**3. The production process of monoclonal antibodies**

In the following section we described two techniques, including hybridoma and phage display used for the production of mAbs.

#### **3.1 Hybridoma technique**

Monoclonal antibodies are generated from a single B lymphocyte clone and bind to the same epitope of an antigen. The hybridoma technique was first used in 1975 to generate mAbs by Milstein and Köhler. Several steps are involved in this method. First, mice are immunized with specific antigens emulsified with appropriate adjuvant. The booster injection is normally done after two weeks and the animal is then sacrificed when enough amount of antibody is produced. Blood collection is performed to assay the sufficient amount of the antibody production using techniques including ELISA and flow cytometry. After sacrificing, the spleen is isolated and then tissue digestion could be applied with an enzymatic or mechanical method leading to release of B cells. B cells could be extracted using density gradient centrifugation [8].

The next step is making a fusion between B lymphocytes and myeloma cells (that are immortal like cancer cells). Prior to fusion, myeloma cells should be prepared by culturing with 8 – azaguanine, making them sensitive to hypoxanthineaminopterin-thymidin (HAT) medium. The fusion process is carried through using polyethylene glycol (PEG), resulting in cell membrane fusing. After the fusing process, there will be a variety of cells including fused B cells with myeloma cells, unfused B cells, unfused myeloma cells, B cells fused to B cells, myeloma cells fused to myeloma cells. Therefore, a selective medium known as hypoxanthine, aminopterin and thymidine (HAT) medium should be used to select only the B cells fused with myeloma cells [10]. Two components of this medium, hypoxanthine and thymidine, are the metabolites of the salvage pathway of nucleoside synthesis. Therefore, only the cells that have the necessary enzyme for the salvage synthesis of nucleic acids, named hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), will be able to survive. Unfused myeloma cells lack HGPRT, so they cannot replicate their nucleic acid and they will not be able to grow in HAT medium. On the other hand, unfused B cells have a limited life span and therefore cannot grow appropriately. Consequently, only fused B cell-myeloma cells known as "hybridomas" are able to grow in the medium. It should be noted that another pathway of nucleic acid synthesis named "de novo" pathway, is also inhibited due to the presence of aminopterin in HAT medium. So, only the HGPRT-positive cells could be grown in this selective medium [8, 10].

To separate antibodies with different specificity and also for further hybridoma growth, the mixture of cells is diluted in microtiter wells in which their walls are coated with murine macrophages or feeder fibrocyte cells providing the growth factors needed for antibody-producing cells. Then, the antigen-binding ability of secreted antibodies by different clones of B cells could be assessed by ELISA, antigen microarray assay, radio-immuno assay (RIA), or immune-dot blot and finally, the stable clone will be selected. The fused hybridomas and produced mAbs can be stored away in liquid nitrogen [8].

Although this process may be well suited for development of therapeutic antibodies, however, there are some important problems with using this technique. The hybridoma process takes approximately between 6 and 8 months to obtain a sufficient amount of mAbs, so its development procedure is very long. On the other hand, because of the murine origin of the antibodies, they can trigger the HAMA response in the host which could accelerate mAb clearance and undesirable allergic reactions upon repeated administration. This issue was resolved by developing antibody engineering methods toward producing less immunologic chimeric or humanized antibodies. These engineered antibodies were created using murine variable regions or CDRs as well as human constant regions aiming to decrease HAMA response and maintain target specificity. Currently, fully humanized antibodies are

**7**

strains.

