REVIEW OF LITERATURE

Review of Liter ature

2.1 Antibodies an Introduction

More than a century ago, Behring & Kitasato (1890), discovered that a

component of serum, later termed antibodies, can transfer specific immunity to

other animals. Since then , antibodies have been extensively characterized in

molecular detail. The typical antibody or immunoglobulin (lg) consists of two

antigen-binding fragments (Fabs) , which are linked via a flexible region (the

hinge) to a constant (Fe) region (Fig . 2.1). This structure comprises two pairs of

polypeptide chains , each pair containing a heavy and a light chain of different

sizes. Both heavy and light chains are folded into immunoglobulin domains. The

'variable domains' in the amino-terminal part of the molecule are the domains

that recognize and bind antigens; the rest of the molecule is composed of

'constant domains' that only vary between lg classes. The Fe portion of the lg

serves to bind various effector molecules of the immune system, as well as

molecules that determine the bio-distribution of the antibody.

CcrnFm ert end Fo ~tor t4ndng

Cl:f'l loq:o

3

2

Fig. 2.1 : T he modular structure of im munog lobuli ns. This figure shows a single immunglobulin (I g) molecule . All immunoglobulin monomers are composed of two identical light (L) chains and two identical heavy (f-1) chains. Light chains are composed of one constant domain (CL) and one variable domain (VL). whereas heavy chains are composed of three constant domains (CHI . Cf-12 and Cf-13) and one variable domain (Vf-1). The heavy chains are covalently linked in the hinge region and the light chains are covalently linked to the heavy chain. The variable domains of both the heavy and li ght chains compose the antigen-binding part of the molecule. termed Fv. Within the variable domains there are three loops designated complementarity determining regions (CDRs) I. 2 and 3. which confer the highest diversity and defi ne the specificity of antibody binding. The Fe portion is glycosylated and contains the sites for interaction with effector molecu les. such as the C I complex of the complement system and a variety of Fe receptors including the neonatal Fe receptor (FeRn).

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Paul Ehrlich through his famous 'Seitenketten-Theorie' (side chain theory)

explained the antigen-antibody interactions, a century ago (1900) and later

coined the imaginative phrase 'magic bullets' for antibodies that target and

neutralize their antigens. Seventy-five years later, Georges Kohler and Cesar

Milstein (1975) invented a means of cloning individual antibody molecules, thus

paving the way for tremendous advances in the fields of cell biology and clinical

diagnostics. Antibody-secreting hybridomas are derived from a myeloma cell that

can grow indefinitely and an immune 8 lymphoblast expressing a specific

antibody gene. The hybridoma cells can be stored in liquid nitrogen for many

years making them virtually immortal (Goding, 1996). Attempts to use the same

technology for generating human mAbs have been hampered by the lack of a

suitable human myeloma cell line. Till date the best results were obtained using

heteromyelomas (mouse X human hybrid myelomas) as fusion partners.

Alternatively, human antibody-secreting cells are immortalized by infection with

the Epstein-Barr virus (EBV). However, EBV-infected cells are very difficult to

clone (James et al, 1987) and usually produce only low yields of immunoglobulin.

An alternative strategy has been developed and used to generate human

monoclonal antibodies by the replacement of the mouse antibody gene repertoire

with that of humans is generation of transgenic animals (Fishwild, et al. 1996;

Mendez, et al.1997; Green, 1999; Tomizuka, et al. 2000). Transgenic mice have

been shown to be able to produce functionally important human antibodies with

very high affinities, after immunization. Cloning and production can be carried out

using the usual hybridoma technology. For example, high-affinity human mAbs

obtained against the T -cell marker CD4 are potential therapeutic agents for

suppressing adverse immune activity (Fishwild, et al. 1996). Another human mAb

with an affinity of 5 X 1 o-11 M for human epidermal growth factor receptor (EGFR)

has been shown to prevent formation and eradicate human epidermoid

carcinoma xenografts in athymic mice (Yang, et al. 1999).

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2.1.1 Antibodies: tools of biomedical research

Today biomedical research is inextricably linked to the use of antibodies although

it is only over the past 25 years that antibody production, characterization and

detection have come within easy reach of every researcher around the globe.

Antibodies are targeted reagents that recognize and bind to specific antigens.

Antigens, especially proteins, possess numerous epitopes, thereby producing

polyclonal antibodies following injection into an animal where each antibody in

the resulting antiserum is specific for a given epitope. A major breakthrough in

the production of large amounts of antibodies directed against a single epitope

came with the development of the hybridoma technology. Use of both polyclonal

and monoclonal antibodies has revolutionized many areas of basic research.

Ouchterlony double-immunodiffusion assay was the norm for antibody detection

until the mid-1970s. In 1976, Burridge demonstrated that specific antigens could

be detected directly in SDS-gels using polyclonal antibodies followed by

incubation with 1251-labeled goat anti-rabbit lgG and autoradiography. Then

in1979, Towbin and coworkers developed a technology of electrophoretically

transferring proteins from one and two dimensional gels to nitrocellulose sheets.

It made antibody detection simple and fast (a few hours of work). Once in the

nitrocellulose paper, the proteins could then be stained using a variety of antigen­

detection methods, including 1251-, fluorescein or horseradish peroxidase (HRPO)

labeled secondary antibodies. Immunofluorescence was developed in the 1940s

by Coons using antibodies directly conjugated to fluorescein (Coons, et al. 1942).

Antibody coupled fluorescencent tages are used in immunofluorescence

microscopy. Subcellular localization of antibody-antigen interactions by

immunofluorescence microscopy has led to important new insights into

structure-function relationships and protein-protein interactions within both the

nuclear and cytoplasmic compartments.

2.1.2 Antibody Diagnostics

Antibody-based immunoassays are the most commonly used type of diagnostic

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assays and are one of the fastest growing technologies for the analysis of

biomolecules. The first major antibody-based immunoassay was the competitive

binding assay, using radioisotope (Berson and Yalow 1959) and later enzyme

labeled (Engvall and Perlmann 1971) immunoassays. This paved the way for a

tremendous expansion in the use of these technologies, particularly in biomedical

research and clinical chemistry. Previously polyclonal antibodies were used in

these assays but the advent of monoclonal antibodies added high specificity into

these assays making them more and more reliable. Wide (1971 ), developed a

method which first allowed to captured the antigen using a solid-phase-bound

antibody and then quantification was done using a second labeled antibody. This

two-site or sandwich immunoassay (Belanger et al. 1975; Maiolini and Masseyeff

1975), increased the specificity compared with the previous simple single-site

assays.

A feather in the hat of immunodiagnostics was added with the development of

the Fluorescence activated cell sorter (FACS) technique. The technique was

developed in 1969 (Hulett et al. 1969), but it was only by 1972, that scientists

began to use FACS to answer immunologically relevant questions. FACS

analysis and sorting studies using mAbs to define the surface markers on normal

and neoplastic cell populations created the basis for routine clinical diagnostic

assays that now range from leukemia classification to monitoring CD4 T cell loss

as HIV disease progresses. Protein microarray technology is also expected to

use either complete antibody molecules or their fragments to make the detection

chips.

2.1.4 Catalytic antibodies

The possibility of the use of antibiotics as catalysts (abzymes) was initially

suggested by Pauling in 1948. In 1969, Jencks proposed the hypothesis that

antibodies obtained during immunization with chemically stable transition-state

analogs could catalyze the corresponding chemical reactions. This was

independently confirmed by Lerner and his coworkers and Schultz in 1986. For

this abzymes were generated against the transition state analoges. Initially this

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approach was tried with acyl transfer reactions and simple hydrolytic reactions.

Phosphates, phosphonates and phosphonamidateswhich are known to be

potent inhibitors of some acyl transfer enzymes were choosen to be the suitable

transition state analogs (Wolfenden, 1976; Bartlett et al. 1983). An efficient

catalytic antibody was generated for the trans-esterification reaction in water

between sec-phenethyl alcohol and an enolic ester to form the corresponding

chiral ester (Wirsching et al, 1991 ). Strategies allowing incorporation of co-factors

into antibody combining sites further expanded the scope of antibody catalysis.

Apart from transition state analogs, antibodies were elicited to multisubstrate

analogs in which the binding site for the cofactor and substrate are generated in

a single immunization. This principle was used to achieve sequence-specific

cleavage of a peptide bond with Zn (II) as a cofactor (Iverson and Lerner 1989).

During the thirty-year period of directed synthesis of abzymes, antibodies

capable of catalyzing more than 100 different reactions have been generated.

A new strategy 'reactive immunization' (Barbas et al., 1997), provides a means to

select antibody catalysts in vivo on the basis of their ability to carry out a

chemical reaction. Designed reactive immunogens are used for immunization

and chemical reaction(s) such as the formation of a covalent bond which occurs

in the binding pocket of the antibodies during their induction. Aldolase antibodies

that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction,

with the enamine mechanism characteristic of natural Class I aldolase enzymes

have been generated by immunization of mice with 1, 3-diketone hapten-carrier

protein conjugate (Wagner et al., 1995).

2.1.5 Therapeutics antibodies

Antibody-based therapies are aimed at the elimination or neutralization of the

pathogenic infection or the disease target, for example, bacteria, viruses or

tumour targets. Therapeutic antibodies function by (a) blocking the action of

specific molecules, (b) targeting specific cells and (c) by functioning as signalling

molecules.

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Target specificity in the treatment and prophylaxis of diseases such as infection,

cancer and autoimmune disorders has become more viable through the

development of monoclonal antibodies. During the 1980s, resources were

directed towards the evaluation of the in vivo use of mouse monoclonal

antibodies in humans, aimed at both imaging and therapy (Larrick & Fry 1991 ).

