The The process starts with immunological recognition

The immune system is a defense
system which mediates the protection of the host from infectious agents by a
variety of effector cells and molecules. To accomplish its purpose, the immune
system has to perform different tasks. The process starts with immunological
recognition (detection of the foreign pathogen), which is performed by the
white blood cells of the innate immune system and by the lymphocytes of the
adaptive immune response 1. The innate response forms the
earliest barrier to the infection and does not need prolonged period for
induction. It mediates a broad and not antigen-specific response able to detect
and destroy the pathogenic agent within hours or even minutes. Conversely, the
adaptive response  is only present in
vertebrates and depends on the specific recognition functions of the
lymphocytes which are able to distinguish the particular pathogen and focus the
immune response on it 1. The immune system also has the
ability to self-regulate, which is a very important feature since its failure
can lead to the generation of allergies and autoimmune diseases. The last task
of the immune system is the generation of the immunological memory, which
contributes to the protection of the host against recurring disease caused by
the same pathogen. This ability permits the host to develop an immediate and
stronger response against the same infection agent 1.

The adaptive response is
triggered by the recognition of the pathogen by the antigen recognition
molecules named immunoglobulins. These proteins belong to the glycoproteins
family and can be found as membrane-anchored receptors on B-cells or as soluble
antibodies secreted by plasma cells. Each B-cell expresses immunoglobulins with
single antigen specificity and so, when a B-cell encounters an antigen in an
environment that provides requisite co-stimulatory signals, the B-cell receptor
(BCR) induces B-cell proliferation (called B cell clonal expansion). This
process is followed by somatic hypermutation of the variable domains of the
antibodies. B-cells which express BCR bearing somatic mutations that increase
affinity for the antigen compete with the others B-cells for binding to the respective
antigen. Thus, B-cells bearing the highest affinity antibodies undergo
preferential expression. This process is called affinity maturation. B-cells
expressing somatic mutated with high antigen affinity BCRs can differentiate
into long-lived memory B-cells (responsible for mediating rapid immune response
after a recurring infection with the same pathogen) or differentiate into
plasma cells 2. These cells produce and secrete antibodies with
the specificity and ability to bind to the pathogen in the extracellular spaces
of the body, consisting in their main effector function 1. As such, the repertoire of BCRs
expressed in each individual is continuously shaped by exposure to exogenous
antigens.

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1.1.1
Monoclonal antibodies

Antibodies are natural proteins
produced by plasma cells and provide protection against infection and foreign
pathogens by acting as soluble effector molecules. Antibodies belong to a class
of glycoproteins named immunoglobulins, and mediate a variety of functions such
as neutralization, agglutination and activation of complement and effector
cells 3. There are five major classes of
immunoglobulins depending of the heavy-chain isotypes (IgM, IgD, IgG, IgA and
IgE), which are characterized by different effector functions (Fig 1). Their
heavy chains are denoted by the following lower-case Greek letter (µ, ?, ?, ?
and ?, respectively).

                        

Fig
1. Schematic
representation of different classes of immunoglobulins IgG, IgE, IgD , IgA and
IgM.

IgG is the most abundant
immunoglobulin and has four sub-classes in humans (IgG1, IgG2, IgG3 and IgG4) 1. They are large molecules with a
molecular weight of approximately 150 kDa and are composed by two different
polypeptide chains: heavy and light chains. The heavy or H chain has a
molecular weight of approximately 50 kDa whereas the light or L chain has 25
KDa respectively. IgG molecules are monospecific, composed by two identical
heavy and two identical light chains linked by disulfide bonds, which
contributes to their ability to bind specifically to the same molecular
structure. Another relevant feature of an IgG molecule is its bivalency,
accomplished by two identical antigen-binding regions which allow the binding
to two identical structures. There are two types of light chains, termed as
lambda (?) and kappa (k). Until now no functional difference
between them has been found 1.

