an
allotransplant. (Xeno is Greek for foreign allo
is Greek for different).
105- Xenotransplantation.
- Tissue rejection, along with ethical and legal
dilemmas, are major obstacles. - Complement masking or shield proteins stop the
complement system from attacking our own cells.
Some transgenic animals can produce organs with
the shield proteins on the surface.
106- Xenotransplantation.
- Pigs are the best possible organ donors. Their
major organs are similar in size and shape to
humans. Few diseases are transferred from pigs to
humans. - Pigs have been produced with shield proteins and
may reach clinical trials. - In 1999, 160 people received pig cells without
bad reactions.
107- Drug Delivery.
- Why Good Delivery Systems Are Needed.
- Biosensors.
-
- Biotech Revolution Nanotechnology.
108- Drug Delivery.
- Why Good Delivery Systems Are Needed.
- Drugs are only useful if an effective method of
delivery is available. - Aerosols can be inhaled through the nose and pass
into the lungs. - A transdermal patch can transfer large peptides
that do not normally diffuse through the skin
into the skin and then into the bloodstream.
109- Drug Delivery.
- Biosensors.
- Biological components such as a cell, antibody,
or protein and are linked to a very small
transducer. The transducer receives the signal
and transforms it into an understandable form. - Transducers bind to the molecule, which produces
an electrical or optical signal that can be
detected.
110- Drug Delivery.
- Biosensors.
- Things that can be measured include blood
components, environmental pollutants, toxins, and
biological warfare agents. - Nanotechnology may make biosensors so small that
they can be easily concealed, or even placed into
the body.
111- Drug Delivery.
- Biotech Revolution Nanotechnology.
- The study, manipulation, and manufacture of
extremely small tools, structures, and machines
at the molecular and atomic levels. - A field that involves engineers, physicists,
chemists, and molecular biologists.
112- Drug Delivery.
- Biotech Revolution Nanotechnology.
- Possible applications include
- Nanowiresvery small wires to transit
electricity. - Nanobotsrobots that assemble products.
- Nanomaterial productioncreate materials such as
powders. - Drug deliverytiny containers may deliver drugs
to the correct places. - DNA computersuse DNA as hardware and software.
113- Drug Delivery.
- Biotech Revolution Nanotechnology.
- Biological molecules may provide frameworks
instead of material like silicon, to perform
computing tasks such as mathematics. - Scientists have demonstrated that 1000 DNA
molecules can solve, in four months, complex
problems that would take a computer 100 years to
solve.
114Immunity and Cancer
Antibody
Macrophage
Cancer cell
Helper T cell
Natural killer cell
Cytotoxic T cell
115Immunotherapy
Radioisotope
Herceptin
Growth factor
Herceptin blocks receptor
Antibody
Antigen
Breast cancer cell
Lymphoma cell
Lymphoma cell destroyed
Growth slows
116Dendritic Cells That Attack Cancer
Dendritic cell matures and is infused back into
patient
Complex binds to dendritic cell precursor
Tumor antigen
T cell
Tumor antigen is linked to a cytokine
Complex is taken in by dendritic cell precursor
Dendritic cell displays tumor antigen and
activates T cells
Cancer cell
T cells attack cancer cell