But soon mouse monoclonal antibodies showed limited use as therapeutic

agents because of their short serum half-life, an inability to trigger human effector

functions and the production of human anti mouse-antibodies (Khazaeli, et al

1994) (the HAMA response). lmmunogenicity of mouse antibodies, was then

attempted to be minimized by the use of genetic engineering to generate

chimeric antibodies, that is antibodies with human constant regions and mouse

variable regions (Morrison, et al 1984; Boulianne, et al 1984 ). Unfortunately

chimeric antibodies also showed human anti-chimeric antibody responses

(HACA) (Bell and Kamm, 2000). Despite this fact four chimeric Mab's and a Fab

derived from a chimeric Mab, have reached the market as therapeutic agents

(van Dijk et al 2001; van de Winkel, 2001; Brekke, 2003; Sandlie, 2003). Further

minimization of the mouse component of antibodies was achieved through CDR

(complementarity-determining region) grafting (Jones, et al 1986). In such

'humanized' antibodies (90-95% human), only the CDR loops that are

responsible for antigen binding are inserted into the human variable-domain

framework. In another approach the use of mice which are transgenic for the

human lg locus gave promising results (Green, 1999). Immunization of such a

transgenic mouse results in a human antibody response, from which hybridomas

that produce human antibodies are generated. Abgenix of (Fremont, California),

was the first company to turn an ordinary mouse called XenoMouse into a

human antibody factory. According to the Abgenix's web site, 11 XenoMouse­

generated antibodies have moved into clinical trials, including ABX-EGF

(panitumumab ), an anticancer drug targeting the epidermal epidermal growth­

factor receptor. Medarex of (Princeton, New Jersey), have also developed a fully

human transgenic mouse platform. Medarex's HuMab-Mouse technology allows

for faster production of fully human MAbs. Today there are approximately 200

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antibodies in clinical trials and the US Food and Drug Administration has

approved several antibodies against cancer (Cragg, et al 1999; Farah, et al

1999), transplant rejection (Berard, 1999), rheumatoid arthritis and Crohn's

disease (Maini, et al. 1999; Sandborn, & Hanauer, 1999) and antiviral

prophylaxis (Saez-Liorens, et al.1998). So far, 20% of all biopharmaceuticals in

clinical trials are monoclonal antibodies, making this the second largest

biopharmaceutical product category after vaccines. As the development of new

potential therapeutic agents into commercial products takes about 10 years, the

FDA-approved antibodies (Table 2.1 ), and some of those in the end-stages of

development pipelines, are chimeric or humanized antibodies that were

developed with early antibody engineering technologies. The more recently

developed reagents, on the other hand, are completely human antibodies that

are derived from phage antibody libraries and transgenic mice Saez-Liorens, et

al.1998; Huls, et al.1999; Nagy, et al. 2002; Mukherjee, et al. 2002).

Table 2.1 FDA Approved Monoclonal Antibodies

Product Year approved Type of molecule Disease indication

OKT-3 1986 Murine (anti-CD3) Organ transplant rejection

ReoPro 1994 Chimeric Coronary intervention (anti-platelet gpllb/llla) and angioplasty

Panorex 1995 Murine (anti-EpCAM) Colorectal cancer (Germany only)

Rituxan 1997 Chimeric (anti-CD20) Non-Hodgkin's lymphoma

Zenapax 1997 Humanized Refractory unstable angina (anti-IL-2 receptor)

Herceptin 1998 Humanized Metastatic breast cancer (anti-ERBB2)

Remicade 1998 Chimeric (anti-TNF-a) Crohn's disease

Simulect 1998 Chimeric Kidney transplant rejection (anti-IL-2 receptor)

Synagis 1998 Humanized Respiratory syncitial viral (anti-F-protein) disease

Mylotarg 2000 Humanized Chemotherapy for acute (anti-CD33) myeloid leukemia

Cam path 2001 Humanized B-cell chronic lymphocytic (anti-CD52) leukemia

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Zevalin 2002 Mouse (anti-CD20) B-cell non-Hodgkin's lymphoma

Xolair 2002 Humanized Allergy (Australia only) (anti-lgE Fe)

2.2 Single chain variable fragments (scFv)

Single chain antibody variable (V) regions (scFvs) are novel recombinant

proteins composed of two antibody variable regions (from the light (L) and heavy

(H) chains, VL and VH, respectively) linked to each other by a peptide of

approximately 15 to 25 amino acid residues, such that a continuous polypeptide

chain is formed (Bird, et al 1988; Huston, et al 1988).

The idea that the variable regions of an antibody could be tethered from the C­

terminus of one chain to the N-terminus of the other, without disrupting antigen

binding, originated from an understanding of the structure of antibody variable

regions, and the underlying requirements for accurate folding and function.

Analysis of the sequence data on the variable regions of immunoglobulins by

Kabat and his colleagues (Kabat, et al 1983, 1987) indicated that each variable

region consisted of three hypervariable regions for the heavy and light chains,

while the remainder was relatively less variable. The hypervariable regions were

proposed to form the antigen-binding portion of the molecules with the less

variable region acting as the structural framework. Subsequent elucidation of the

crystal structures of several antibody variable regions showed that the framework

variable regions fold into almost identical structures (the antibody folds), and the

hypervariable regions bind the antigen (Kabat, et al 1987). Each domain of the

variable region was found to fold into nine strands of closely packed ~-sheets.

This overall folding pattern was preserved from one antibody to another, even

though there was variation in sequence of the variable domains, particularly in

the hypervariable regions (Padlan, 1977; De Prevdl, and Fougereau, 1976). The

less variable framework sequences determined the folding of the variable domain

and the hypervariable sequences were found in loops at one end of this domain.

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The hypervariable sequences of each domain were thus shown to occur at the

ends of ~-strands at the same side of the molecule. In cases where the structure

of antibody- antigen co-crystals have been solved, these aligned hypervariable

sequences were shown to make contacts with the antigen. This information

allowed a more complete way of humanizing rodent antibodies by replacement of

the V-region framework, relying on the architecture of V domains as a framework

of ~-sheets topped with antigen binding loop.s (Kabat, et al 1987). By grafting the

loops, the antigen binding site could be transferred from a rodent to a human

antibody. This triggered a cascade of reports of engineering of whole antibody

molecules for diagnostic and therapeutic applications. Further developments lead

to the cloning of the antibody genes from lymphocytes of immunized mice, their

expression in bacteria and screening of specific antibodies via antigen binding

(Ward, et al 1989; Huse, et al 1989). Fv fragments, non-covalent heterodimers

of VH and VL domains were less stable and prone to dissociation. Stable Fv

fragments were then engineered either by linking the domains with a hydrophilic

and flexible peptide (Bird, et al 1988; Huston, et al 1988) or by introducing

disulphide bonds between the domains (Giockshuber, et al 1990).

Since scFvs came to existence in late 1980s, they have been put to test in every

field wherever whole antibody molecules are being used. Their novel

characteristics related to their being smaller in size with the same specificity as

their parent antibody have made them have higher priority over whole antibody

molecules in many areas. Single-chain Fvs are finding applications in many

areas, having been raised against haptens, tumour associated markers, cardiac

myosin, viral proteins, major histocompatibility complex (MHC) molecules, fibrin,

nucleic acids, and plant proteins.

2.2.1 scFv diagnostics

The advent of cloning and expression of antibody fragments [like Fab (antigen­

binding fragment of immunoglobulin) or scFv] has so far not had a great impact

on immunoassay technology. There is, for example, no direct need for human

antibodies in assay development, which has been one of the main obstacles for

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the use of antibodies in immunotherapy and conventional mouse hybridoma

technology is working very well for assay development. The possibilities in

antibody engineering are, however, tremendous and have an effect on the

development of antibodies for immunoassays, as it permits changes in the affinity

(Marks, et al. 1992; Soderlind, et al. 1999) and fine specificity (Ohlin, et al. 1996;

Hemminiki, et al. 1998) of antibodies, and the expression of antibody fragments

as fusion proteins coupled to marker molecules (Casadei, et al. 1990).

Antibody engineering might also affect assay development as a result of the

introduction of antibody molecular libraries (Borrebaeck, 1998). A molecular

library is a body of entities designed to handle large numbers of molecules

efficiently at the same time. When Smith and coworkers (Smith, 1985)

demonstrated the use of the filamentous fd phage as a display vehicle, the

potential of phage libraries was fully realized. Furthermore, novel antibody library

designs (Soderlind, et al. 2000; Sblattero and Bradbury, 2000; Knappik, et al.

2000; Jirholt, et al. 1998) constantly fuel the assay development arena with

powerful approaches to increase the performance of antibody-based analysis.

The major advantages of phage display in immunoassay development are that it

is possible to obtain rare specificities, for example, against conserved epitopes

and carbohydrates (Soderlind, et al. 2000) from any specie and especially since

the antibodies have not gone through negative selection in an animal. The latter

point makes it possible to isolate antibody specificities that could not have been

obtained by conventional approaches, such as hybridoma technology. One

example of this was demonstrated by Nissim et al (1994) who made phage

antibodies against BiP (heavy-chain-binding protein), which cannot normally be

generated as they are held up in the endoplasmic reticulum of B cells. scFvs

have also replaced primary antibodies from tissue section detection systems. A

scFv, faithfully mimicing the specificity of the parental antibody makes a useful

tool for identifying cells in tissue sections with new means of identifying bound

scFv. This is achieved simply by epitope tagging, (Kolodziej and Young, 1991 ).

scFs have been generated with both carboxy- terminal and amino-terminal

recognition tags. For example, scFv derived from the anti-hen egg lysozyme

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antibody 01.3 comprises NH2VH o1.3-linker-VK ou-c-myc peptide-C02H

(McCafferty, et al 1990). The monoclonal antibody 9EIO (Evan, et al 1985)

recognizes the fused c-myc peptide, and thus form part of the detection system.

