Antibody Engineering 

      Authors: Andrea Galassi, Seth Parker, Sarah Haeger, Brian Evanko

 

Applications of Antibody Engineering

     Antibody engineering is used in the treatment of numerous diseases and many diagnostic tests. Each antibody binds very specifically to only one type of cell or molecule and antibody technology allows researchers to target specific cells (Maynard).  The applications of antibody engineering are successful due to this specific binding of antibodies and antigens. Production methods of antibodies continue to progress, making it possible to generate antibodies specific to almost any antigen. Emerging applications of antibody engineering include cancer therapy and radioimmunodetection of  tumors. Antibodies present many exciting possibilities to the future of medicine.

 

1. Antibody Binding Specificity
2. Production of Antibodies
   1. Antibody Repertoire Libraries
   2. Hybridoma and Phage Display Technology
3. Cancer Therapy
   1. Previous Methods
   2. Antibody Methods
4. Medical Imaging
   1. Previous Methods
   2. Antibody Methods
5. Other uses of Antibodies

  

 

1. Antibody Binding Specificity

     Antibody engineering has become an emerging field because of antibodies' ability for specific binding. The reason antibodies have such specific binding properties is because of their structure. Antibodies are composed of two identical heavy chains and two identical light chains. The light chains make the arms of the Y-shaped antibody which have identical antigen binding sites at the end of each arm.  The heavy chain makes up the tail of the y-shaped antibody which contains a binding site for molecules that activate other parts of the immune system.

 

 

(Immunoglobulins and the Immunoglobulin Fold)

Figure 1- This figure shows the structure of an antibody.

The light chains are where the antigen binding sites are located.

 

     Antibodies are created in the body during an immune response. During an immune response antigens bind to B lymphocyte cells in the body. There are millions of different kinds of B cells in the body, each specific for a type of antigen that may be encountered in a lifetime. They lie dormant until an antigen binds to them. Once an antigen binds to its specific B-cell the B-cell differentiates into two types of cells, plasma cells and memory cells. Plasma cells are antibody producing cells that secrete large amount of soluble antibodies that are able to bind to the specific antigen and memory cells are cells that live for a long time that are able to respond quickly if the encounter the same antigen as before. The ability for the antibody to recognize the specific antigen it is targeted for allows antibodies to be very specific when targeting antigens (Immunoglobulins and the Immunoglobulin Fold).

  

2. Production of Antibodies

     Antibodies that target a specific antigen are found rather than designed. The gene for the antibody of interest is narrowed down from an antibody repertoire library containing 10⁵-10¹⁰ distinct immunoglobulin genes. There are three types of antibody repertoire libraries: Immune Libraries, Naïve Libraries, and Synthetic Libraries (Maynard).

 

          Immune Libraries

      Challenging the immune system of mammals, especially mice, with a particular antigen and collecting B-cells forms immune libraries. These B-cells’ immunoglobulin genes have already undergone affinity maturation. This means that the immune response initiated in the mammal causes a very high percentage of B-cells to express genes for antibodies that target a specific antigen. Immune libraries can be fairly small because most the genes they contain are relevant to this one antigen (Maynard). However, the antibodies produced are made by animal immune systems and trigger an unfavorable immune response in humans (Siegel). Because it is unethical to immunize humans with antigens, techniques have been developed to humanize the genes in immune libraries, but this is not ideal. Immune libraries are also not effective at targeting self antigens (Maynard).

 

Naïve Libraries

      Naïve libraries do not require immunization and are therefore derived from human B-cells. Unlike immune libraries, these libraries are very large and have a wide variety of antibodies represented. With such a large selection, an antibody to almost any antigen can be derived. This also means a naïve library can be used repeatedly to select a different kind of antibody every time (Siegel). Unfortunately, the lack of immunization means that the immunoglobulin genes have not undergone affinity maturation, resulting in antibodies with lower antigen affinity. With effort, this can be partially compensated for by increasing the size of the library (Maynard).

