Introduction together. The polynucleotides are antiparallel to each
DNA (deoxyribonucleic acid) carries genetic information in all living organisms, it contains genetic instructions for development and function. DNA is a double-stranded polynucleotide made up of many repeating monomer units called nucleotides. A DNA nucleotide is composed of a phosphate group, a pentose sugar called deoxyribose and a nitrogenous base (as shown in Figure 1- Structure of a nucleotide). There are four possible nitrogenous bases in DNA nucleotides; guanine, adenine, thymine and cytosine. Each nucleotide is joined by a condensation reaction and a phosphodiester bond is formed. Many millions of nucleotides units form the polynucleotide. 1
Figure 1 – Structure of a nucleotide 2
Alternating phosphate groups and pentose sugar form the backbone of the DNA. Complementary base pairing occurs; this is when purine bases bond with pyrimidine bases via hydrogen bonding. Adenine pairs with thymine and guanine pairs with cytosine. The base pairing creates combinations that contribute genetic differences in humans. It links two polynucleotide chains together. The polynucleotides are antiparallel to each other. The molecule is twisted to form the double helix structure (Figure 2- Structure of DNA) and is maintained by hydrogen bonding 3.
The order of the bases creates a code, every three bases form a triplet code called a codon and consequently determine the type of amino acid produced. Additionally, the order of amino acids formed will determine the primary structure of the protein. The genetic code is universal as the same codes are used in all living organisms.
Figure 2 – Structure of DNA 4
DNA encloses instructions to make a protein by protein synthesis. Firstly, a DNA molecule is copied to make an RNA molecule in the nucleus via transcription.
An enzyme called DNA helicase, breaks the hydrogen bonds between nucleotides at specific regions of the DNA molecule, in an ATP dependant reaction. The two strands in the helix unwind and separate, exposing the nucleotide bases. One of the stands will act as a template for initiation. Initiation is when the enzyme RNA polymerase links to the template strand at a sequence called the promoter. The Promoter is a sequence that starts transcription.
In elongation; the RNA polymerase catalyses the reaction of the addition of free complementary nucleotides. RNA bases are adenine, guanine, cytosine and uracil. Uracil pairs with adenine and guanine pairs with cytosine. The RNA polymerase moves along the DNA allowing free RNA nucleotides to align themselves opposite complementary bases in the DNA template strand. This forms a newly synthesised mRNA strand. Behind the RNA polymerase the DNA strand and mRNA strand join to reform the double helix (Figure 3 – Transcription). Elongation continues until the RNA polymerase reads a terminator sequence close to the end. The terminator sequence stops transcription. Once the terminator sequence has been transcribed an RNA molecule is released. The RNA molecule is call mRNA (messenger RNA). The mRNA carries the DNA code out of the nucleus through a nuclear pore to the cytoplasm. 5
Figure 3 – Transcription 6
A protein is a polypeptide chain made up of many repeating monomer units of amino acids. In the process of translation, the mRNA molecule contains instructions to build a protein made up of a specific sequence of amino acids. The mRNA attaches to a ribosome so the protein can be made.
Amino acids are carried to the ribosome by a tRNA molecule (transfer RNA). Each tRNA molecule has its own amino acid that is determined by the anticodon on the tRNA and is specific. Three bases of the codon bind to three bases of the anticodon by complementary base pairing. Another codon is read and the individual amino acids carried by the tRNA molecules are close enough to form a peptide bond between adjacent amino acids. The tRNA molecules leave the ribosome leaving the attachment site vacant. The ribosome continues to move along the mRNA molecule to the next codons 7. Amino acids are added each time a codon is read (this process is show in figure 4 – Translation). The sequence is read until a ‘stop’ codon is read. Consequently, the finished polypeptide chain is released from the ribosome.
Figure 4 – Translation 8
Antibodies are glycoproteins. They are large, Y-shaped and are used by the immune system to identify and attack foreign objects like bacteria and viruses 9. All antibodies are formed in the same way from two heavy and two light polypeptide chains joined by a disulphide bond 10. The top section of the antibody is called the variable region as this can change to make the antibody specific. Antigen binding sites are found at the end of arms of the antibody, it changes to bind to antigens specifically, this is also known as the Fab region (fragment antigen binding region). The bottom section is called the constant region as It remains the same, it is also known as the Fc region (fragment crystallisable region) made of two heavy chains. The Fc region determines the antibodies effect on the body. (The structure can be seen in Figure 5- Antibody Structure).
