employed as regions determining the specificity (Riechmann et al. 1988; Foote and

Winter 1992). CDRs have been identified as the regions with highest variability

values in multiple alignment of antibody sequences as per Kabat definition (Wu and

Kabat 1970). Another definition for regions determining specificity based on the

structure of antibody has been used (Chothia et al. 1989). The advantage of using the

latter definition is that the CDRs are shorter; therefore, the humanized antibody will

have less xenogeneic component. However, the use of Kabat definition generally

leads to less iteration in the humanization design (Presta et al. 1993).

Either mature or germline gene sequences of human antibody are used for

grafting the identified CDRs of the mouse antibody. Mature sequences carry somatic

mutations which are not under species selection, resulting in potential immunogenic

residues. Thus, human germline genes have increasingly been utilized as source of

FR donors (Neuberger and Milstein 1995; Tan et al. 2002; Hwang et al. 2005). There

are certain advantages in using germline gene sequences as human FR acceptor.

Primary advantage can be attributed to the fact that due to the absence of somatic

mutation, it may be less immunogenic. Further, the physical maps of the germline

human H and L chain loci and the functional germline gene repertoire they encode

have been thoroughly characterized. In addition, the use of human germline genes

encoding light and heavy chains has more plasticity to accommodate diverse CDRs

with fewer back mutations (Wedemayer et al. 1997; Zimmermann et al. 2006). In

fact, “superhumanization” protocol takes into account the homology of CDRs of

nonhuman and germline human template regardless of FR homology (Tan et al.

2002).

In general, the affinity of humanized antibody decreases after CDR grafting as a

consequence of incompatibilities between nonhuman CDRs and human FRs. Hence,

it is critical to identify amino acid residues that must be retained during grafting of

CDRs to prevent affinity loss of the humanized antibody. There are some residues

underlying the CDRs in variable part of both light and heavy chains of

immunoglobulins that are responsible for stabilizing the hypervariable loop struc-

ture. Since these residues fine-tune the antibody affinity, this region is designated as

vernier zone (Foote and Winter 1992; Makabe et al. 2008). Another important class

of residues is interchain packing residues that lie at the interface between variable

light and heavy chains (Chothia et al. 1985, 1989). Further, canonical residues and

presence of additional unusual residues close to the antigen binding site should also

be retained during humanization of the antibody (Shearman et al. 1991; Graziano

et al. 1995). The first humanized Mab Daclizumab for kidney transplant rejection

was approved for clinical use by the FDA in 1997 (Vincenti et al. 1998; Table 22.2).

After this, till 2017, several humanized antibodies got license for clinical use for a

variety of disorders that has been summarized in Table 22.2. For example,

bevacizumab (Avastin^®) inhibits angiogenesis by neutralizing vascular endothelial

growth factor (VEGF). It has been licensed to treat various cancers including

colorectal, lung, breast, and glioblastoma. Another humanized IgG1 neutralizing

MAb, palivizumab (Synagis^®), that binds to the fusion protein of respiratory syncy-

tial virus (RSV) inhibits the virus entry into the cell. It has been used to prevent RSV

infection in infants.

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Therapeutic Human Monoclonal Antibodies

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