Another scFv, made from mAb H 1 '7E2 (Travers and Bodmer 1984) has been

produced (c-myc-epitope-tagged) which recognizes the tumor-associated marker

placental alkaline phosphatase (PLAP) (Savage, et al 1993). Because an scFv,

lacks the CH1, hinge, CH2. and CH3 domains of the parental lgG the potential of

undesirable cross-reactions are reduced and the new detection tag peptide

detect tissue layer (for PLAP) with high specificity. scFvH17E2.2 recognises

H.Ep#2 tumor cells (which express PLAP), in cytospin preparations by detection

of bound c-myc-epitope-tagged scFvH17E2 with mAb 9EIO. In frozen tissue

sections from H.Ep#P tumor xenografts grown in nude mice, tumor islands of

PLAP-positive cells are detected with this new scFv detection peptide. For /·.;\1 er ::r ~ ...... ~ .rr • .

~ diagnosis of tumor sections a different approached has been taken. An scFv, /e( ~ c:J-. recognizing the hapten, 4hydroxy-3 nitrophenylacetic acid (NP) and its iodinated \·~;\ 5. ~ derivative, 4hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) (Spooner, et al 1993) \(!~~\-~~~--

\ "-..::...: .. _ .. :r: has been made. The molecule comprises the anti NP/NIP VH domain from the

~ mAb B1.8 and the VA domain from the mouse plasmacytoma J558L. Since it is

easy to conjugate NIP to antibodies hence scFVNP (NH2-VHNp-linker-VA. JssaL­

C02H) has been used to develop a model system for an in vivo two step

targeting strategy. It is used as an universal detection secondary reagent for in

vitro diagnosis. For example, human colon adenocarcinoma LoVo cells,

(Drewinko, et al 1976) grown in the wells of a microtiter plate, were incubated

with mAb AUA1, (Arklie, et al 1981) NIP38-AUAI (AUAI conjugated with 38

molecules of NIP), or NIP35-HMFGI [mAb HMFGI (Taylor-Papadimitriou, et al

1981) conjugated with 35 molecules of NIP]. Previously, bound material was

detected by sheep anti-mouse antisera conjugate to horseradish peroxidase

(Sham-HRPO) or by serial incubation with commercially available biotinylated

goat anti-mouse lambda (GamX) and streptavidin-HRPO. When ScFvNP protein

was used as a secondary detection reagent hapten-derivatised NIP38-AUAI was

recognized, but non-hapten- conjugated AUAI was not. Bifunctional scFv-fusion

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molecules capable of binding antigen and also of interacting with standard

immunohistochemical reagents have been shown to have value for diagnostic

pathology. An antigen-specific scFv, fused with an enzyme activity, is an

example of a new type of immunohistochemical reagent that is capable of

detecting an antigen (which is routinely used for histopathological diagnosis) on

cell or tissue samples in one step. Thus gene encoding an scFv, derived from

mAb FRP5, specific for the extracellular domain of the c-erbB-2 receptor, has

been fused to the E coli phoA gene that encodes for bacterial alkaline

phosphatase."' The resultant product, scFv FRP5 -PhoA, binds the human erbB-2

protein, albeit with reduced affinity, and is detected by incubationwith a substrate

for alkaline phosphatase.

In other highly sensitive applications scFvs are derivetized with biotin since the

water-soluble vitamin biotin has very high affinity (RI = 1 o-15M) for avidin. Once

formed, the association between avidin and biotin is resistant to extremes of pH,

buffer salts, organic solvent concentration, and other denaturing agents.

Commonly, one of the antibody detection reagent used for diagnosis is

derivatised with biotin and the signal is detected by incubation with a stable

preformed avidin:biotinylated enzyme complex (ABC) (Hsu, et al 1981 ).

Multivalent and multispecific antibodies with defined stoichiometry could provide

valuable tools for biological and medical research and for the diagnosis and

therapy of cancer. In another attempt a single chain antibody (scFv) was fused

with streptavidin. This chimeric protein, expressed by the vector pSTE-215

(plasmid for streptavidin tagged expression), forms tetrameric complexes, binds

with antigen and also contains the biotin binding site which is then used for

further complex formation (Diibel, et al 1995). The scFv fusion protein could be

purified by affinity chromatography using biotin analogues. Along with this the

scFv fusion protein was used for direct detection of its antigen in ELISA and

Western blots when stained with biotinylated horseradish peroxidase.

The immunoglobulin variable region genes of a murine anti-insulin lgG-producing

hybridoma were cloned into a bate rial expression vector (Lake, et al 1994 ). The

scFv bound the insulin with 3.5 times less affinity than the parent antibody. This

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study was actually aimed at determining the immunoglobulin variable region

residues involved in binding to insulin. Mutagenesis of the variable heavy chain

complementary determining regions (CDR) indicated that CDR1 and CDR2 were

important for binding to insulin. Position 99 in the CDR3 of heavy chain was the

critical position for binding of the scFv to insulin.

In the SimpliRED HIV test, the key murine lgG (monoclonal IC3/86; AGEN

Biomedical, Australia) which is directed against glycophorin A on the human

erythrocyte surface has been reduced to an Fab fragment and linked to

immunodominant HIV peptide epitopes. These reagents are able to detect

circulating serum antibodies against HIV. One group replaced the Fab molecule

from this detection kit and (Lilley et al .. 1994) demonstrated that E. coli-produced

IC3/86 scFv peptide epitope fusions can, in the presence of specific antibodies,

effectively mediate the agglutination of human erythrocytes in whole blood.

Recently scFv recognizing DNA damage has been reported. Variable regions

were amplified from hybridoma cells expressing monoclonal antibody C3B6,

recognizing the thymidine( 5-4 )thymidine [T(6-4 )T] photoproduct. These scFvs are

useful tools for molecular recognition and structure-activity investigation (Zavala,

et al 2000). scFvs are being reported also against cytokines for, e.g. Laman et al

(2002) have reported the production of scFvs against human granulocyte colon

stimulating factor (G-CSF) using a murine scFv combinatorial library.

Antibody microarrays (one emerging class of proteomic technologies) having

broad applications in proteome analysis, disease diagnostics and quantitative

analysis. Compared to DNA microarrays, protein targets have significantly more

complex interactions with their ligands such as antibodies. Introduction of

antibody microarrays for clinical diagnostics is challenging the conventional

immunoassays. Now several companies are working to bring antibody chips to

the market. BD Biosciences Clontech (Palo Alto, CA) developed a monoclonal

antibody chip containing 500 monoclonal antibodies immobilized onto a glass

surface, allowing for the comparison of 500 proteins in two biological samples.

The Antibody Arrayk systems from Hypromatrix (Millbury, MA) contain hundreds

of high quality antibodies against well-studied proteins. The ideal proteomics-

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based analytical tool would consist of a microarray of a large number of high

affinity, high specificity protein ligands, and one for each protein in the proteome

of interest (Borrebaeck, 2000). Although several tens of thousands of monoclonal

antibodies are commercially available, this number is insufficient for large-scale

protein profiling. Also, natural antibodies are too large, heterogeneous, and it is

hard to create a large comprehensive set. Therefore, recombinant antibodies are

more promising (Tomlinson, et al 2001 ). Formats based on the antibody scaffold

include single chain antibody fragments (scFv) or Fab fragments (Hallborn,and

Carlsson, 2002). Because of the potentially high affinity and specificity,

recombinant antibodies and their small molecular weight, dense and oriented

attachment to a support surface is facilitated (Borrebaeck, 2000; Kusnezow,

Hoheisel, 2002). Hence recombinant antibodies based on a single molecular

scaffold, which are readily available in large display libraries, particularly meet

the requirements of being used as probe for antibody microarrays, which are

needed for global proteome analysis (Steinhauer, et al 2002). Cambridge

Antibody Technology (CAT) (Cambridge, UK) and Dyax (Cambridge, MA, USA)

are making shorter versions of antibodies using an established technique known

as phage display. With this technique, CAT can screen about 20,000 different

antibody fragments per month (Service, 2001 ).

2.2.2 Catalytic scfvs

Catalytic antibodies are fairly well established in the field of chemistry. Since their

discovery in the mid-1980s (Tramontano et al., 1986; Pollack et al., 1986), a few

exceptions apart, or lately developed new immunization strategies (reactive

immunisation as described by Barbas et al., 1997), anti-hapten catalytic

antibodies are obtained through immunisation against a stable transition state

analogue or via the switch-and-bait strategy. In any case, screening (of

monoclonal antibodies, mAbs) or purification (of polyclonal antibodies) is

routinely based on the binding abilities of the antibodies towards these haptens

and not according to their catalytic activity. This screening method can also

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prevent selection of catalytic antibodies of higher efficiency (comparable to those

of enzymes), since in these strategies, antibodies are raised and screened for

their main property, direct binding, but aimed at a totally different activity:

catalysis. It is observed many times that transition state analogue binding and

catalytic activity are definitely not related (Baca et al., 1997; Taran et al., 1998;

Desilva et al., 2000). Among the selected mAbs, higher catalytic efficiency is

often borne by the poorest binders. A simple solution would be to screen the

antibodies directly for their catalytic activity at the earliest stages. Some direct

screenings for the catalytic activities have been undertaken and many interesting

techniques have been found in order to elaborate efficient high-throughput

screenings for catalytic activities. Yet, despite many efforts (Geymayer et al.,

1999; Klein and Reymond, 2001 ), these strategies did not prove to have wide

applicability. Almost all catalytic antibodies produced to date have been isolated

as hybridoma-derived monoclonal antibodies. This method of producing catalytic

antibodies has a number of advantages, especially in the ability to produce large

amounts of pure antibody from ascetic fluids. However, hybridoma technology

cannot be used directly to prepare mutants in experiments designed to improve

the catalytic activity of an existing antibody or to probe its mechanism (Stewart et

al., 1995). Therefore, to evolvethe already made abzymes towards increasingly

higher catalytic activity, their differential affinity must be maximized. However an

immune response solely against TSA (transition state analog) makes it difficult to

control antibody recognition of both molecular species (the transition state and

the ground state of the substrate), and thus optimize the differential affinity.

