**3. Approved therapeutic monoclonal antibodies**

By the end of 2016, 20 fully human mAbs were approved for therapeutic use by the US Food and Drug Administration (FDA) and often approved by the European Medicines Agency (EMA) too. The approved fully human mAbs are derived basically from two technologies: phage display or transgenic mice expressing human antibody genes [132, 133]. Among approved mAbs, five were generated by phage display with selection of antigen-specific binders from two different libraries, Cambridge Antibody Technology - CAT (MedImmune, subsidiary of AstraZeneca) based on scFv fragments or Dyax Corp's human Fab fragment libraries [25, 134]. The other 15 mAbs were developed in transgenic mice, with integrated human immunoglobulin loci.

Considering the therapeutic mAbs approved which used phage display technology to acquire the human antibody composition, there are examples of different targets for immune intervention: two (adalimumab and belimumab) are in use for autoimmune diseases, one (raxibacumab) is for control of *Bacillus anthracis* infection and two others (ramucirumab and necitumumab) address growth factor receptors for cancer treatment.

The first fully human mAb approved for therapy in 2002 (FDA) and 2003 (EMA)—adalimumab (Humira®, AbbVie Inc., formerly Abbott Laboratories)—was generated by phage display technology [25, 134]. Adalimumab is an anti-TNFα mAb developed in 1993 through a collaboration between BASF Bioresearch Corporation and CAT using an anti-TNF murine antibody (MAK195) from BASF as a template for guided selection of human antibody V-domains with CAT's antibody phage display technology [25]. Adalimumab was first approved for treatment of rheumatoid arthritis (RA) under the brand name Humira (human monoclonal antibody in rheumatoid arthritis) [25, 135]. Besides RA, adalimumab is currently used in the treatment of plaque psoriasis, ulcerative colitis, Crohn's disease, non-infectious uveitis, hidradenitis suppurativa, psoriatic arthritis, juvenile idiopathic arthritis and ankylosing spondylitis [136].

using nickel resin. Isolated DNAs were cloned into a vector, amplified and sequenced. A limited affinity selection efficiency was observed so that the method needed improvement [128]. The selection of diverse anti-FLAG Tag peptides from a peptide-DNA library was performed using monoclonal antibody anti-FLAG M2 [129]. The same group also demonstrated that folded proteins can be displayed on the DNA display platform by the introduction of linkers between streptavidin and fused proteins [130]. It could be shown that the GST gene could be enriched by this method by the selection of the DNA-protein conjugates with glutathionecoupled beads. The application of DNA display technology to select Fab fragments using the STABLE method was described later [131]. The selection of a Fab fragment gene was performed using a new approach: randomized hydrophobic core in the constant region with heat

Advantages of this technology compared to other cell free *in vitro* display technologies such as ribosome and mRNA display is that RNase-free conditions are not needed for the selection step because a reverse transcription step is unnecessary. Furthermore, the removal of a stop codon between nucleic acid and peptide is not necessary. DNA display technology is simpler than other fully *in vitro* display technologies, with lesser steps [128–130]. One disadvantage of this technology is its novelty, devoid of a robust platform and knowledge in comparison to

By the end of 2016, 20 fully human mAbs were approved for therapeutic use by the US Food and Drug Administration (FDA) and often approved by the European Medicines Agency (EMA) too. The approved fully human mAbs are derived basically from two technologies: phage display or transgenic mice expressing human antibody genes [132, 133]. Among approved mAbs, five were generated by phage display with selection of antigen-specific binders from two different libraries, Cambridge Antibody Technology - CAT (MedImmune, subsidiary of AstraZeneca) based on scFv fragments or Dyax Corp's human Fab fragment libraries [25, 134]. The other 15 mAbs were developed in transgenic mice, with integrated

Considering the therapeutic mAbs approved which used phage display technology to acquire the human antibody composition, there are examples of different targets for immune intervention: two (adalimumab and belimumab) are in use for autoimmune diseases, one (raxibacumab) is for control of *Bacillus anthracis* infection and two others (ramucirumab and

The first fully human mAb approved for therapy in 2002 (FDA) and 2003 (EMA)—adalimumab (Humira®, AbbVie Inc., formerly Abbott Laboratories)—was generated by phage display technology [25, 134]. Adalimumab is an anti-TNFα mAb developed in 1993 through a collaboration between BASF Bioresearch Corporation and CAT using an anti-TNF murine antibody (MAK195) from BASF as a template for guided selection of human antibody V-domains with CAT's antibody phage display technology [25]. Adalimumab was first

treatment application for selective pressure.

**3. Approved therapeutic monoclonal antibodies**

necitumumab) address growth factor receptors for cancer treatment.

other *in vitro* display technologies.

60 Antibody Engineering

human immunoglobulin loci.

Nine years after the approval of the first human mAb developed by phage display technology (adalimumab), in 2011 belimumab (Benlysta, GSK) was approved for the treatment of systemic lupus erythematosus [25, 134, 137]. Belimumab was selected using CAT library as a result of the collaboration between CAT and Human Genome Sciences (now GlaxoSmithKline), and targets the soluble form of human B-lymphocyte stimulator (BLyS) [25, 134]. Under a license to use the CAT library, Human Genome Sciences (now GSK) discovered Raxibacumab (Abthrax) also using phage display. Raxibacumab binds the *Bacillus anthracis* protective antigen (PA) and was approved by FDA in 2012 for the prophylaxis and treatment of anthrax [25, 134, 138, 139].

In addition to the three previously described antibodies, which were discovered using scFvfragment CAT library by antibody phage display, two mAbs—ramucirumab and necitumumab—were derived from the same technology but from a different library, a human Fab-fragment library constructed by de Haard and colleagues at Dyax [25, 134]. Ramucirumab (Cyramza, Eli Lilly) is an anti-VEGFR-2 (vascular endothelial growth factor receptor 2) mAb approved for the treatment of cancer (gastric or gastroesophageal junction, non-small cell lung and colorectal) in 2014 [140]. Necitumumab (Portrazza, Eli Lilly) targets epidermal growth factor receptor (EGFR) and blocks binding to epidermal growth factor (EGF) [25, 134]. This mAb gained first marketing approval for FDA and EMA in 2015 and 2016, respectively, for the treatment of non-small cell lung cancer [25, 141, 142].

Ranibizumab (Lucentis, Genentech), an anti-VEGF-A (vascular endothelial growth factor A) Fab fragment, was generated by the humanization of the murine mAb A4.6.1 [134]. Based on a random mutation library, clones were selected by phage display [143]. Afterward, an alanine scanning study and information of the crystal structure of the Fab-VEGF complex were used for affinity maturation [144, 145]. This mAb was approved in the US in 2006 for the treatment of age-related macular degeneration [25, 134], and posteriorly for the treatment of macular edema after retinal vein occlusion, diabetic macular edema and diabetic retinopathy [25].