           

Synthetic Libraries

     This final class of libraries relies less on organisms’ immune systems and more on creative biochemical engineering. Randomly cloning and combining different immunoglobulin gene segments creates genes that can produce a wide variety of antibodies. These fully synthetic libraries generally do not produce antibodies with high antigen affinity however, so a better option is a semi-synthetic library. Semi-synthetic libraries are made by taking existing antibody genes and using PCR to randomize key areas called hypervariable regions. This creates antibodies with specificities for many different antigens. Because these libraries are made without much interaction from the human immune system, they produce better self-targeting antibodies necessary for targeting cancer cells (Maynard) 

 

 

     After producing an antibody library there are two methods for storing the genetic information and producing antibodies in large quantities. The first method uses hybridomas to store every individual B-cell while the second method uses bacteriophages to store only the immunoglobulin genes (Siegel). 

 

 

 

Figure 2 - This figure demonstrates the difference between using hybridoma

tissue culture methods and phage display libraries. (Siegel) 

 

 

Hybridomas

     Hybridomas were first created in 1975. They consist of animal B-cells fused with myeloma (cancer) cells (Maynard). The B-cell provides the immunoglobulin gene and the myeloma provides the ability to infinitely duplicate.  Hybridomas are meant to differentiate and secrete antibodies, which can then be used for practical applications. Unfortunately they can only be made from animal B-cells, so while they can store immune libraries they cannot store synthetic or naïve libraries. Antibodies made from hybridomas will therefore produce an immune response in humans if they are unaltered. Hybridomas are also problematic because they cannot be made from plasma (immune memory) cells and a great deal of time is required to isolate a hybridoma for a specific antibody. Although technology is beginning to move beyond hybridomas, they have been crucial to the advancement of monoclonal antibodies (Siegel).

 

 

Phage Display Libraries

     Phage Display Libraries are a collection of immunoglobulin genes rather than entire B-cells. Unlike hybridomas, they can store genes for immune libraries, naïve libraries, or synthetic libraries (Siegel). Immune and naïve libraries are derived from B-cells, so the genes are transferred to bacteriophages in several steps. First, mRNA from the B-cells undergoes PCR with reverse transcriptase to form a complementary, or cDNA, library. The cDNA is then amplified and cloned into bacteriophages, so that each phage contains one immunoglobulin gene. The reverse transcription is not necessary for synthetic libraries because they are already expressed as individual genes (Phage).

      Phage Display Libraries can be refrigerated for storage and expanded by infecting bacteria. The phage DNA is engineered so that each phage has the antibody protein it encodes expressed on the outside of its protein coat. This property allows a specific immunoglobulin gene to be isolated from a phage display library far faster than from hybridomas. To isolate a gene in a copy of a phage display library, the bacteriophages are put in an environment where the targeted antigens are bound to a surface, cell, or particle. The phages displaying the correct antibody bind to the antigens and the rest of the library can be easily removed, leaving behind only the desired phages. A few more rounds of “panning” narrow the antibody down completely. When a final candidate has been selected, the phage undergoes analytical tests, such as ELISA, or “enzyme-linked immunosorbent assay” (Phage) 

      Infecting a bacterial colony with the phage produces antibodies on the outside of phage particles. This form can be useful. It is also possible to engineer the phage to infect bacteria and produce antibodies that are independent of the phage protein. This does not produce the entire antibody, just the regions most necessary for antigen binding. Which regions are selected and how they are linked together varies depending on what the antibody will be used for (Maynard). 

 

3. Cancer Therapy

 Previous Method: Chemotherapy:

     All cancers are similar in that they are caused by cells growing out of control.  Chemotherapy is the first choice for treatment when dealing with most cancers (Chemotherapy). The first drug used to treat cancer was mustard gas, when it was accidentally discovered to lower white blood cell counts. Doctors in the 1940s believed that this may have a similar effect on the rapidly dividing cancer cells (Chemotherapy). Slowly, improvements were made, other drugs were developed, and the drug was delivered by vein rather than by inhalation.Chemotherapy works by damaging the cellular DNA of any dividing cell, after damaged the cell dies (How).