Phage display technology to evolve an abzyme in vitro on the basis of the

evolutionary dynamics of enzymes provided the solution to this issue. scFvs

show a smaller version of the whole antibody molecule with the same binding

specificities. The relatively small size of the molecule makes it an attractive target

for structural studies by X-ray crystallography and nuclear magnetic resonance.

scFv proteins are expressed efficiently by bacteria and, in general, retain the

antigen binding characteristics of the parental antibody from which they have

been derived (Huston et al., 1988; Bird et al., 1988). The first construction and

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characterization of catalytic scFv was reported by Gibbs et al. (1991 ). This

protein was expressed in E. coli as inclusion bodies and showed the same

catalytic parameters as the parent monoclonal antibody after refolding. Using the

transition state analog (TSA) p-nitrobenzyl phosphonate as a hapten, Tawfik et

al., (1997a) generated esterolytic antibodies which efficiently hydrolyse both the

p-nitrobenzyl and p-nitrophenyl esters in addition to binding to their respective

phosphonate esters. A group of these monoclonal antibodies has been found to

display one of the highest esterase activities demonstrated thus far. Antibody

phage libraries can be made from both immune and nonimmune sources.

Chemical selection with reactive compounds using antibody phage libraries in

vitro allows for the access to catalytic antibodies that are not limited by animal

sources or immune responses. Aldolase antibodies are broad in scope, the

efficiency with which any given aldol is processed varies significantly. To access

aldolase antibodies with altered substrate specificity and turnover, a strategy

based on screening designed phage libraries, using different diketone derivatives

was developed. In this approach, libraries were prepared by recombining the

catalytic machinery of aldolase antibodies with a naive V gene repertoire (Tanaka

et al., 2000). This strategy was used for preparing catalysts that efficiently

processed cyclohexanone-aldols since retro-aldol reactions are relatively slow

compared to those involving acetone-aldols. Mechanism-based inhibitors react to

form covalent adducts with the enzymes that process them along a defined

reaction pathway allowing for the direct selection of catalytic antibodies that

utilize particular features of a designed mechanism. Phage display systems

facilitate this approach since phage that display the desired catalytic antibody

can be directly trapped after reaction with a mechanism-based inhibitor allowing

for effective searches through large combinatorial libraries. A mechanism-based

inhibitor of ~-lactamase, keyhole limpet hemocyanin (KLH) conjugate of a penam

sulfone, KLH-2 was used to obtain ~-lactam hydrolytic antibodies (Tanaka et al.,

1999a). Reactive oxygen species, including the superoxide anion (02 -), H20 2 ,

organic peroxides and the hydroxyl radical, are normal products of aerobic

metabolism known to destroy key biological molecules and cause damage to cell

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membranes (Vaughan, 1997). Examples of such oxidative-stress-related

diseases include reperfusion injury, brain ischaemia, tumour, cataract and

various types of inflammation and physiological aging. Living organisms have

evolved a family of antioxidant enzymes to cope with oxidative stress, including

superoxide dismutase, which catalyses the dismutation of 02- to H202, the

selenium containing enzyme glutathione peroxidase (GPX), which catalytically

destroys hydroperoxides, and catalase, which catalyses the breakdown of H202.

The GPXs are substantially more efficient on a molar basis than other enzymes

(Michiels et al, 1994). A selenium containing scFv that mimics glutathione

peroxidase (Se-scFv2F3) has been cloned by gene engineering methods (Ren et

al, 2001 ).

2.2.3 scFv therapeutics

Phage-display libraries of human antibody fragments is today the most used and

established technology for the development of new human antibodies. One of the

great advantages of the library approaches is that they allow for the selection of

antibodies of high specificity and affinity towards a variety of different antigens.

Another advantage is the ability to select specific antibodies against toxins,

drugs, cytokines and other targets that cannot, for various reasons, be injected

into an animal to raise an immune response, as the target might kill the animal

and that it might not be immunogenic at all.

As the development of potential new therapeutic agents into commercial

products takes about 10 years and some of those in the end-stages of

development pipelines, are chimeric or humanized antibodies that were

developed with early antibody engineering technologies. The more recently

developed reagents, on the other hand, are completely human antibodies that

are derived from phage antibody libraries and transgenic mice. These antibody­

derived fragments do part of the job normally performed by intact antibodies,

such as blocking the action of a toxin, blocking the interaction between a cytokine

and its receptor. These antibody fragments can also carry effector molecules to

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their targets. The blocking effect of antibodies simply requires the inhibition of

subsequent ligand or receptor binding, and does not necessarily require effector

functions. A protective toxin-neutralizing effect has recently been described in the

neutralization of botulinum neurotoxin by antibody fragments. The anti-botulinum

toxin antibodies were derived from different phage-display libraries obtained from

humans or immunized mice (J. Marks, 2002). In another attempt, Maynard and

colleagues described high affinity antibodies against Bacillus anthracis (Maynard,

et al. 2002). These antibodies were fragments derived from variable chain genes

of a mouse monoclonal antibody and expressed in E. coli as single-chain Fv

fragments (scFvs). Administration of these antibody fragments to mice before

injection of anthrax toxin, showed protection against the toxin.the use of

polyclonal passive human serum therapy is a routine practice in treating

infectious diseases. If in this approach monoclonals are to be used,

administration of highly specific and high-affinity monoclonal antibodies is

required, which has the limitations of production costs associated with

manufacturing of these human antibodies. Here, blocking antibody fragments

synthesized in E. coli, offers a possible solution to this problem which could be

used to combat, for example, viral infections.

Tumour-necrosis factor-a (TNF-a), interleukins and complement proteins are

cytokines associated with inflammation and autoimmune diseases. Humanized

antibodies are already in us.e in their treatment and now scFvs are also available

as an alternative. Adalimumab, anti-TNF-a antibody, is the first phage-display­

derived human antibody brought into the clinic, generated by 'guided selection'

using a mouse monoclonal antibody (Jespers, et al. 1994 ). The method is based

on the selection of a human variable-domain repertoire coupled to one of the

original mouse variable domains, so as to 'guide' the human variable domain

repertoire towards the same specificity as the original mouse variable domains.

This antibody has been affinity optimized by iterative rounds of selection and

mutagenesis. Adalimumab has completed Phase Ill clinical trials and is currently

in registration for FDA approval. J695 (Cambridge Antibody Technology) is

another human antibody derived from a phage-display library against interleukin-

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2, in clinical trials at present and have shown potential in the treatment of

inflammatory diseases.

I While clinical results are showing positive results with new monoclonals, genetic

engineering and combinatorial library technology have provided a new

generation of antibody constructs. The first clinical test of this class of reagents

was reported with a single-chain Fv (scFv) antibody, MFE-23, detecting

carcinoembryonic antigen (CEA), with uptake into the tumor (Napier, et al. 1996).

Among various approaches of anti-cancer therapy one is the generation of

antibodies directed against the major histocompatibility complex (MHC) class II

proteins to specifically target and eliminate cancer cells. Monoclonal antibodies

have already been made following this approach. Recently, an anti- MHC class

II human antibody derived from an antibody phage-display library was shown to

induce apoptosis of activated lymphoid cells (Nagy, et al. 2002). Antibody

fragments have shown to be better than the complete antibodies in penetrating

the tissue for effective antibody targeting. According to recent research high

affinity fragments are retained in the periphery of the tumour, whereas the

medium-affinity antibodies penetrate throughout the tumour (Adams, et al. 2001 ).

Furthermore, bivalent low-affinity fragments penetrate better and more uniformly

than high-affinity fragments (Nielsen, et al. 2000). Since known antibody

fragments can be selected for optimal affinity and specificity by in vitro selection

processes (Schier, et al. 1996; Hanes, et al. 2000) combinatorial phage libraries

is again an advantage. However small antibody fragments also show rapid

clearance from circulation, and as a result the fraction of the injected dose that

reaches its target is at present too low for a therapeutic benefit, even for bivalent

fragments.

A new approach to increasing the half-life of antibodies, is the pegylation of

antibodies and antibody fragments, which simultaneously reduces their

immunogenicity (Chapman, 2002). The effect is achieved by chemical coupling

of polyethylene glycol (PEG) to amino groups in the protein structure. The main

property contributing to this effect is the increase in the size of the molecule

above the glomerular-filtration limit. However, modifications of amino groups

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within antibodies affecting the effector functions (Anderson, & Tomasi 1988) as

well as the antigen-binding capacity (Suzuki, et al. 1984) have also been

reported. Another alternative was to direct the PEG attachment to sites on the

antibody molecules that are distant from the CDR regions and crucial sites for

effector functions (Chapman, 2002). Indeed, an anti-TN F-a human Fab fragment

(COP 870; Celltech) had its circulating half-life prolonged to 14 days by site­

specific pegylation in the hinge region (Choy, et al. 2002).

The intracellular expression of recombinant antibody fragments e.g. Fab or scFv

molecules termed intrabodies is an exciting therapeutic approach. These single­

chain antibodies, synthesized by the cell and targeted to a particular cellular

compartment, are used to interfere in a highly specific manner with cell growth

and metabolism. The antibodies are directed to the relevant cellular compartment

using classical intracellular-trafficking signals. Single-chain antibodies targeted to

the lumen of the ER (endoplasmic reticulum), inhibit the transport across the

plasma membrane and thus secretion of proteins to the cell surface.

