Introduction on Monoclonal Antibodies

*Mona Sadeghalvad and Nima Rezaei*

## **Abstract**

Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B lymphocytes against a particular antigen. mAbs are identical in several properties such as protein sequence, antigen-binding site region, binding affinity for their targets, and identical downstream functional effects. These characteristics of mAbs highlight their differences with the polyclonal antibodies which have heterogenous activities and recognize different epitopes on an antigen. Murine mAbs was the first generation of mAbs developed by hybridoma technology however, because of their murine origin, they can trigger the anti-mouse antibody response in the host which could accelerate mAb clearance and undesirable allergic reactions upon repeated administration. This issue was resolved by developing engineering methods toward producing less immunologic chimeric or humanized antibodies. mAbs applications have become a novel way of targeting antigens in a wide variety of diseases such as autoimmunity, malignancies, and asthma. In addition, high specificity and high affinity binding properties of mAbs make them effective biological reagents in immunodiagnostic assays. They can be used in diagnosis of infectious diseases and detection of certain antigens or in serological assessments for detection of antibodies against a certain antigen. This chapter summarizes the general properties of mAbs, their production processes, and their important diagnostic and therapeutic applications.

**Keywords:** monoclonal antibodies, mAb, chimeric mAb, humanized mAb, fully humanized mAb

#### **1. Introduction**

Antibodies or immunoglobulins (Ig) are glycoproteins produced by differentiated B lymphocytes named "plasma cells" in response to exposure to antigens. The diversity of antibody responses to different antigens is because of the gene recombination process in the hyper-variable regions of antibodies. During the recombination process in their genes, antibodies undergo gene rearrangement that allows them for diverse binding [1]. High specificity and diversity of antibodies have made them popular molecules with very high efficiencies in several therapeutic or diagnostic applications.

Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B lymphocytes against a particular antigen. Monoclonal antibodies are identical in several properties such as protein sequence, antigen-binding site region, binding affinity for their targets, and identical downstream functional effects. These characteristics of mAbs highlight their differences with the


#### **Table 1.**

*Different types of monoclonal antibodies. Murine mAbs were the first generation of mAbs with higher immunogenicity in humans. Gene engineering methods provide the less immunogenic mAbs by replacing human components in mAb structure. mAb: Monoclonal antibody.*

**3**

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

cardiovascular, and infectious diseases [3].

applications, including therapeutic and diagnostic uses.

**therapeutic mAb**

(VH and VL, respectively) [4].

**2. Antibody structure and functions: immunoglobulin G as the** 

An antibody molecule has a Y-shaped structure with a total molecular weight of ~150 kDa, composed of four polypeptide chains including two identical heavy (H) and two light (L) chains (**Figure 1**). Covalent bonds (mainly disulfide interactions) provide the stability of heavy and light chains next to each other. Each heavy or light chain is composed of constant (CH and CL, respectively) and variable domains

Each antibody has two identical arms known as "antigen binding fragments" or Fabs, acting as antigen-binding sites. Each Fab consists of a variable region known as Fv (formed by the VH and VL domains), and the constant region (formed by the CH and CL domains). Fv is a highly variable region and responsible for specific binding of antibody to the antigen, contributing to direct effects of antibody such as inhibiting or neutralizing the antigen. There are three hyper variable regions known as complementarity determining regions or CDR1, CDR2, and CDR3 in the variable regions of light and heavy chains, allowing diverse antigenic specificities to be recognized [4]. The Y structure's stem, known as the "fragment crystallizable region" or Fc, is a constant region of the antibody molecule. The Fc region determines the class of the antibody and its functional properties. There are five classes

epitopes on an antigen.

polyclonal antibodies which have heterogenous activities and recognize different

Using mAbs has become a novel way of targeting antigens in a wide variety of diseases and conditions since the first mAb was approved in 1986. Orthoclone OKT3® (muromonab-CD3) was the first mAb approved by the Food and Drug Administration (FDA). OKT3 was produced based on murine hybridoma technology by Kohler and Milston for the treatment of acute transplant rejection [2]. Currently, mAbs are the important group of therapeutic molecules in clinical trials for treating different disorders such as inflammatory and autoimmune diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel diseases), malignancies (e.g. leukemia, melanoma, breast cancer, and multiple myeloma),

