single chain antibody

• The immune system of vertebrates has the amazing capacity to generate specific molecules against virtually all possible foreign substances.

• These adapters, known as antibodies become available within a time span of days and can further be modified to increase their specificity and/or their affinity.

• The composition and overall structure of antibodies is remarkably well conserved among the mammals (Padlan, 1994; Kabat et al., 1991).

• The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs)

• Two H chains, each consisting of one V and five C domains, where the V domain is unpaired and constitutes a single-binding module.

• The structural basis for this property is a result of

Additional recombination events in the IgNAR V-D-J cluster and

the introduction of junctional diversity, which results in significant heterogeneity of CDR3 sequences

•Shark IgNARs demonstrate a surprising ability for specific and high affinity binding to diverse antigens by using just two variable domains per molecule, each carrying only two CDRs

• Dromedaries (Camelus dromedarius & Llama glama ),alpacas posses antibodies composed of heavy chains only (Hamers-Casterman et al.,1993).

• About 50% of the antibodies in camelids are of the ordinary mammalian heavy/light-chain typ

CAMELIDS have both the conventional heterotetrameric

antibodies common to all vertebrates and the homodimeric heavy-chain antibodies.

In IgG2, the missing CH1 seems to be replaced by an extended hinge region.

• In the absence of light chains, the fragment-antigen-binding (fab) part of these antibodies is reduced to a single domain, the so called VHH (variable domain of heavy chain antibodies) domain or nanobody.

• Recombinant VHH, or nanobodies, can be as small as 15 kDa.

• There are three subclasses of functional IgGs present in serum of Camelidae:

• IgG1 is a heterodimer consisting of heavy and light chain homodimers.

IgG2 and IgG3 consist only of heavy chains and thus are called heavy chain antibodies (hcAbs)

IgG2 fraction

IgG3 fraction

Upon reduction yield only heavy chains of 46K & 43K respectively.

UPON REDUCTION

• Heavy-chain antibodies can also occur in human serum or in mouse myeloma mutants (Seligmann et al., 1979).

• Non functional due to an extensive deletion from within the VH region until the hinge. Their occurrence in serum is considered as a pathological disorder.

SDS±PAGE of dromedary serum immunoglobulins G. The first lane contains a molecular weight marker (sizes are indicated on the left). The next two lanes contain the IgG1 fraction under non-reducing and reducing conditions, respectively, purified on proteins A and G. The pure IgG2 fraction was applied on the following two lanes under non-reducing and reducing conditions. The last two lanes contain the IgG3 fraction, again before and after reduction with dithiotreitol

The Antigen-binding Domain Of Heavy Chain Antibodies

• Since the heavy-chain antibodies do not contain light chains the antigen-binding site is reduced to a single domain.

• Compared to the conventional VH, at least five residues of otherwise conserved sequences are substituted in VHHs

• These VH–VHH landmarks are: Leu11Ser, Val37Phe or Val37Tyr, Gly44Glu, Leu45Arg or Leu45Cys and Trp47Gly (Muyldermans et al., 1994; Vu et al., 1997).

• The substitution of Leu11Ser in dromedary VHH is seen as an adaptation to accommodate the absence of the CH1 domain.

The Antigen-binding Domain Of Heavy Chain Antibodies

• While the Leu11Ser substitution is widespread among the

VHH of dromedaries, it was noted that a considerable fraction of the llama VHH still maintained a Leu at position 11 (Vu et al., 1997).

• the high expression yields and ease of purification;

• a highly soluble and stable, single-domain immunoglobulin fold

• the generation of antigen-specific, high-affinity binders

• the recognition of unique conformational epitopes with the dominant involvement of its long CDR3

• the close homology to human VH fragments.

Single chain variable fragment (Scfv)

• A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.


• The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.

• The first scFv fragments were cloned by two labs in the same year, Bird et al. and Huston et al in 1988

The two possible structures of a single-chain variable fragment, with the antigen binding sites including the N-termini on the left and the C-termini on the right. The linker peptides are shown as arrows.


•These fragments can often be purified or immobilized using Protein L, since Protein L interacts with the variable region of kappa light chains. • scientists incorporate a six histidine tag on the c-terminus of the scFv molecule and purify them using immobilized metal affinity chromatography (IMAC). •For unknown reasons, some scFv can also be captured by Protein A


Examples Pexelizumab, a scFv binding to component 5 of the complement system and used to reduce side effects of cardiac surgery. C6.5, a diabody targeting HER2/neu found in some breast cancer.

Bispecific T-cell engager antibodies (BiTEs) are single chain antibodies designed for polyclonal activation and redirection of cytotoxic T-cells to tumor cells.

BiTEs combine the minimal antigen-binding domains of two monoclonal antibodies, fused with a short flexible linker. One of the antibody's arms recognizes CD3, a cluster of differentiation for T-cells, and the other one detects tumor cells .

The BiTEs antibodies have a high potential to activate T-cells.

• Using this approach, blinatumomab (MT103), CD19-specific BiTE antibody has been developed for the treatment of B cell malignancies .

In vivo applications of ScFv

• ScFv fragments retain the binding specificity of the parent antibody and offer several advantages compared to full-length mAbs.

• They display improved pharmacokinetic properties, such as better tissue penetration and rapid blood clearance, which may be beneficial in radiotherapy and diagnostic applications.

• When scFvs are joined to radionuclides in radiotherapy applications, their increased rate of clearance from blood

minimizes exposure to healthy tissue.

Drawbacks

• Low retention on the target and rapid off rates, due to

their monovalency.

• Their small sizes, though a desirable property for tissue penetration, such as in cancer therapy, also leads to a short in vivo half-life, limiting the exposure of the target molecule to the scFv.

• These issues have been addressed by genetically manipulating and engineering scFvs to yield multivalent and multifunctional multimers (diabodies, triabodies and tetrabodies) that have higher avidity and lower blood clearance, without compromising tissue penetration abilities.

ScFvs Under Clinical Test

• Since the discovery of HCAb in camels almost 22 years ago VHH fragments or nanobodies have been used in a growing number of applications.

• VHHs have many advantages for biotechnological applications. They can be economically produced in microorganisms and have a high stability.

• Mainly their small size and monomeric nature make them superior in many ways compared to conventional antibodies.

• ScFv have become the de facto building block for engineered antibody fragments.

• Studies have demonstrated the efficacy and safety of scFvs in preventing amyloid protein aggregation in preclinical models.

single chain antibody

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