To date, this technique has been used to functionally inactivate three cell-surface

receptors that are implicated in human cancer. An ER-targeted intrabody was

used to down regulate the a subunit of the receptor for human interleukin 2, IL-

2Ra (Richardson, et al. 1995). IL-2Ra plays a key role in T -cell-mediated

immune responses, and is constitutively over expressed in some T- and B-cell

leukemias, most notably in adult T-cell leukemia. T-cell lines that stably

expressed an ER targeted single-chain antibody (scFvTac) against IL-2R{X

exhibited a complete loss of cell-surface IL-2R{X expression and were no longer

responsive to IL-2. The receptor chain was detected inside the cells as an

immature form that was sensitive to endoglycosidase H; this finding is consistent

with its retention in a pre-Golgi compartment (Richardson, et al. 1995).

lntrabodies, such as scFvTac, offer significant potential for immunomodulation

and for the control of IL-2R-dependent tumor-cell growth in vivo, especially when

used in combination with targeted gene-delivery systems that allow the genetic

manipulation of specific cell types (Salmons, and Giinzburg, 1993; Russell, et al

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1993; Michael, and Curiel, 1994 ). ErbB2 a member of the epidermal growth

factor receptor (EGFR)- related family of receptor tyrosine kinases is

overexpressed in a variety of human tumors, including breast and ovarian

carcinoma, where it correlates with an unfavorable prognosis. Two ER- targeted

intrabodies are being shown to markedly decrease the cell-surface expression of

ErbB2 in NIH3T3 fibroblasts that express an oncogenically activated form of

ErbB2 (Beerli, et al. 1994 ). A complementary study has shown the

downregulation of ErbB2 in the human ovarian-carcinoma cell line SKOV3

(Deshane, et al. 1994 ), using an ER directed anti- ErbB2 single chain antibody.

Transient expression of the intrabody gene in SKOV3 cells led to the loss of

ErbB2 expression at the cell surface, and induced a temporary arrest of cellular

proliferation. New single-chain antibodies have been made to perturb the function

of p21ras. a guanine nucleotide-binding protein that is strategically involved in the

control of cell growth and differentiation. Members of the ras gene family have

been implicated in many types of human cancer. When the mRNA encoding an

anti-p21 ras intra body was microinjected into Xenopus oocytes, it was shown to

inhibit insulin-induced meiotic maturation of the cell, a process known to be

p21ras_dependent (Biocca, et al. 1993; Biocca, et al 1994). lntrabodies that

interfere with the function of cytosolic kinases, GTPases or other molecules

involved in signal transduction could prove extremely valuable in unravelling the

complex and interconnecting pathways that serve to deliver extracellular signals

to the nucleus. Intracellular antibodies have important therapeutic potential in the

defence against microbial pathogens, particularly the human immunodeficiency

virus (HIV-1). The exploration of genetic approaches to combat HIV-1 infection has

gained impetus following the clinical failure of reverse transcriptase and protease

inhibitors (Condra, et al. 1995). Other approaches that were initially promising,

such as the use of soluble CD4 to block virus entry into cells, have similarly not

translated into an effective therapy. Whereas a single-chain antibody directed

against the HIV-1 exterior-envelope glycoprotein gp120 has been shown to

interfere with virus assembly in HIV-1-infected cells (Marasco, et al. 1993). The

envelope protein mediates the attachment of the virus to its cellular receptor (the

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CD4 molecule), and is required both for cell-free and cell-to-cell transmission of

the virus. Expression of the scFvl05 intrabody in the ER of infected cells led to

intracellular retention of the envelope precursor gp160, indicating that cell­

surface translocation and proteolytic cleavage of the precursor were inhibited as

a result of its interaction with scFvl05 in the ER. The biological consequences of

this were a marked reduction in the envelope-mediated cytopathic effect

(syncytium formation) and a drop in infectivity (by three orders of magnitude) of

the virus particles released (Marasco, et al. 1993; Chen, et al. 1994 ). Though

intrabody research is still in its early stages, with their in vivo efficacy being so far

untested, the in vitro experimental results make them an attractive candidate for

therapeutic use. This is true with other scFvs too that are under trial at the

moment and taken together definitely hold a better potential for the future.

2.3 Phage display

Technologies have been emerging for making antibodies in vitro by mimicking

the selection strategies of the immune system. Repertoires of antibody fragments

are displayed on the surface of filamentous bacteriophage, each displaying a

single antibody species; the phage is selected by binding to antigen; and finally

soluble antibody fragments are secreted from infected bacteria. As in the immune

system, the V genes can be subjected to random mutation, and mutants may be

selected with higher binding affinities. This allows the isolation of human antibody

fragments of defined specificity, against both foreign and self-antigens.

2.3.1 Selection technology

Phage display essentially mimics the functioning of a B cell. Filamentous phage

was first used to display small peptides by fusion to the minor coat protein (pill:

three or five copies per phage particle) (Smith, G.P. 1985). Two sites of pill are

used for fusion: in the flexible spacer between the two domains of pill (Smith,

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1985), or close to the N-terminus (Parmley and Smith, 1988) or the N-terminus

(Cwirla, et al 1990). The phage are enriched by binding of peptide monoclonal

antibody. Through growth of the enriched phage and further selection by binding

to antibody, very rare phage can be isolated (Smith, G.P. 1985). Folded antibody

fragments (McCafferty, et al 1988) and other proteins (Bass, et al 1990;

McCafferty, et al 1992) can also be displayed on phage. The antibody fragments

can be displayed as single chain Fv fragments, in which VH and VL domains are

connected on the same polypeptide chain by a flexible polypeptide spacer

(Huston et al., 1988; Bird et al., 1988), as Fab fragments, in which one chain is

fused to pill and the other is secreted into the periplasm (McCafferty, et al 1988,

Hoogenboom, et al 1991; Barbas, et al 1991; Garrard, et al 1991; Breitling, et al).

With fusions to the N-terminus of pill, phage remains infective (McCafferty, et al

1988, Hoogenboom, et al 1991 ). However, if the N-terminal domain of pill is

excised and fusions made to the second domain, the phage is not infective, and

wild type pill has to be provided by helper phage (Bass, et al 1990, Barbas, et al

1991; Garrard, et al 1991 ). The pill fusion and other proteins of the phage can be

encoded entirely within the same phage replicon (McCafferty, et al 1988, Smith,

G.P. 1985), or on different replicons (Bass, et al 1990; Collet, et al 1992). When

two replicons are used, the pill fusion is encoded on a phagemid, a plasmid

containing a phage origin of replication. Phagemids are packaged into phage

particles by "rescue" with a helper phage such as M 13K07 that provides all the

phage proteins, including pill, but due to a defective origin is itself poorly

packaged in competition with the phagemids (Vieira, et al 1987). The pill fusion

is often proteolysed. This is expected to give a population of phage particles,

each displaying zero, one, two, three (and perhaps four and five) antibody

fragments. The average valency of the population is further reduced by use of

helper phage, in which the helper pill competes for incorporation into the phage

particle. Such phage have been estimated on average to display less than a

single fusion protein per particle; they have been termed "monovalent" phage

(Garrard, et al 1991, Lowman, et al 1991 ). Other helper phages (M 13L1glll) that

lack pill have been designed to rescue phage particles that incorporate only the

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pill fusion from the phagemid; these are therefore multivalent (Griffiths, et al

1993). Hence use of different helpers alter the valency of the phages. The major

coat protein of the phage (pVIII: 3000 copies per phage particle) is also used to

display peptides and antibody fragments (Kang, et al. 1991; Huse, 1991; Chang,

et al. 1991; Huse, et al. 1992). Pentapeptides (ll'ichev, et al 1989; ll'ichev, et al

1990) and hexapeptides (Greenwood, et al 1991) are fused close to the N­

terminus of pVIII, but phage encoding longer peptides are not viable unless wild

type pVIII is provided (Greenwood, et al1991; Felici, et al1991). The phage

population is multivalent. With helper pVIII, up to about 900 peptides

(Greenwood, et al 1991) and 24 antibody fragments (Kang, et al. 1991) can be

incorporated per phage particle. Fusions to pill rather than pVIII have to date

been preferred for antibody display.

The immune system is capable of selecting one or more B cells from repertoires

of- 5 x 108 cells in mice and - 1012 cells in humans and is able to selectively

enrich for B cells displaying antibodies with slightly improved binding affinities,

allowing affinities to be built up in a step-wise manner through rounds of mutation

and selection. Phage selection appears to be at least, powerful as immune

selection. Phages displaying antibodies are selected by binding to antigen coated

plates (Barbas, et al 1991; Marks, et al. 1991 ), column matrices (McCafferty, et al

1988), cells (Marks, et al. 1993), or to biotinylated antigen in solution followed by

capture (Hawkins, et al. 1992). The phages bound to the solid phase are washed

and then eluted by soluble hapten {Ciackson, et al. 1991 ), acid (Barbas, et al

1991) or alkali (Marks, et al. 1991 ). Phages can be enriched 20-1000 fold by a

single round of selection (McCafferty, et al 1988; Garrard, et al 1991; Marks, et

al. 1991 ). Moreover, the enriched phages are grown in bacterial culture and

subjected to further rounds of selection. In this way, enrichment factors of only

50-fold in each round can build up to 107 fold enrichments over four rounds of

selection (Marks, et al. 1991 ). The efficiency of selection depend on many

factors, including the kinetics of dissociation during washing, and whether

multiple antibody fragments on a single phage can simultaneously engage with

(solid phase) antigen. Antibodies with fast dissociation kinetics (and weak binding

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affinities) shall be retained by use of short washes, multivalent display and a high

coating density of antigen at the solid phase. The high density not only stabilizes

the phage through multivalent interactions, but favor rebinding of phage that has

dissociated. Nevertheless, binding affinities (for a single antibody fragment) of

105 M-1 are barely sufficient to hold multivalent phage to solid phase (Ciackson,

et al. 1991 ). Conversely the selection of antibodies with slow dissociation kinetics

(and good binding affinities) is promoted by use of long washes (Bass, et al

1990), monovalent phages (Bass, et al 1990), and a low coating density of

antigen (Marks, et al. 1992). In principle, phages with very high affinities (> 1010

M-1) are difficult to elute, but a change in pH may suffice to dissociate the

complex (Lowman, et al 1991; Roberts, et al. 1992).