 

   

Figure 3 - Normal Cell Division Cycle

 

     There are several other actions currently used to treat cancer including surgery and radiation. The drawbacks of both of these are that they only act in a specific area, and due to cancer’s ability to migrate and spread these methods are both slightly ineffective. Chemotherapy is beneficial in that it is a systemic treatment, meaning that the medicine travels throughout the body to reach the cancer cells wherever they have spread (Chemotherapy). Another beneficial aspect of chemotherapy is that the drug can be delivered in various ways including by shot, by IV, or by pill. This means that chemotherapy can be taken at home with strict instructions.

                Although chemotherapy is highly effective in killing cancer cells, it is not cell specific and will target any cell that is dividing. This means that some healthy cells will be killed and will have highly detrimental effects. Some cells that are affected include hair, bone marrow, skin and mouth, stomach, and intestinal (Chemotherapy). Chemotherapy causes hair loss on the whole body caused by the killing of hair cells; exhaustion, easy bruising and bleeding, and a higher risk of infection caused by the killing of bone marrow cells; dryness and sores in the mouth and on the skin caused by the killing of mouth and skin cells; nausea, vomiting, and diarrhea caused by the killing of stomach and intestinal cells (Chemotherapy). Also, because chemotherapy drugs affect the bone marrow cells, white blood cell count will decrease which consequently decreases the body’s ability to fight infections.

                The cost of chemotherapy varies depending on the type of cancer and how long of a treatment is required (Chemotherapy). Some chemotherapy drugs, such as 5-FU and leucovorin are relatively inexpensive, while newer drugs are very expensive—especially when used in combinations (High). An eight week treatment with 5-FU and leucovorin will cost around $100-$300, whereas adding oxaliplatin or irinotecan will increase the cost to more than $10,000 (High). Adding bevacizumab (Avastin) or cetuximab (Erbitux) will raise the cost to $20,000-$30,000 (High). These estimates are for the drugs alone, excluding the hospital fees and external costs of missed work.

          

 

New Method: Antibodies: 

 

         Monoclonal antibodies have been discovered recently to be useful for cancer therapy, with some treatments gaining FDA approval. It has been determined that some antibodies bind specifically to tumor-associated antigens which are present in higher concentration on tumor cells than normal cells (Maynard).  The antibodies then begin to accumulate in malignant tumor tissue which can then recruit cytotoxic immune cells or can be combined with a drug delivery mechanism to deliver chemotherapy drugs.  One monoclonal antibody, rituximab, has been used for the treatment of large cell lymphoma in conjunction with chemotherapy. Studies show that after one year of treatment, 69% of patients treated with rituximab in conjunction with chemotherapy were free of lymphoma versus only 49% of patients treated solely with chemotherapy were free of lymphoma (Monoclonal Antibodies).  The use of monoclonal antibodies for cancer treatment is very promising.

 

Figure 4- Targeting and death of tumor cells by monoclonal antibodies

 

 

 

Cancer targeting monoclonal antibodies

     One type of monoclonal antibody that has been FDA approved is AVASTIN or Bevacizumab which is used to treat metastatic colorectal cancer, non-small cell lung cancer, and metastatic breast cancer (AVASTIN Bevacizumab: Reach Beyond Convention”. ). Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that binds to the vascular edothelial grown factor (VEGF) and inhibits its activity. This drug is able to target VEGF specifically because it contains human framework regions and complementarity determining regions of a murine antibody that specifically binds to vascular endothelial growth factor.

     The bevacizumab antibodies prevents VEGF from interacting with its receptors on the surface of endothelial cells which causes the endothelial cells to proliferate and new blood vessels to form. In mice with xenotransplant models of colon cancer the drug  reduced microvascular growth and slowed progression of the metastatic cancer. It has been found that the treatment of cancer is best paired with AVASTIN and chemotherapy for best results. (AVASTIN(Bevacizumab) For Intravenous Use)

     Although monoclonal antibodies have shown promising results in the treatment of cancer, dangerous side effects can occur. In AVASTIN some dangerous side effects that have been seen are gastrointestinal perforation  would healing complications, and hemorrhage. In clinical trials for AVASTIN all three dangerous side effects resulted in fatality (AVASTIN Bevacizumab: Reach Beyond Convention”. ).  