Murine mAbs was the first generation of monoclonal antibodies developed by hybridoma technology. They have no human components in their structure and could result in producing human anti-mouse antibodies (HAMAs). HAMA response caused hypersensitivity reactions (e.g. anaphylaxis and serum sickness) in the recipients, resulting in fast clearance of antibodies or reducing their effectiveness [4]. Genetic engineering approaches and using transgenic animals were developed to overcome these troubles; So that a transformed cell line could produce the altered antibody structurally closer to human antibodies. These modified antibodies are known as chimeric mAbs because their constant region is human while their variable region is murine (**Table 1**). This technology was developed for the first time in 1980s by scientists in Cambridge, UK. After that, humanized and fully human mAbs were developed to reduce mAb immunogenicity and their side effects. Humanized antibodies have human light and heavy chains but hypervariable regions are still murine while fully human antibodies are totally humanized. However, they are still immunogens and may have important adverse effects caused by production of antidrug antibodies (ADAs) [5]. This chapter summarizes the general properties of mAbs, their production processes, and their important

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

*Monoclonal Antibodies*

Murine mAb Murine mAbs was the first

antibodies (HAMAs). Suffix: -Omab

ovarian cancer)

remain murine Suffix: -Ximab

Suffix: -Zumab

100% human Suffix: -Umab

e.g.: Ibritumab, Ofatumumab

*human components in mAb structure. mAb: Monoclonal antibody.*

*Different types of monoclonal antibodies. Murine mAbs were the first generation of mAbs with higher immunogenicity in humans. Gene engineering methods provide the less immunogenic mAbs by replacing* 

**Description Structure**

generation of monoclonal antibodies developed by hybridoma technology. They have no human components in their structure and could result in producing the human anti-mouse

e.g.: Abagovomab (anti CA-125 in

In chimeric mAb, constant regions are humanized but variable regions in both heavy and light chains

Hyper variable regions are murine

e.g.: Natalizumab, Gemtuzumab

e.g.: Rituximab, Infliximab

**Type of mAb**

Chimeric mAb

Humanized mAb

Fully human mAb

**2**

**Table 1.**

polyclonal antibodies which have heterogenous activities and recognize different epitopes on an antigen.

Using mAbs has become a novel way of targeting antigens in a wide variety of diseases and conditions since the first mAb was approved in 1986. Orthoclone OKT3® (muromonab-CD3) was the first mAb approved by the Food and Drug Administration (FDA). OKT3 was produced based on murine hybridoma technology by Kohler and Milston for the treatment of acute transplant rejection [2]. Currently, mAbs are the important group of therapeutic molecules in clinical trials for treating different disorders such as inflammatory and autoimmune diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel diseases), malignancies (e.g. leukemia, melanoma, breast cancer, and multiple myeloma), cardiovascular, and infectious diseases [3].

Murine mAbs was the first generation of monoclonal antibodies developed by hybridoma technology. They have no human components in their structure and could result in producing human anti-mouse antibodies (HAMAs). HAMA response caused hypersensitivity reactions (e.g. anaphylaxis and serum sickness) in the recipients, resulting in fast clearance of antibodies or reducing their effectiveness [4]. Genetic engineering approaches and using transgenic animals were developed to overcome these troubles; So that a transformed cell line could produce the altered antibody structurally closer to human antibodies. These modified antibodies are known as chimeric mAbs because their constant region is human while their variable region is murine (**Table 1**). This technology was developed for the first time in 1980s by scientists in Cambridge, UK. After that, humanized and fully human mAbs were developed to reduce mAb immunogenicity and their side effects. Humanized antibodies have human light and heavy chains but hypervariable regions are still murine while fully human antibodies are totally humanized. However, they are still immunogens and may have important adverse effects caused by production of antidrug antibodies (ADAs) [5]. This chapter summarizes the general properties of mAbs, their production processes, and their important applications, including therapeutic and diagnostic uses.

## **2. Antibody structure and functions: immunoglobulin G as the therapeutic mAb**

An antibody molecule has a Y-shaped structure with a total molecular weight of ~150 kDa, composed of four polypeptide chains including two identical heavy (H) and two light (L) chains (**Figure 1**). Covalent bonds (mainly disulfide interactions) provide the stability of heavy and light chains next to each other. Each heavy or light chain is composed of constant (CH and CL, respectively) and variable domains (VH and VL, respectively) [4].

