**1. Introduction**

The sale of therapeutic monoclonal antibodies (mAbs) is increasing yearly in relation to other class of biological products [1], and pharmaceutical/biotechnological companies are pursuing all opportunities to develop this product. Therapeutic mAbs are indicated to diverse clinical conditions such as treatment of cancers, immune-mediated disorders and infectious diseases, among others. Every year, new mAbs are approved while a crescent number of other mAbs advance to the next phase of clinical study [2].

of mAbs of non-human origin. In relation to immunogenicity, approximately 9% of humanized antibodies induced human anti-humanized antibodies (HAHA) in different clinical trials [5]. A less frequent humanization technique aiming to retain the original antibody affinity by altering only the mouse surface-exposed residues of the framework (veneering) was used with success for an anti-NaPi2b oncologic antibody where the humanized version (Rebmab200) demonstrated a slightly improved affinity in relation to the murine version (MX35) [14, 15].

Display Technologies for the Selection of Monoclonal Antibodies for Clinical Use

http://dx.doi.org/10.5772/intechopen.70930

49

Concomitant to the utilization of antibody humanization techniques started the development of *in vitro* display technologies and the exploration of the molecular diversity of the antibody genes present in a determined library. The first technology available was phage display by presenting exogenous peptides on filamentous bacteriophage (phage) surfaces [16]. The peptide sequences were fused to the amino-terminus of the p3 protein of phages and the fusion protein was expressed on the phage surface, further purified by affinity through the binding to a specific antibody. Selected phages were amplified in bacteria. Then, a biopanning process, a classical cyclic procedure to select phage clones presenting peptides by affinity binding was described [17]. The occurrence of the phage display technology to select mAbs depended on the development of two techniques. Firstly, the expression of functional antibody fragments (scFv and Fab fragment) in the periplasm of *Escherichia coli* was reported [18, 19]. The second technique to mention is the PCR for amplification of antibody genes (heavy and light chains) from hybridomas, a pool of prokaryotic and eukaryotic cells transfected with antibody genes, human peripheral blood cells or tissues rich in B cells [20–22]. The amplification of human immunoglobulin genes directly from immune or naïve human materials opened the possibility to select human antibodies from these sources, either for diagnostic or clinical purposes. In phage display technology, amplified antibody genes are cloned into appropriate phage display vectors to construct the library. Antibody fragments present in the library are expressed on the phage surface and then are submitted to biopanning to select phages by the binding to a specific peptide epitope or antigen. After some cycles of biopanning, the phages encoding the antibody fragments are analyzed individually to select specific clones. Soluble antibody fragments are expressed allowing the characterization of the antibodies and isolation of lead clones [23, 24]. Using phage display technology, six human mAbs were approved for therapeutic use and other candidates are in advanced phases of clinical studies [25]. Other *in vitro* display technologies were developed such as yeast display, ribosome display, bacterial display, mRNA display, mammalian cell surface display and DNA display and, although mAbs with therapeutic potential were obtained by using these other *in vitro* display technolo-

In the present chapter, we describe discovery technologies to select human therapeutic mAbs.

*In vitro* display technologies such as phage display, yeast display, ribosome display, bacterial display, mammalian cell surface display, mRNA display and DNA display represent *in vitro* selection platforms of specific molecules presented in a determined library. These technologies are used mainly to isolate peptides and antibody fragments in scFv, single domain

**2. Display technologies to obtain recombinant monoclonal antibodies**

gies, no one reached the clinical approval so far.

Generation of mAbs started with the discovery of the hybridoma technology by Köhler and Milstein [3] in 1975. The fusion of B cells from immunized animals with myeloma cells originated hybrid cells producing unlimited quantities of antibodies with unique specificities. The potential of this technique for clinical and diagnostic use became evident with the approval of the first therapeutic mAb 10 years later, Orthoclone OKT3 targeting the CD3 receptor present in T lymphocytes to control renal transplantation rejection [4]. The clinical success conducted immediately to the development of other mAbs derived from this technology. However, due to the non-conforming sequence of murine mAbs, the generation of an immune response in humans was observed and the use of higher and multiple doses for prolonged time was not possible. Murine mAbs can induce immunogenicity, human anti-mouse antibodies (HAMA, including human anti-idiotype antibodies) affecting the safety and therapeutic efficacy [5]. The evaluation of mAb immunogenicity is crucial during clinical trials and it is recommended by regulatory agencies.

With advances in the understanding of antibody structure and in molecular biology techniques, the field of antibody engineering emerged with objective to change antibody properties by altering its primary sequence. Antibody humanization techniques use antibody engineering approaches to produce antibodies with less immunogenicity and preservation of affinity and specificity of the parental antibody of non-human origin [6]. The first humanization technique led to the combination of variable region domains of a murine antibody with human constant region domains resulting in chimeric antibodies with 70% of human content [7]. Chimeric antibodies maintained the specificity of parental murine antibody and demonstrated decreased immunogenicity, however, they generated human anti-chimeric antibodies (HACA) in approximately 40% of patients [5]. Efforts to obtain mAbs with a minimum of immunogenicity resulted in the development of a technique where sequences responsible for the antibody specificity to the antigen called complementarity-determining regions (CDRs) were transplanted to human framework sequences. This technique was designated CDR grafting and generated humanized antibodies [8–10]. However, it was observed that most of the antibodies obtained by CDR grafting did not preserve the affinity of the parental murine antibody. This fact is due to the influence of the human framework on the structure of the transplanted mouse CDRs [11, 12]. Through 3-dimensional modeling, key residues were identified in the murine framework sequences that interacted with CDRs and the antigen representing the integrity of the antigen-binding site. In a maneuver of back mutating the identified critical framework residues to the mouse framework sequence, antibodies with affinities close to the parental murine antibody were obtained. Using this approach Zenapax® (daclizumab) was approved by FDA in 1997 for therapeutic use for the prevention of renal transplantation rejection [13]. Soon the antibody humanization technique became viable for the clinical application of mAbs of non-human origin. In relation to immunogenicity, approximately 9% of humanized antibodies induced human anti-humanized antibodies (HAHA) in different clinical trials [5]. A less frequent humanization technique aiming to retain the original antibody affinity by altering only the mouse surface-exposed residues of the framework (veneering) was used with success for an anti-NaPi2b oncologic antibody where the humanized version (Rebmab200) demonstrated a slightly improved affinity in relation to the murine version (MX35) [14, 15].