**uses**

*Introduction on Monoclonal Antibodies DOI: http://dx.doi.org/10.5772/intechopen.98378*

**3.2 Phage display technique**

generated in transgenic mice models (e.g. HuMabMouse and XenoMouse) using hybridoma technology. For this purpose, the mouse immunoglobulin gene loci have

The phage display method is one alternative to traditional hybridoma technology for generating monoclonal antibodies. This method was developed in 1985 by George P. Smith, who demonstrated that a peptide of interest could be displayed on the surface of filamentous phages following inserting the DNA fragment into the coat protein gene of phage. Then, a process known as "panning or biopanning" is explained by Parmley and Smith; the process describing a selection and affinity enrichment in order to isolation of peptide-phage fusions based on their specific binding affinity. Finally, phage display technology was used for the first time by McCafferty and Winter for generating antigen specific mAbs by creating combina-

This method involves integration of a gene sequence coding for a particular antibody into the DNA sequence of a filamentous bacteriophage leading to the expression of interest protein on the surface of the bacteriophage capsid. These phage libraries could be generated from healthy donors (creating Naïve libraries) or individuals who carry a particular disease, such as metastatic cancer or particular infection, or have been immunized with a particular antigen (creating immunized libraries). M13 is a filamentous bacteriophage that is widely used for antibody production via phage display. This phage infects *Escherichia coli* (*E. coli*)

The discovery of smaller recombinant antibody fragments such as Fv (variable region consisting of VH and VL), Fab, single-chain variable domain (scFv), and diabodies (bivalent scFvs) has played an important role in the advancement of antibody phage display technology [11, 12]. Compared to full antibodies, these fragments are more inclined to expression in bacteria. These fragments can be cloned into a bacteriophage (next to the coat protein known as PIII protein) using a vector. Bacteriophages are then used to infect *E. coli* to generate a library containing approximately 1010 cells. Later, bacteriophage containing the antibody segments were secreted from *E. coli*. These cells can then be isolated and sequenced. This technology enables fast and large-scale production of antibodies without animal use and it is easy to screen a large diversity of clones. However, it has some drawbacks,

such as more expensive costs and more difficult techniques [11, 12].

**4.1 Therapeutic applications of mAbs in cancer therapy**

**4. Applications of monoclonal antibodies: therapeutic and diagnostic** 

Monoclonal antibodies could be designed specifically against a target antigen found on cancer cells. Several therapeutic mAbs have been approved against different cancer types after the discovery of proto-oncogenes and specific tumor antigens [13]. In 1994, an antibody named MAB 17-1A was approved against epithelial cell surface antigen for identification of adenocarcinomas. It was efficient in reducing the mortality and occurrence rate of colorectal cancer [14]. Rituximab, an anti-CD20 chimeric antibody, was approved in 1997 for treating non-Hodgkin B cell lymphoma. Rituximab interacts with CD20 antigen expressed on B cell tumors and then eliminates malignant cells through an effective immune response [15].

been replaced with human loci within the transgenic mouse genome [8].

torial antibody libraries on filamentous phages [11].

generated in transgenic mice models (e.g. HuMabMouse and XenoMouse) using hybridoma technology. For this purpose, the mouse immunoglobulin gene loci have been replaced with human loci within the transgenic mouse genome [8].

## **3.2 Phage display technique**

*Monoclonal Antibodies*

**3.1 Hybridoma technique**

ent centrifugation [8].

selective medium [8, 10].

stored away in liquid nitrogen [8].

Monoclonal antibodies are generated from a single B lymphocyte clone and bind to the same epitope of an antigen. The hybridoma technique was first used in 1975 to generate mAbs by Milstein and Köhler. Several steps are involved in this method. First, mice are immunized with specific antigens emulsified with appropriate adjuvant. The booster injection is normally done after two weeks and the animal is then sacrificed when enough amount of antibody is produced. Blood collection is performed to assay the sufficient amount of the antibody production using techniques including ELISA and flow cytometry. After sacrificing, the spleen is isolated and then tissue digestion could be applied with an enzymatic or mechanical method leading to release of B cells. B cells could be extracted using density gradi-