2.3.2 Technology for making V- gene repertoires

In the immune system the sequence diversity of antibody binding sites is not

encoded directly in the germline but is assembled in a combinatorial manner from

V gene segments. In human heavy chains, the first two hypervariable loops (HI

and H2) are drawn from less than 50 VH gene segments (Tomlinson, et al.

1992), which are combined with D segments and JH segments (Ravetch, et

al.1981) to create the third hypervariable loop (H3). This loop is exceptionally

variable in sequence and length (2-26 residues) (Wu, et al. 1993) because the

joining of the segments is imprecise, different reading frames of the D segment

may be used, nucleotides can be inserted and deleted at the junctions, and the D

segments can recombine as D-D fusions (Sanzl. 1991 ). In human light chains,

the first two hypervariable loops (L 1 and L2) and much of the third (L3) are drawn

from probably less than 30 V"A (Williams and Winter, 1993) and less than 30 VK

gene segments. These segments are combined with J"A and JK segments to

complete the third hypervariable loop (L3). This loop has limited variability. It

ranges in size from 7 to 11 residues in 'A light chains (Combriato and Klobeck,

1991) and is most commonly 6 residues in K light chains (Kabat, et al. 1991) but

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can vary between 5 and 8 residues. Thus, most of the sequence diversity (and

structural diversity) is encoded by the heavy chains.

Y- ~~;_u~ _l! ~P':!Hsi Tl'

F'olymerase chain reaction, with pnmers matching the 5' and 3' ends of

rearranged VH and VL genes, has provided the means to amplify, clone, and

express V-genes from lymphocytes (Orlandi, et al. 1989), thereby making diverse

V gene repertoires for expression. The V- genes are amplified from both eDNA

and genomic DNA, with back primers at the 5' end of the exon encoding the

mature V-domain and forward primers based within the J-segment (Ward, et al.

1989; Orlandi, et al. 1989). However, for amplificaton from eDNA, "back" primers

have also been based in the leader exon (Jones and Bendig, 1991 ), and forward

primers within the constant region (Sastry, et al. 1989). To maximize

complementarity, degeneracy is incorporated into the primers (Orlandi, et al.

1989; Sastry, et al. 1989), or different primers are designed for different families

of V genes (Marks, et al.1991 ). For cloning of the amplified DNA into expression

vectors, rare restriction sites are introduced within the PCR primer (Orlandi, et al.

1989), as a "tag" at one end, or by further PCR amplification with a tagged primer

(Ciackson, et al. 1991 ). "Primary" repertoires of genes harvested from a

lymphocyte population are likely to contain somatic mutations, although most

published human VH and VK gene sequences encode few (< 5) amino acid

substitutions (Tomlinson, et al.1992). Repertoires of "synthetic" rearranged V

genes have also been derived in vitro from V gene segments. Most of the human

VH-gene segments have now been cloned, sequenced (Tomlinson, et al.1992),

and mapped (Matsuda, et al. 1993); these cloned segments (including all the

major conformations of the H1 and H2 loop) have been used to generate diverse

VH gene repertoires with PCR primers encoding H3 loops of diverse sequence

and length (Hoogenboom and Winter, 1992). VH repertoires have also been

made with all the sequence diversity focussed in a long H3 loop of a single length

(Barbas, et al. 1992). Human VK and VA. segments have been cloned and

sequenced (Williams and Winter, 1993) and are therefore available for making

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synthetic light chain repertoires. Synthetic V gene repertoires, based on a range

of VH and VL folds, and L3 and H3 lengths, encode antibodies of considerable

structural diversity.

Combining the VH and VL repertoire

Repertoires of antibody fragments have been constructed by combining VH and

VL gene repertoires together in several ways. Each repertoire can be created in

different vectors, and the vectors recombined in vitro (Hogrefe, et al. 1993) or in

vivo (Waterhouse, et al. 1993); alternatively, the repertoires may be cloned

sequentially into the same vector (Barbas, et al. 1991) or assembled together by

PCR and then cloned (Ciackson, et al. 1991 ). A technique of "in-cell PCR

assembly" has also been used for combining the VH and VL genes within the

lymphocyte by PCR, and then cloning the repertoires of linked genes (Embleton,

et al. 1992). Repertoires of VH domains have also been combined with a single

VL gene (Hoogenboom and Winter, 1992; Barbas, et al. 1992). The route by

which repertoires are combined dictates the structural diversity and repertoire

size. For example, combining VH and VL repertoires in vivo, by combinatorial

infection (Waterhouse, et al. 1993), allow the creation of libraries of > 1012

different VHNL combinations.

Advantage of Immunization

Immunization increases the number of cells making an immune response,

especially the levels of mRNA. Resting B cells make about 100 copies of lg

mRNA per cell, whereas a hybridoma (and also presumably a plasma cell)

makes about 30,000 copies (Schibler, et al. 1978). Spleen, lymph nodes, tonsils,

and bone marrow (but not peripheral blood lymphocytes) provide a rich source of

plasma cells and lg mRNA. Repertoires of VH or VL genes amplified from the

mRNA of spleen cells of an immunized mouse are therefore greatly enriched in V

genes encoding part of an antigen binding site (Hawkins and Winter, 1992). In

random combinatorial libraries (Huse, et al. 1989), the VH and VL gene

repertoires are combined at random, and the original combinations of the

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immune lymphocyte are destroyed. Nevertheless, the artificial pairings from

phage display libraries, enriched by immunization, can provide antibody

fragments with good affinities. Antibody fragments have also been isolated from

immunized humans with binding activities against several viral antigens, for

example, HIV gpl20 (Barbas, et al. 1992; Barbas, et al. 1993), respiratory

syncytial virus (RSV) (Barbas, et al. 1992), and hepatitis virus (Zebedee, et al.

1992).

By-Passing Immunization (natural repertoire)

Human peripheral blood lymphocytes (PBLs), provide a diverse source of

rearranged V genes by using "family based" PCR primers to amplify each of the

human VH, VK, and V~ families (Marks, et al. 1991 ). The repertoires of VH and VL

genes are combined at random, destroying the original combinations and

specificities of the PBLs and generate new specificities (Marks, et al. 1991 ).

From this library, it is possible to isolate phage with binding activities against

many different antigens. For example, antibodies were isolated against the

foreign antigens bovine serum albumin (BSA), turkey egg lysozyme, the hapten

ph Ox (Marks, et al. 1991 ), and bovine thyroglobulin (Griffiths, et al. 1993),

against the human self-antigens tumor necrosis factor a (TNF-a), thyroglobulin,

a monoclonal antibody, carcinoembryonic antigen (CEA), mucin and CD4

(Griffiths, et al. 1993).

By-Passing Immunization (synthetic repertoire)

Synthetic V gene repertoires are build from cloned human Vwgene segments. A

repertoire (2 x 107 clones) was first constructed using a short H3 loop of five or

eight random residues with each of 49 VH segments, and combined with a fixed

light chain. Antibodies of high specificity were selected against two haptens,

phOx and NIP (with affinities of up to 106 M-1) and human TN F-a (Hoogenboom,

and Winter, 1992). By adding a range of H3 loops of different lengths, up to 12

residues, a single library was created from which a range of more than 20

binding specificities were selected, including against haptens; the foreign

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antigens lysozyme, keyhole limpet haemocyanin, streptavidin, and

immunoglobulin binding protein (BIP); and the self-antigens, the oncogene

protein rhombotin and the tumor suppressor protein p53. The epitope of an

antibody binding to p53 was mapped and found to be new. The antibodies

appeared to be specific and could be used as reagents for immunofluorescence

staining of p53 in the nuclei of cells, and for Western blotting of cell lysates for

BIP.

2.3.3 Phage antibody screening procedures

It is necessary to screen large numbers of antibodies to identify those variants

with the most optimal characteristics. The best screening assays are fast, robust,

amenable to automation (e.g. 96-well format), and use unpurified phage

antibodies, or the soluble antibody fragments from the bacterial supernatant.

Binding of poly- or monoclonal phage antibodies to the antigen has been tested

with diverse assays, ranging from a simple ELISA with coated antigen (Marks, et

al. 1991) to bioassays that screen for direct neutralization upon binding, and

whole cell ELISA or flowcytometry. Typically, for a first screen, ELISA-based

assays are used in combination with restriction-fingerprinting of the antibody­

DNA to identify different clones (Marks, et al. 1991 ). Further, specificity of

antibodies is tested using immunoprecipitation (de Wildt, et al. 1996) or

immunocytochemistry or histochemistry (Van Ewijk, et al. 1997; Carnemolla, et

al. 1996). To speed up screening procedures, phagemid vectors that incorporate

a dual purpose have been developed. These allow both monovalent display of

antibody fragments and the production of soluble antibody fragments for

screening without the necessity to subclone the antibody V-genes. In such

systems, an amber codon is positioned between the antibody and pill genes

(Hoogenboom, et al. 1991 ). A variety of tags have been described that can be

appended to the antibody fragment for detection, including the myc-derived tag

recognized by the antibody 9E1 0 (Marks, et al. 1991 ), and the Flag sequence

(Lah, et al. 1994; Lindner, et al. 1997). This set-up allows the use of unpurified

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phage antibodies or antibody fragments, present in crude supernatant or

periplasmic extracts, for screening assays. For example it is possible to fuse, in

between the antibody and gill, a histidine-encoding tag, for purification of

antibodies using Immobilized Metal Affinity Chromatography (Hochuli, et al.