     Antibodies may be used as a more effective cancer treatment than chemotherapy in the future. Since monclonal antibodies have the ability to target cells directly that exhibit the designated antigen, this treatment may one day be used instead of chemotherapy which effects all cells in the body.  Since there are still dangerous side effects of using monoclonal antibodies as treatment an antibody would have to be developed that didn't cause complications.

 

4. Imaging Technology 

Previous Techology: MRI/CAT Scan

     Previous imaging techniques include Magnetic Resonance Imaging (MRI) and CAT Scan (CT). Both of these methods are effective in the imaging and diagnosis of diseases of the body such as cancer and cardiovascular disease.

Figure 5- Multislice CT scanner

 

     An MRI produces a high quality image of inside the human body (Hornak). MRI uses the principles of nuclear magnetic resonance (NMR) which is a spectroscopic technique used to obtain microscopic chemical and physical information about molecules (Hornak). MRI was developed in 1977 by Raymond Damadian (Hornak). The magnetic resonance produces NMR slices through the human body that can each be analyzed by doctors for diagnosis (Hornak). A couple disadvatages of MRI are that people with pacemakers, who are morbidly obese, or claustrophobic will not be able to have an MRI taken. Due to the long amount of time it takes for an MRI exam to complete, the patient must sit perfectly still for long periods of time or risk the chance the images will become distorted.

     Computed Tomography, or CT, is useful in that it takes many thin sliced images of an organ and layers them onto a 3-dimensional plane. A motorized table moves the patient through the imaging machine while a source of x-ray rotates around the patient emitting a narrow, fan-shaped beam (Brenner). These x-rays produce data that is collected by the x-ray detectors rotating in synchrony with the x-ray emitters (Brenner). A computer compiles all these thin-layered images later for analysis. The main disadvantage of a CT scan is that it has limited contrast and cannot show different tissues as clearly.

 

Figure 6- Example of a compilation of layered images taken by an MRI

 

     The costs of both medical imaging techniques are relatively inexpensive, averaging in the $200-$750 range depending on what is imaged. The basic charge for an MRI is around $100 an hour (MRI) and around $150 an hour for a CT scan (SPECT/CT). Hospital and doctor fees are not factored into this cost.

 

 

New Technology: Radioimmunodetection

     Monoclonal antibodies are also used for the radioimmunodetection of tumors.  Through this technology, antibodies are tagged with radioisotopes, whose presence in tissue can be detected with medical imaging scanners (Goldenberg).  Once the antibodies have been injected into the body, they target tumor cell surface marker antigens.  The resulting image can detect cery small tumors and can tell more about the stage of the cancer.  Using antibodies to detect the most common tumor cell surface marker, the carcinoembryonic antigen, has been shown to detect tumors as small as 0.5cm (Maynard).  The use of radioimmunodetection could result in earlier detection of small tumors in early treatable stages.

  

5. Other Uses of Antibodies: 

Drug-Targeting Monoclonal Antibodies

     Monoclonal antibodies have become very important in engineering targeting drugs because cell surfaces have many receptors that can be recognized by engineered antibodies. When developing a monoclonal antibody to be used as a medication there are several factors to take into consideration to develop successful drug. Most importantly, the antigens that the monoclonal antibodies recognize should be localized on the surface of the targeted cell. This would cause the antibodies to have tumor tissue specificity. Also, the antibody should be localized at the target site and the toxicity of the engineered antibody must be taken into consideration. The drug complex then should be biodegradable and non-immunogenic as to not trigger an immune response upon treatment. Ideally, the drug should be released when the target cell and carrier molecule interact (Panchagnula, R., and C. S. Dey).

 

FDA Approved Monoclonal Antibodies

     Because of the specificity of antibodies, antibody engineers have currently been using antibodies to target cancer cells and viruses.  There have been many kinds of monoclonal antibodies that have been engineered to treat different conditions that have been approved by the FDA. These treatments can be effective because they can very specifically target the cells needed because of antibody properties.

 

 

 Figure 7-this figure shows a list of several monolonal antibodies that have been approved by the US FDA

and what conditions they treat by their specific target of action(Carter).