The next step is making a fusion between B lymphocytes and myeloma cells (that are immortal like cancer cells). Prior to fusion, myeloma cells should be prepared by culturing with 8 – azaguanine, making them sensitive to hypoxanthineaminopterin-thymidin (HAT) medium. The fusion process is carried through using polyethylene glycol (PEG), resulting in cell membrane fusing. After the fusing process, there will be a variety of cells including fused B cells with myeloma cells, unfused B cells, unfused myeloma cells, B cells fused to B cells, myeloma cells fused to myeloma cells. Therefore, a selective medium known as hypoxanthine, aminopterin and thymidine (HAT) medium should be used to select only the B cells fused with myeloma cells [10]. Two components of this medium, hypoxanthine and thymidine, are the metabolites of the salvage pathway of nucleoside synthesis. Therefore, only the cells that have the necessary enzyme for the salvage synthesis of nucleic acids, named hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), will be able to survive. Unfused myeloma cells lack HGPRT, so they cannot replicate their nucleic acid and they will not be able to grow in HAT medium. On the other hand, unfused B cells have a limited life span and therefore cannot grow appropriately. Consequently, only fused B cell-myeloma cells known as "hybridomas" are able to grow in the medium. It should be noted that another pathway of nucleic acid synthesis named "de novo" pathway, is also inhibited due to the presence of aminopterin in HAT medium. So, only the HGPRT-positive cells could be grown in this

To separate antibodies with different specificity and also for further hybridoma growth, the mixture of cells is diluted in microtiter wells in which their walls are coated with murine macrophages or feeder fibrocyte cells providing the growth factors needed for antibody-producing cells. Then, the antigen-binding ability of secreted antibodies by different clones of B cells could be assessed by ELISA, antigen microarray assay, radio-immuno assay (RIA), or immune-dot blot and finally, the stable clone will be selected. The fused hybridomas and produced mAbs can be

Although this process may be well suited for development of therapeutic antibodies, however, there are some important problems with using this technique. The hybridoma process takes approximately between 6 and 8 months to obtain a sufficient amount of mAbs, so its development procedure is very long. On the other hand, because of the murine origin of the antibodies, they can trigger the HAMA response in the host which could accelerate mAb clearance and undesirable allergic reactions upon repeated administration. This issue was resolved by developing antibody engineering methods toward producing less immunologic chimeric or humanized antibodies. These engineered antibodies were created using murine variable regions or CDRs as well as human constant regions aiming to decrease HAMA response and maintain target specificity. Currently, fully humanized antibodies are

**6**

The phage display method is one alternative to traditional hybridoma technology for generating monoclonal antibodies. This method was developed in 1985 by George P. Smith, who demonstrated that a peptide of interest could be displayed on the surface of filamentous phages following inserting the DNA fragment into the coat protein gene of phage. Then, a process known as "panning or biopanning" is explained by Parmley and Smith; the process describing a selection and affinity enrichment in order to isolation of peptide-phage fusions based on their specific binding affinity. Finally, phage display technology was used for the first time by McCafferty and Winter for generating antigen specific mAbs by creating combinatorial antibody libraries on filamentous phages [11].

This method involves integration of a gene sequence coding for a particular antibody into the DNA sequence of a filamentous bacteriophage leading to the expression of interest protein on the surface of the bacteriophage capsid. These phage libraries could be generated from healthy donors (creating Naïve libraries) or individuals who carry a particular disease, such as metastatic cancer or particular infection, or have been immunized with a particular antigen (creating immunized libraries). M13 is a filamentous bacteriophage that is widely used for antibody production via phage display. This phage infects *Escherichia coli* (*E. coli*) strains.

The discovery of smaller recombinant antibody fragments such as Fv (variable region consisting of VH and VL), Fab, single-chain variable domain (scFv), and diabodies (bivalent scFvs) has played an important role in the advancement of antibody phage display technology [11, 12]. Compared to full antibodies, these fragments are more inclined to expression in bacteria. These fragments can be cloned into a bacteriophage (next to the coat protein known as PIII protein) using a vector. Bacteriophages are then used to infect *E. coli* to generate a library containing approximately 1010 cells. Later, bacteriophage containing the antibody segments were secreted from *E. coli*. These cells can then be isolated and sequenced. This technology enables fast and large-scale production of antibodies without animal use and it is easy to screen a large diversity of clones. However, it has some drawbacks, such as more expensive costs and more difficult techniques [11, 12].