1988; McCafferty, et al. 1994 ).

2.3.4 Making High Affinity Antibodies

Mutations

In the immune system, the higher affinity antibodies are made after repeated

rounds of immunization, arising either as mutants of a primary response

antibody, or as entirely new antibodies (Such antibodies may arise by somatic

mutation of very low affinity antibodies (Berek and Milstein, 1987). The increase

in binding affinity of primary response antibodies sometimes modest, with anti­

NP hybridoma showing a five-fold improvement in affinity (Cumano and

Rajewsky, 1986), or large, with anti-phOx hybridomas showing improvements of

1 00-fold (Foote and Milstein, 1991 ). Site-directed mutagenesis of an anti­

pazophenylarsonate antibody suggests that somatic mutation at a few sites can

together contribute to an improvement factor of > 200 to binding affinity (Sharon,

1990). In phages, antibody fragments are designed with higher binding avidities,

for example, as single chain dimers (Griffiths, et al. 1993) or "diabodies"

(Holliger, et al. 1993). Presumably other multimeric fragments could be designed

to mimic lgM. Furthermore, mutations are introduced at random in vitro (Hawkins,

et al. 1992; Gram, et al. 1992) using error prone polymerase (Leung, et al. 1989)

or in vivo by use of mutator strains of bacteria (Schaaper, 1988; Yamagishi, et al.

1990), and the phage are selected for higher affinities. To make higher affinity

mutants, it is desirable most of the times to increase the frequency of random

mutation or to combine rounds of mutation and selection, for example, by

growing phage in bacterial mutator strains. Alternatively, it is recommended to

start with lower affinity antibodies (as may occur in repertoire shift), in the event

that a higher affinity binding site is trapped at a local optimum and becomes

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incapable of further affinity maturation (Macken and Perelson, 1989). Phage

display appears to have potential advantages over the immune system for the

creation of secondary (mutated) repertoires. Firstly, the size of the secondary

repertoires can be much larger than in immune systems. Secondly, random

mutation can be focused to the antigen binding loops or outside, for example, at

framework residues that influence loop conformation (Foote and Winter, 1992).

Indeed, mutations outside the contact surface with antigen can often have

profound effects on binding affinity (Sharon, 1990; Lavoie, et al. 1992).

Chain Shuffling

Chain shuffling was first used to analyze the promiscuity of VH and VL pairings in

repertoires from immunized mice (Ciackson, et al. 1991; Kang, et al. 1991 ). It

was then used for the affinity maturation of a human antibody fragment (affinity 3

x 106 M-1) for phOx isolated from a V gene repertoire. The VH gene was paired

with VL genes from the original repertoire, and the new (light chain shuffled)

repertoire was displayed on phage. A light chain partner was isolated that

conferred improved binding affinity (6 x 107 M-1). Likewise the new VL gene was

paired with the original repertoire of VH genes, (but now combined with the H3

loop of the original VH gene), and after selection a fragment was isolated with a

further improved affinity (1 09 M-1 ). Indeed the affinities of the original and shuffled

fragments were found similar to those of mouse hybridomas of the primary and

later responses to the same hapten. The 20-residue changes suggest that large

changes in affinity (500-fold here) require many random mutations (Marks, et al.

1992). Chain shuffling is therefore used to tap the somatically mutated V genes

and make higher affinity binding sites. However, chain shuffling is also used for

more extensive diversification. For example, the heavy and light chains of mouse

monoclonal antibodies against the hapten phOx and human TNF-a

(Hoogenboom and Jespers, unpublished data) were sequentially replaced to

create entirely human antibodies of the same specificity, a process termed

epitope imprinted selection.

Large Repertoires

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It is logical to assume that the larger the library, the greater the chance of findir

antibodies that bind to any given epitope, and similarly better the chances 1

getting higher affinity (Perelson and Oster, 1979). However, the limiting factor

making large primary libraries has been the efficiency of introduction of plasm

or phage DNA into bacteria. In practice, this limits the library size to 107-1 (

clones, even taking advantage of 2 phage vectors with excisable filamentous

phage replicons (Hogrefe, et al. 1993). Generating more of the possible chain

combinations simply increases the library size. This has prompted a new

approach called combinatorial infection (Waterhouse, et al. 1993). For example,

105 different light chains were cloned for display as a Fab-plll fusion in a phage

vector, and then the phage were used to infect> 1012 bacteria harboring a library

of 107 different heavy chains in a plasmid, this will theoretically create 1012

possible Fab fragments (Hoogenboom, et al. 1991 ). If the two chains were

recombined efficiently in vivo onto the same phage replicon by use of loxP sites

(Waterhouse, et al. 1993), this would create a phage library with huge diversity.

2.4 Expression libraries in Escherichia coli

Initially antibody genes were taken from hybridomas, cloned into plasmid vectors

and expressed as complete antibodies in mammalian cells or as fragments in

bacteria (Cabilly, et al. 1984; Boss, et al. 1984; Skerra and Pluckthun, 1988).

Here the specificity was predetermined in the starting antibody genes (as it was

generated from a specific antibody secreting hybridoma) and both the variable

domains were secreted together into the periplasmic space, where protein folding

as well as heterodimer (Fv fragment) association occurred (Piuckthun, 1988).

Then stable Fv fragments were engineered by linking the domains with a

hydrophilic (Bird, et al. 1988) and flexible peptide (Huston, et al. 1988) to create

single chain Fv fragments or by introducing disulphide bonds between the

domains (Giockshuber, et al. 1990). Huston et al (1988) demonstrated for the

first time, expression of a functional scFv in E. coli. This time too, variable

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domain genes were generated from anti-digoxin monoclonal antibody secreting

hybridoma.

For the first time, Ward et al (1989) expressed the immunoglobulin variable

domain (dAb) library in Escherichia coli. Using the polymerase chain reaction, a

diverse repertoire of rearranged VH genes was generated from the spleen

genomic DNA of mice immunized with lysozyme. Amplified DNA was cloned for

expression into a vector which had peiB signal sequence at N-terminal (for

transport of the protein into periplasmic space) and incorporated a C-terminal

peptide tag to facilitate the detection of the expressed variable domains. About

two thousand colonies were toothpicked into wells of ELISA plates and

supernatants were tested for binding to lysozyme-coated plates. Binding

activities were detected against the antigen, and two VH domains were

characterized with affinities for lysozyme in the 20 nM range. This research group

coined the name 'single domain antibodies (dAbs)' for these antigen binding

peptides.

At the same time Huse et al, (1989) described the generation of large

combinatorial libraries of the immunoglobulin repertoire in phage lambda. This

technology changed the way antibody fragments were expressed in a library and

screened for their binding activities. After this, phage display became the

technique for cloning antibody genes directly from the lymphocytes of immunized

animals, bypassing the hybridoma route. Recently a group headed by Stacy et al,

(2003), reported the direct isolation of recombinant human antibodies against

group B Neisseria meningitidis from scFv expression libraries. This is the first

example of screening and successful isolation of human antibodies against a

bacterial pathogen directly from an antibody expression library without prior in

vitro selection. scFv antibody expression libraries have generated from peripheral

lymphocytes of four vaccinated individuals (vaccinated with outer membrane

vesicles (OMV) from N. meningitidis strain 44/76). Forty thousand clones were

screened for antibodies binding N. meningitidis. Of the 430 specific clones

detected, 225 candidates were isolated and re-screened against a different N.

meningitidis strain which gave 4% cross-reactive clones. Antibodies further

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characterized by DNA sequencing, ELISA and surface plasmon resonance

(SPR) analysis, showed broad V-gene diversity and nanomolar scFv affinities

(Stacy et al, 2003).

2.5 Granulocyte, Macrophage-Colony Stimulating

Factor (GM-CSF)

Colony-stimulating factors (CSFs) are glycoprotein molecules that support growth

of hematopoietic colonies in vitro. Among these, granulocyte-macrophage

colony- stimulating factor (GM-CSF) and interleukin-3 (IL-3) have broad activity in

the proliferation and differentiation of erythroid, megakaryocytic, and myeloid

lineage progenitor cells. Macrophage colony-stimulating factor (M-CSF) and

granulocyte colony-stimulating factor (G-CSF) act selectively on cells of the

macrophage and granulocyte lineage, respectively. Human, endogeneous GM­

CSF (granulocyte macrophage-colony stimulating factor), a glycoprotein with an

apparent molecular weight of 22 000 Da, stimulates the development of both

granulocytes and macrophages in bone marrow.

The GM-CSF gene comprises four exons spread over approximately 2.5 kbp of

DNA (Miyatake eta/., 1985; Stanley eta/., 1985). It is located on the long arm of

human chromosome 5 at 5q22-31 (Huebner et a/., 1985, 1990; Le Beau et a/.,

1986; van Leeuwen et a/., 1989; Frolova et a/., 1991 ). The most interesting

feature of its genetic location is its close proximity to the gene for multi-CSF (IL-

3). The multi CSF gene is located approximately 10 kb from the 5' of GM-CSF

gene in the human genome (Yang eta/., 1988;; Frolova eta/., 1991). GM-CSF

secretion was first identified in activated CD4+ helper T cells. However, a number

of different cell types are now known to produce GM-CSF following various kinds

of stimuli. These cell types include monocytes/macrophages, B and T

lymphocytes, neutrophils, eosinophils, mast cells, keratinocytes, fibroblasts,

stromal cells, endothelial cells, osteoblasts, various solid tumors and different

epithelial cell types. GM-CSF secretion by these cells is usually minimal but can

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rise upon mechanical injury, ultraviolet irradiation or the presence of microbial

products. During inflammation, various cytokines also influence GM-CSF

production. IL-1, IL-2, TNF- alpha and IFN-gamma all induce GM-CSF secretion

whereas, IL-10 and TGF-beta suppress GM-CSF secretion. GM-CSF can be

produced by a number of different cell types under different circumstances

(Metcalf, 1984 ).