 

Costs of Treatment

     Since monoclonal antibody target therapy is relatively new, this treatment is generally expensive. When compared to the price of cancer therapies, monoclonal antibodies are relatively more expensive.

 

Monoclonal Antibody

Service Items   Time   Criteria   Price Anti-regular peptide antibody (customer provide antigen) 4-5 months 1.  Customer provide peptide(4mg) $3,700 2.  Immunization of two mice, fusion, screening, subclone and initial characterization 3.  With up to three monoclones and their supernatant for confirming detection 4.  Generated antibody recognizes immunogen in ELISA assay 5.  Purify Antibody from 100 ml of hybridoma culture supernatant or 1 ml of ascites fluid     Anti-regular peptide antibody (RayBiotech provide antigen) 4-5 months 1.  Design, synthesize and conjugate  peptide(15-20mer,5-10mg) $4,600 2.  Immunization of two mice, fusion, screening, subclone and initial characterization 3.  With up to three monoclones and their supernatant for confirming detection 4.  Generated antibody recognizes immunogen in ELISA assay 5.  Purify Antibody from 100 ml of hybridoma culture supernatant or 1 ml of ascites fluid     Anti-phospho-peptide antibody 5-6 months 1. Design, synthesize, specific site phophorylation and conjugate  peptide(11-15mer, 5 mg) $5,600 2.  Immunization of two mice, fusion, screening, subclone and initial characterization 3.  With up to three monoclones and their supernatant for confirming detection 4.  Generated antibody recognizes immunogen in ELISA assay 5. Antibody purification by affinity column(1-5mg) may be obtained upon request     Anti-protein antibody 5 months 1.  4mg of purified Protein from customer $3,990 2.  Immunize two mice, fusion, screening, subclone and initial characterization 3.  with up to three monoclones and their supernatant for confirming detection 4. Generated antibody recognizes immunogen in ELISA assay 5. Antibody purification by affinity column(1-5mg) may be obtained upon request

 

Figure 8- This figure shows the price of RayBiotech’s monoclonal antibody production,

and the amount of time it takes to produce a certain antibody. (Products-Custom Antibody Service)

 

     As it can be seen from this chart, the costs of monoclonal antibodies are very expensive. These prices are only for a small amount of the antibody as well. Since monoclonal antibodies are a generally new form of treatment, they can be very expensive.

 

Virus Targeting Monoclonal Antibodies:

     Recently, scientists have been working to devolop virus targeting monoclonal antibodies. There have been promising studies in monoclonal antibodies targeting viruses. It was found that an engineered monoclonal antibody was able to cure West Nile virus in infected mice. This experiement was the “first successful demonstration of the use of a humanized antibody as a post-exposure therapy agaisnt a viral disease” (Purdy). In the experiment when the engineered monoclonal antibody was used as a treatment in mice infected with West Nile virus, over 90% of the mice survived. Normally, without any treatment, the mice only had a 10% survival rate. This antibody was successful because it either bound to the West Nile Virus’ envelope protien or it bound to the virus in a way the virus was nutralized. (Purdy).

     From these successful trials in mice, scientists have gone on to create a monoclonal antibody to treat West Nile virus in humans. This drug, MGAWN1 , has been approved by the FDA for clinical investigation. The humanized monoclonal antibody has been shown to neutralize the virus in vivo. (MacroGenetics IND for West Nile Virus Monoclonal Antibody is Active).

     Most monoclonal antibodies engineered to target virus infected cells are still in their clinical stages. Even so, there have been other monoclonal antibodies that have been engineered to target cells infected with HIV, respiratory syncytial virus, swine vesicular disease virus, and many others. In respiratory syncytial virus infections, a monoclonal antibody drug Palivizumab has been engineered to treat this virus. Palivizumab is a humanized monoclonal antibody that targets an epitope in the virus’s  A antigenic site of the F protein (SYNAGIS (PALIVIZUMAB) for Intramuscular Administration).

     Virus targeting monoclonl antibodies may be able to become a treatment for dangerous viruses in the future. The recent experimental work could lead to a more effective treatment for HIV. Virus targeting antibodies is just one appliation that the future of antibody entineering may hold.

 

               

 

Works Cited

 

 

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