Each antibody has two identical arms known as "antigen binding fragments" or Fabs, acting as antigen-binding sites. Each Fab consists of a variable region known as Fv (formed by the VH and VL domains), and the constant region (formed by the CH and CL domains). Fv is a highly variable region and responsible for specific binding of antibody to the antigen, contributing to direct effects of antibody such as inhibiting or neutralizing the antigen. There are three hyper variable regions known as complementarity determining regions or CDR1, CDR2, and CDR3 in the variable regions of light and heavy chains, allowing diverse antigenic specificities to be recognized [4]. The Y structure's stem, known as the "fragment crystallizable region" or Fc, is a constant region of the antibody molecule. The Fc region determines the class of the antibody and its functional properties. There are five classes

#### **Figure 1.**

*The schematic structure of an antibody. An antibody molecule is composed of four polypeptide chains including two identical heavy (H) and two light (L) chains. Each heavy or light chain is composed of constant (CH and CL, respectively) and variable domains (VH and VL, respectively). Variable domains form the antigen binding site. CDR: complementarity determining regions; S-S: disulfide bond; C: constant; V: variable.*

of antibodies including immunoglobulin G (IgG), IgM, IgD, IgE, and IgA with distinct effector mechanisms for recognition and elimination of the antigens. In addition, the Fc region can interact with a variety of receptors such as Fc receptors or FcRs (expressed on the immune cells) and the components of the complement system (such as C1q). Fc recognition by the immune system components results in initiating the effector functions of antibodies such as antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) (**Figure 2**) [4].

Therefore, several functions are explained by which antibodies can eliminate a particular antigen and both variable and constant regions of antibodies contribute to this response. The stability and flexibility of antibodies and their effector functions such as activating ADCC, CDC, as well as interaction with C1q are important factors determining the suitability of immunoglobulins for the development of therapeutic mAbs. The majority of the clinically available mAbs are IgG. IgG is a glycoprotein with a size of 150 kDa consisting of two heavy and two light chains as described before. A conserved glycosylation site is present at amino acid Arginine297 (N297) in the CH2 domain, playing an important role in the structural conformation of the Fc and its binding to FcRs and complement component C1q [6].

Totally, IgG consists of four subclasses of IgG1, IgG2, IgG3 and IgG4 which differ in their heavy constant region (CH), as well as the hinge structure (the region where Fabs are bound to the Fc region). The difference between hinge regions confers many of the unique characteristics to each IgG subclass, including flexibility, stability and distances between the two Fabs. In addition, the amino acid differences between the binding sites of each subclass could explain the differences in the

**5**

properties [6, 7].

**Figure 2.**

*cytotoxicity.*

the serum [8, 9].

IgG1 > IgG3 > IgG4 > IgG2 respectively) [6].

(MS) and acute myeloid leukemia (AML), respectively [6].

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

phage display used for the production of mAbs.

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

effector functions of the IgG subclasses. These variations between IgG subclasses correlate with their selection for therapeutic purposes. Of the IgG subclasses, IgG3 has a longer hinge region compared with other subclasses, making them inappropriate for target binding. On the other hand, IgG3 cannot be purified with protein A and also has the shortest half-life (approximately 7 days) and high allotypic polymorphism compared with other subclasses. So, engineering techniques are required for modifications to the amino acid content of the IgG3 hinge region for development of therapeutics purposes. Meanwhile, most of the mAb therapeutics on the market are composed of IgG1, IgG2 or IgG4 with slow clearance and long half-life

*Two important effector functions of antibody. ADCC is an extracellular killing mechanism leading to antigen elimination. IgG has a bifunctional structure related to the fragment antigen-binding (fab) and fc portions of antibody. ADCC is initiated by the engagement of fab with the antigen from one side, and fc interaction with Fc*γ*R on effector cells, from another site. Subsequently, degranulation of effector cells (mainly NK cells) leads to target cell lysis. NK: natural killer cell; MQ: macrophage; Eos: eosinophil; ADCC: antibody-dependent cell* 

IgG1 has high stability and exhibits potent effector functions including ADCC, CDC, and C1q binding being the majority of therapeutic mAbs. IgG1 has the higher affinity for the FcRs compared with the other subclasses (the affinity for Fc receptor:

IgG2 has low affinity for interaction with antigen and also exhibits reduced functional activity compared to IgG1. IgG2 antibodies have three isoforms (known as IgG2-A, IgG2-A/B, and IgG2-B) based on types of disulfide bonds between the antibody chains. These isoforms could be converted to each other. This phenomenon, which is referred to disulfide shuffling, could regulate the activity of IgG2 in

IgG4 has a low affinity for C1q and therefore, this subclass of IgG could emerge as a therapeutic mAb when the host effector function is not desirable. In addition, the exchange of Fab arm is a normal biological process that can occur in IgG4 and is not desirable due to its adverse effects. Natalizumab (Tysabri) and gemtuzumab ozogamicin (Mylotarg) are the examples of therapeutic IgG4 for multiple sclerosis