A full length IgG comprises the
Fc constant domain and the antibody binding fragment (Fab), both located at
carboxyl-terminal and amino-terminal respectively. Contrarily to Fab region, Fc
fragment does not directly contribute to the binding to the antigen, but it is instead
specialized for activating different effector mechanisms. Moreover, it is
important for antibody stabilization and half-life time as well as to antibody-dependent
cell-mediated-cytotoxicity 4. The binding region (Fab) is composed by two
constant regions (CH1 and CL) and two variable domains (VH and VL), which is
responsible for the specific binding properties (Fig 2).

Fig 2.
Representation of full-length IgG and antibody fragments. The IgG contains  two  light chains with one V-region (VL)
and one C-region (CL), and two  heavy chains with one V region (VH)
and three C-regions (CH1, CH2 and CH3). Fab
fragments are composed by one constant region and the variable regions of the
heavy and light chains. scFv comprises both variable regions from light and
heavy chains.

 

The most variable parts of the
V-regions are delimited to hypervariable regions or complementarity-determining
regions (CDRs). There are three CDRs (CDR1, CDR2 and CRD3) and are located
between four flaking framework regions, which compose about 85% of the V-region.
They present less variability than CDRs and its main function consists on the maintenance
of the overall structure of the V-region and support the binding between the
CDRs and the antigen 5.

1.1.2   Applications
of monoclonal antibodies

During the last years monoclonal
antibodies have been widely used in research, biotechnology and medicine, being
one of the most important biopharmaceutical products nowadays. Due to the
current high demand on the discovery of monoclonal antibodies, this research
area has been developed and progressed in several therapeutic areas such as
oncology, inflammation, cardiovascular, autoimmune diseases and viral and
bacterial infections, becoming therefore potential diagnostic and therapeutic
agents 6. From a Biopharmaceutical company perspective and considering
that several companies work on the same therapeutic areas (and in many cases on
the same clinical targets), the search for innovative approaches has become
indispensable. To that end, various strategies have been designed to optimize
manufacturing processes as well as reducing costs and time to market, assuring
at the same time the desired properties of the products.

There are currently two main
strategies being used with the purpose of generating novel and effective monoclonal
antibodies: animal immunization and surface display methodologies. Wild type or
transgenic animal immunization followed by hybridoma screening is an effective
method for generation of antibodies. However, this in vivo method can be less effective when poorly immunogenic
epitopes (intractable targets) or self-antigens are used. Besides, some
therapeutic applications may require specific properties such as size, valency
and stability, which are difficult to obtain by hybridoma technology 7. Display technologies, on the other hand, are based
on in vitro selection from naïve or
immune-libraries having the ability to overcome the limitation of immune
tolerance. Display technologies have not only the ability to generate
antibodies in vitro from DNA
libraries but also to improve antibody affinity, specificity and stability.
Moreover, the fact of being an in vitro
methodology, it entails that selection and screening are both performed under
controlled conditions. Consequently, this allows the selection of certain
specificities against defined antigens conformations or epitopes, that
otherwise could not be addressed by conventional immunization methods 6.

Additionally to full monoclonal
antibodies, the interest in the production of antibody fragments has also grown
greatly, due to not only its therapeutic use but also in immune detection,
purification and bioseparation applications. Although antibody fragments have
reduced stability without the Fc fragment they still exhibit antigen binding
ability and also show increased tissue penetration together with lower
retention time when compared with full antibodies 4.The two antibody fragment formats most commonly
used are scFv and Fab fragments.  scFv
fragments own the advantage of maintaining the antigen binding site and require
less complex expression demands than Fab fragments, but are less stable and
susceptible to form aggregates, which may result in  lower affinity than Fab fragments 8, 9. Furthermore , often after conversion of scFv to
Fab or IgG formats the binding ability together with  neutralization/activation activity is lost 10. Regarding the Fab fragments, since its expression
entails the assembly of two separate polypeptide chains with disulfide bond,
they tend to fold less efficiently than scFv which  might lead to lower expression levels and
solubility in E. coli 8.