Figure 5– Antibody structure 11
Antibodies in the body
Antibodies are produced by white blood cells called B- lymphocytes 12. Pathogens have a substance on their surface called an antigen that stimulates an immune response, examples are viruses and bacteria.
When a foreign pathogen enters the body, its antigen binds to the B-lymphocyte surface. It causes the B-lymphocytes to divide and create clones called plasma cells. These plasma cells secrete millions of antibodies into the bloodstream 12. Each type of antibody is different depending on the pathogen they are targeting. They have an antigen binding region exclusive to the antigens on the bacteria or virus. Once attached to the antigens they begin to attack the pathogen by neutralising it. This can arise in several ways. Firstly, it can make the pathogen immobile preventing it from travelling and entering host cells. Antitoxins can change the chemical composition of the antigen by counteracting the toxins produced by the antigen 12
How antibodies are made in the lab
Polyclonal antibodies are all different to each other. They can be produced in a lab by a process where a safe injection containing a specific antigen is injected into a mouse or rabbit. This triggers an immune response and antigen-specific plasma cells are produced with specific antibodies. The antibodies can be removed from the mouse by a blood sample. It is cheap to produce a polyclonal population.
A monoclonal population is where all the antibodies are identical and they can be produced by Hybridoma. Hybridoma is when a specific antigen is injected into a mouse. Antibody-producing plasma cells are extracted from the mouse’s spleen and diffused with a tumor cell called melanoma. The cells multiply and produce specific, identical antibodies each time the cell divides. This is an expensive technique but is essential for diagnostics and therapeutics. 13
Monoclonal antibodies recognize specific proteins on cells, particularly cancer cells. Each monoclonal antibody detects a protein on the antigen of the cell. Once the antibodies are attached to the proteins, it triggers the immune system and stimulates an immune response in order to attack and kill cancer cells (Figure 6 – Monoclonal antibodies and cancer cells). An example of monoclonal antibodies is trastuzumab that is used to treat breast cancer 14.
Figure 6- Monoclonal Antibodies and cancer cells 15
Different types of antibodies
There are five immunoglobulin classes (isotypes) of antibodies molecules IgG, IgM, IgA, IgE and IgD. The way they are classified is according to the heavy chain they contain.
The first class is IgA, it is found in the respiratory system. They are also found in tears and saliva. They bind to foreign antigens before they attack tissues. It holds the pathogens in secretion so they can be easily removed from the body.
Next is IgG that ultimately provides protection for long periods of time. They remain in the body a long time after a presence of the antigen that triggered the immune response. Their main function is to protect the body from viruses, bacteria and toxins. They are also passed on from mother to a newborn in order to fight infections 16.
IgM’s purpose is to fight infections in the blood and also is the first class of antibody to be produced by the fetus. They clump pathogens together to remove them from the body.
IgD is found on the surface of B- lymphocytes and is used as a receptor for the antigens on the foreign pathogens. It induces the production of antibodies as an immune response.
Lastly, IgE is found in membranes and the skin. They trigger allergic reactions when they bind to cells that the body is allergic to. IgE also protects the body from parasites. 17
Their structures can be seen below in Figure 7 – The different classes of antibodies.
Figure 6 – The different classes of antibodies 18
How antibodies are used in precision medicine
Precision medicine is the approach to treat diseases by looking at individuals’ genes and characteristics. Monoclonal antibodies are all identical and therefore specific, making them ideal for targeting certain cells and antigens. Antibodies are used in ADCs (Antibody- Drug Conjugates) in precision medicine.
Antibody- Drug Conjugates composed of a monoclonal antibody, a cytotoxic drug and a specific linker are part of newly developed, therapeutic treatment for cancer and tumors. They can be used as an alternative to chemotherapy and radiotherapy. The antibody detects a specific antigen found on the surface of the tumor cell; it binds to the antigen by weak and non-covalent forces and is internalized by the cell to destroy it. An appropriate linker should be used that provides specific release of the cytotoxic drug. The drug should only be released at tumor sites. This prevents systemic cytotoxicity and creates discrimination between healthy and diseased tissues. The linkers ensure stability in circulation and efficient release of the drug. The linkers used are selectively-cleavable meaning they will only release the drug in certain environments. An example is Hydrazone that is designed to allow degradation in acidic regions of the cytoplasm. 19. The structure can be seen below in Figure 7 – Structure of an ADC.