**1. Introduction**

48 Antibody Engineering

advance to the next phase of clinical study [2].

The sale of therapeutic monoclonal antibodies (mAbs) is increasing yearly in relation to other class of biological products [1], and pharmaceutical/biotechnological companies are pursuing all opportunities to develop this product. Therapeutic mAbs are indicated to diverse clinical conditions such as treatment of cancers, immune-mediated disorders and infectious diseases, among others. Every year, new mAbs are approved while a crescent number of other mAbs

Generation of mAbs started with the discovery of the hybridoma technology by Köhler and Milstein [3] in 1975. The fusion of B cells from immunized animals with myeloma cells originated hybrid cells producing unlimited quantities of antibodies with unique specificities. The potential of this technique for clinical and diagnostic use became evident with the approval of the first therapeutic mAb 10 years later, Orthoclone OKT3 targeting the CD3 receptor present in T lymphocytes to control renal transplantation rejection [4]. The clinical success conducted immediately to the development of other mAbs derived from this technology. However, due to the non-conforming sequence of murine mAbs, the generation of an immune response in humans was observed and the use of higher and multiple doses for prolonged time was not possible. Murine mAbs can induce immunogenicity, human anti-mouse antibodies (HAMA, including human anti-idiotype antibodies) affecting the safety and therapeutic efficacy [5]. The evaluation of mAb immunoge-

nicity is crucial during clinical trials and it is recommended by regulatory agencies.

With advances in the understanding of antibody structure and in molecular biology techniques, the field of antibody engineering emerged with objective to change antibody properties by altering its primary sequence. Antibody humanization techniques use antibody engineering approaches to produce antibodies with less immunogenicity and preservation of affinity and specificity of the parental antibody of non-human origin [6]. The first humanization technique led to the combination of variable region domains of a murine antibody with human constant region domains resulting in chimeric antibodies with 70% of human content [7]. Chimeric antibodies maintained the specificity of parental murine antibody and demonstrated decreased immunogenicity, however, they generated human anti-chimeric antibodies (HACA) in approximately 40% of patients [5]. Efforts to obtain mAbs with a minimum of immunogenicity resulted in the development of a technique where sequences responsible for the antibody specificity to the antigen called complementarity-determining regions (CDRs) were transplanted to human framework sequences. This technique was designated CDR grafting and generated humanized antibodies [8–10]. However, it was observed that most of the antibodies obtained by CDR grafting did not preserve the affinity of the parental murine antibody. This fact is due to the influence of the human framework on the structure of the transplanted mouse CDRs [11, 12]. Through 3-dimensional modeling, key residues were identified in the murine framework sequences that interacted with CDRs and the antigen representing the integrity of the antigen-binding site. In a maneuver of back mutating the identified critical framework residues to the mouse framework sequence, antibodies with affinities close to the parental murine antibody were obtained. Using this approach Zenapax® (daclizumab) was approved by FDA in 1997 for therapeutic use for the prevention of renal transplantation rejection [13]. Soon the antibody humanization technique became viable for the clinical application Concomitant to the utilization of antibody humanization techniques started the development of *in vitro* display technologies and the exploration of the molecular diversity of the antibody genes present in a determined library. The first technology available was phage display by presenting exogenous peptides on filamentous bacteriophage (phage) surfaces [16]. The peptide sequences were fused to the amino-terminus of the p3 protein of phages and the fusion protein was expressed on the phage surface, further purified by affinity through the binding to a specific antibody. Selected phages were amplified in bacteria. Then, a biopanning process, a classical cyclic procedure to select phage clones presenting peptides by affinity binding was described [17]. The occurrence of the phage display technology to select mAbs depended on the development of two techniques. Firstly, the expression of functional antibody fragments (scFv and Fab fragment) in the periplasm of *Escherichia coli* was reported [18, 19]. The second technique to mention is the PCR for amplification of antibody genes (heavy and light chains) from hybridomas, a pool of prokaryotic and eukaryotic cells transfected with antibody genes, human peripheral blood cells or tissues rich in B cells [20–22]. The amplification of human immunoglobulin genes directly from immune or naïve human materials opened the possibility to select human antibodies from these sources, either for diagnostic or clinical purposes. In phage display technology, amplified antibody genes are cloned into appropriate phage display vectors to construct the library. Antibody fragments present in the library are expressed on the phage surface and then are submitted to biopanning to select phages by the binding to a specific peptide epitope or antigen. After some cycles of biopanning, the phages encoding the antibody fragments are analyzed individually to select specific clones. Soluble antibody fragments are expressed allowing the characterization of the antibodies and isolation of lead clones [23, 24]. Using phage display technology, six human mAbs were approved for therapeutic use and other candidates are in advanced phases of clinical studies [25]. Other *in vitro* display technologies were developed such as yeast display, ribosome display, bacterial display, mRNA display, mammalian cell surface display and DNA display and, although mAbs with therapeutic potential were obtained by using these other *in vitro* display technologies, no one reached the clinical approval so far.

In the present chapter, we describe discovery technologies to select human therapeutic mAbs.