In the following section we described two techniques, including hybridoma and

**Figure 2.**

*Monoclonal Antibodies*

of antibodies including immunoglobulin G (IgG), IgM, IgD, IgE, and IgA with distinct effector mechanisms for recognition and elimination of the antigens. In addition, the Fc region can interact with a variety of receptors such as Fc receptors or FcRs (expressed on the immune cells) and the components of the complement system (such as C1q). Fc recognition by the immune system components results in initiating the effector functions of antibodies such as antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and

*site. CDR: complementarity determining regions; S-S: disulfide bond; C: constant; V: variable.*

*The schematic structure of an antibody. An antibody molecule is composed of four polypeptide chains including two identical heavy (H) and two light (L) chains. Each heavy or light chain is composed of constant (CH and CL, respectively) and variable domains (VH and VL, respectively). Variable domains form the antigen binding* 

Therefore, several functions are explained by which antibodies can eliminate a particular antigen and both variable and constant regions of antibodies contribute to this response. The stability and flexibility of antibodies and their effector functions such as activating ADCC, CDC, as well as interaction with C1q are important factors determining the suitability of immunoglobulins for the development of therapeutic mAbs. The majority of the clinically available mAbs are IgG. IgG is a glycoprotein with a size of 150 kDa consisting of two heavy and two light chains as described before. A conserved glycosylation site is present at amino acid Arginine297 (N297) in the CH2 domain, playing an important role in the structural conformation of the Fc and its binding to FcRs and complement

Totally, IgG consists of four subclasses of IgG1, IgG2, IgG3 and IgG4 which differ in their heavy constant region (CH), as well as the hinge structure (the region where Fabs are bound to the Fc region). The difference between hinge regions confers many of the unique characteristics to each IgG subclass, including flexibility, stability and distances between the two Fabs. In addition, the amino acid differences between the binding sites of each subclass could explain the differences in the

complement-dependent cytotoxicity (CDC) (**Figure 2**) [4].

**4**

component C1q [6].

**Figure 1.**

*Two important effector functions of antibody. ADCC is an extracellular killing mechanism leading to antigen elimination. IgG has a bifunctional structure related to the fragment antigen-binding (fab) and fc portions of antibody. ADCC is initiated by the engagement of fab with the antigen from one side, and fc interaction with Fc*γ*R on effector cells, from another site. Subsequently, degranulation of effector cells (mainly NK cells) leads to target cell lysis. NK: natural killer cell; MQ: macrophage; Eos: eosinophil; ADCC: antibody-dependent cell cytotoxicity.*

effector functions of the IgG subclasses. These variations between IgG subclasses correlate with their selection for therapeutic purposes. Of the IgG subclasses, IgG3 has a longer hinge region compared with other subclasses, making them inappropriate for target binding. On the other hand, IgG3 cannot be purified with protein A and also has the shortest half-life (approximately 7 days) and high allotypic polymorphism compared with other subclasses. So, engineering techniques are required for modifications to the amino acid content of the IgG3 hinge region for development of therapeutics purposes. Meanwhile, most of the mAb therapeutics on the market are composed of IgG1, IgG2 or IgG4 with slow clearance and long half-life properties [6, 7].

IgG1 has high stability and exhibits potent effector functions including ADCC, CDC, and C1q binding being the majority of therapeutic mAbs. IgG1 has the higher affinity for the FcRs compared with the other subclasses (the affinity for Fc receptor: IgG1 > IgG3 > IgG4 > IgG2 respectively) [6].

IgG2 has low affinity for interaction with antigen and also exhibits reduced functional activity compared to IgG1. IgG2 antibodies have three isoforms (known as IgG2-A, IgG2-A/B, and IgG2-B) based on types of disulfide bonds between the antibody chains. These isoforms could be converted to each other. This phenomenon, which is referred to disulfide shuffling, could regulate the activity of IgG2 in the serum [8, 9].

IgG4 has a low affinity for C1q and therefore, this subclass of IgG could emerge as a therapeutic mAb when the host effector function is not desirable. In addition, the exchange of Fab arm is a normal biological process that can occur in IgG4 and is not desirable due to its adverse effects. Natalizumab (Tysabri) and gemtuzumab ozogamicin (Mylotarg) are the examples of therapeutic IgG4 for multiple sclerosis (MS) and acute myeloid leukemia (AML), respectively [6].