Figure 7 – Structure of an ADC 20
Mechanism of Action
The antibody binds to the antigen receptor on the plasma membrane of the tumor cell to produce an ADC antigen complex. This creates a signal in the tumor cell which causes the ADC antigen complex to become completely internalized by a process called endocytosis. Endocytosis is a form of active transport by which a cell allows molecules such as proteins into the cell.
Once inside the tumor cell the ADC the complex becomes degraded or broken down in a lysosome. A lysosome is a membrane-enclosed organelle that contains digestive enzymes called acid-hydrolases. These are capable of breaking down unwanted toxins and chemicals 21. Once the ADC antigen complex has been degraded in the lysosome, the cytotoxic drug is released and enter the cytoplasm of the cell. The cytotoxic drug binds to its molecular target which is either a DNA strand or microtubule. The drug will either create DNA strand breakage or cause microtubule disruption. This will cause apoptosis. 22
Apoptosis is known as cell death. Firstly, the cell shrinks and chromatin condensation occurs. Next, membrane blebbing occurs where there is protrusion in the plasma membrane; the blebbing continues until a nuclear collapse occurs where the nucleus disintegrates. The cell separates and apoptotic bodies form as shown in Figure 8 – Apoptosis.
Figure 8 – Apoptosis 23
Examples of ADCs
Brentuximab vedotin is an example of an ADC used to treat cancer. It treats Hodgkin Lymphoma; a cancer of the white blood cells. Brentuximab is a monoclonal antibody that targets the protein on the antigen of the cancer cells. The protein they target is CD30 that is found on the surface of lymphoma cells and also anaplastic lymphoma cells. The drug is inserted into the bloodstream. 24
Trastuzumab emtansine also known as Kadcyla is another marketed ADC. It is used to treat HER2 positive breast cancer that has spread to other areas of the body. Emtansine the cytotoxic drug is released into the cancer cells and destroys them. The monoclonal antibodies target the protein HER2 on the surface of the cancer cells. HER2 is a dangerous protein as it makes the cancer cells divide and grow. This ADC is also inserted into the blood stream.
A recently marketed ADC called Inotuzamab ozogamicin (Bespona) in another example. It is used to treat B cell – acute lymphoblastic leukemia.
Advantages and disadvantages of ADCs in Precision Medicine
Antibody-Drug conjugates are specific and avoid destroying healthy cells, therefore, more patients will be able to tolerate the drugs. Chemotherapy contributes to hair loss and destruction of the immune system whereas ADCs create less severe side effects as healthy cells are not targeted. The specificity of the linkers increases the efficiency of the delivery of the cytotoxic drug to the cell, protecting the tissues surrounding it.
There are disadvantages to ADCs, including the possibility that other cells in the body contain the same extracellular receptors and proteins as the cancer cells, so they are also targeted. This would create unwanted toxicity in other cell populations in the body. There are side effects of the drug such as nausea, fatigue and fevers. However, these are not as relentless as other cancer treatments. There also a large number of considerations needed for giving a patient the ADC. The ADC must be stable in the bloodstream to prevent systemic toxicity. The ADCs monoclonal antibody must bind to the protein on the surface of the targeted cell and the cytotoxic drug must destroy the cell completely. The ADCs only work on specific cells and therefore, require a lot of research to produce. 25
Advantages and disadvantages of Precision medicine
Precision medicine gives doctors the ability to provide specific treatment to patients using their genetic information. It also provides the possibility of predicting which treatments will work most efficiently for patients. It improves the ability of doctors to prevent, diagnose and treat a wide range of diseases. Early diagnosis is fundamental to the survival of the patient, precision medicine makes this possible. Economically theirs is potential to decrease the cost of health care if treatment of disease is specific and fewer drugs are needed 26.
However, precision medicine is very specific and requires a large amount of research to ensure that the drugs work. The design and analysis of the drugs are more complex than conventional drugs