The structure of human GM-CSF has been determined by partial amino acid

sequence analysis and by deduction from the nucleotide sequence of eDNA

clones (Lee eta/., 1985, Wong eta/., 1985a). The mature protein is preceded by

a hydrophobic leader sequence of 25 amino acid residues in length. The mature

GM-CSF comprises of 127 amino acid residues. The GM-CSF sequence

contains two potential N-lined glycosylation sites. The crystal structure of human

GM-CSF has been determined (Diederichs eta/., 1991) and in common with a

number of other hematopoietic growth factors (Branhuber et a/., 1987: Bazan,

1990a, 1992; Parry eta/., 1991; Powers eta/., 1992), comprises two pairs of anti­

parallel D-helices). The pattern of disulphide bonding has been determined, with

the first and third, and second and fourth residues, respectively, being paired.

The molecular sequence of endogenous human GM-CSF was first identified in

1985; within a few years, three different synthetic human GM-CSFs were

produced using recombinant DNA technology and bacterial (Burgess, et al 1987),

mammalian (Wong, et al. 1985), and yeast expression systems (Cantrell, et al.

1985). Thus GM-CSF was the first human myeloid hematopoietic growth factor to

be cloned. Sargramostim, yeast-derived rHuGM-CSF was produced using

Saccharomyces cerevisiae; bacterially derived rHuGM-CSF was produced using

Escherichia coli and was termed molgramostim whereas mammalian-derived

rHuGM-CSF was produced using Chinese hamster ovary cells (CHO) and was

termed regramostim.

rHuGM-CSF, became available, as a drug for acceleration of myeloid

engraftment in neutropenic patients in late 1990s. This particular clinical use of

rHuGM-CSF was based on the knowledge of its myeloproliferative effects hence

it was used in myelosuppressed patients. As additional information accumulated

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by in vitro research and from results of clinical trials, diverse biologic effects of

rHuGM-CSF started to unfold, suggesting its vital role in various functions of the

immune system, including responses to inflammation and infection, as well as in

hematopoiesis. Consequently, a variety of potential clinical uses for rHuGM-CSF

became a hot spot of research, such as in prophylaxis or adjunctive treatment of

infection in high-risk settings or immunosuppressed patient populations, use as a

vaccine adjuvant, and use as immunotherapy for malignancies.

2.5.1 Use in enhancing hematopoietic recovery

Administration of rHuGM-CSF has been found to induce a dose-dependent

increase in peripheral blood neutrophil counts (Wing, et al. 1989; Kaplan, et al.

1989). When rHuGM-CSF was discontinued, leukocyte counts gradually

decrease to pretreatment levels (Wing, et al. 1989; Metcalf, 1986). Apart from

increasing the number of circulating monocytes, rHuGM-CSF also increases the

function of monocytes and macrophages, including oxidative metabolism,

cytotoxicity, and Fc-dependent phagocytosis (Wing, et al. 1989; Coleman, et al.

1988; Wiltschke, et al. 1995). rHuGM-CSF enhances dendritic cell maturation,

proliferation, and migration (Young, et al. 1995; Szabolcs, et al. 1995, 1996). In

addition, class II major histocompatibility complex (MHC) expression on

macrophages and dendritic cells is increased by rHuGM-CSF, enhancing the

function of antigen presenting cells (Fischer, et al. 1988).

Combined, these effects of rHuGM-CSF increases hematopoietic cell counts and

enhances immune function. The ability of rHuGM-CSF to accelerate myeloid

recovery and to prevent infection has now resulted in multiple approved

applications for sargramostim and molgramostim. The drugs are now being used

in patients after autologous bone marrow transplantation (AuBMT), peripheral

blood progenitor cell (PBPC) transplantation, induction therapy for acute

myelogenous leukemia (AML), engraftment delay or failure after bone marrow

transplantation, and chemotherapy induced neutropenia (Ganser and Heil, 1997;

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Geller, 1996; Lieschke, et al. 1997; Lifton and Bennett, 1996; Lifton, and Bennett,

1996; Montemurro, et al. 1997).

There has always been increasing interest in combining rHuGM-CSF with other

cytokines, especially G-CSF, as a means of improving mobilization without

having to administer chemotherapy. Research has expanded in some of these

settings to investigate use of these combinations of the cytokines for PBPC

mobilization, to prime leukemic cells before or during chemotherapy for AML, and

as an adjunct to increase hemotherapy dose intensity. Lane et al (Lane, et al.

1995) evaluated the PBPC mobilization efficacy of G-CSF, sargramostim, and

sargramostim plus G-CSF in normal donors. The median CD34+ cell yield with

the combination regimen and with G-CSF was significantly higher than for

rHuGM-CSF alone. Investigations are ongoing to determine optimal doses and

sequence of administration of the cytokines in combination (Law, et al. 1996;

Winter, et al. 1996).

GM-CSF receptors on myeloid leukemic cells and their precursors allow them to

proliferate and differentiate on rHuGM-CSF exposure (Griffin, et al. 1986;

Kelleher, et al. 1987; Miyauchi, et al. 1987; Vellenga, et al. 1987). This suggests

that the recruitment of chemoresistant resting leukemic cells into sensitive

phases of the cell cycle by rHuGM-CSF may enhance the antileukemic effect of

chemotherapy. A study done by Buchner et al (1997) to compare use of

chemotherapy alone with use of chemotherapy in conjunction with sargramostim

priming (Bodey, et al. 1993), in test patients showed that 79% of sargramostim­

treated patients and 84% of controls achieved disease remission; persistent

leukemia was observed in 4% and 18% of patients, respectively. In patients

younger than 60 years of age, complete remissions were achieved in 82% of

sargramostim-treated patients and 73% of controls (non sargramostim-treated

patients), with fewer relapses in the sargramostim treated patients during the first

6 months (3% and 22%, respectively) (Buchner, et al. 1997).

Adjunctive use of rHuGM-CSF allows an increase in the dose intensity of

combination chemotherapy regimens including drugs with a primary toxicity of

myelosuppression. The ability of sargramostim to support a multiple cycle high-

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dose chemotherapy regimen was evaluated in a phase Ill, double-blind,

randomized trial of patients with lymphoma (Fischer, et al. 1988) or breast cancer

(Yau, et al. 1996). Sargramostim-treated patients showed a significantly

decreased duration of neutropenia after the first course of chemotherapy in

comparison to patients who received placebo but the difference did not achieve

statistical significance after the second or third courses.

2.5.2 Use against infectious diseases

rHuGM-CSF activates and enhances the ability of neutrophils and macrophages

to phagocytize and destroy bacteria and fungi. Enhancement of the microbicidal

activity of neutrophils by rHuGM-CSF has been shown in vitro against

Staphylococcus aureus, (Roilides, et al. 1990; Verhoef and Boogaerts, 1991)

Torulopsis glabrata, (Kowanko, et al. 1991) and Candida albicans (Smith, et al.

1990; Gad ish, et al. 1991; Richardson, et al. 1992). Neutrophils treated with

rHuGM-CSF have been shown to kill 90% of intracellular C albicans in

comparison to 50% of intracellular yeast cells killed by untreated neutrophils

(Richardson, et al. 1992). Similarly, enhancement of the microbicidal activity of

monocytes by rHuGM-CSF was shown in vitro against C albicans, (Smith, et al.

1990) A fumigatus (Roil ides, et al. 1996; Roilides, et al. 1994 ), Histoplasma

capsulatum (Newman, S.L., Gootee, L. 1992), Cryptococcus neoformans

(Collins, H.L., Bancroft, G.J. 1992), and Trypanosoma cruzi (Reed, et al. 1987).

Sargramostim also promotes killing of Mycobacterium avium complex

(Bermudez, et al. 1990, 1994; Onyeji, et al. 1995; Suzuki, et al. 1994; Roilides, et

al. 1996). The effect of sargramostim on the incidence and severity of fungal

infections was observed in randomized, double-blind studies of the drug in

patients undergoing AuBMT and in patients with AML (Rowe, et al. 1996). In the

phase Ill ECOG trial of elderly patients undergoing chemotherapy for AML,

sargramostim significantly reduced mortality due to fungal infection (Richardson,

et al. 1992; Rowe, et al. 1996).

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Use of sargramostim in patients with HIV infection was focused on its ability to

ameliorate drug-induced myelosuppression (Gill, et al. 1992; Scadden, et al.

1991, 1996). In a phase 1111 study in patients with Kaposi's sarcoma

administration of sargramostim showed prompt increase in absolute neutrophil

count in all patients and an absolute neutrophil count greater than 1000 cells/IJL

within 7 days with no increase in p24 antigen levels (Scadden, et al. 1991 ).

2.5.3 Use as a vaccine adjuvant

The important role of rHuGM-CSF in the maturation and function of antigen­

presenting cells, such as dendritic cells and macrophages, as well as its ability to

affect T-cell immunity, provided the basis for its potential evaluation as a vaccine

adjuvant in new immunotherapy strategies for infectious diseases and cancer.

Oisis et al (Disis, et al. 1996) evaluated the use of sargramostim as an adjuvant

for protein- and peptide-based vaccines in rats, using tetanus toxoid as model for

the foreign antigen system, and peptides derived from a self antigen, rat neu

protein, as the tumor antigen model system. Results of these experiments

showed comparable adjuvant activity to that of Freund's adjuvant and alum.

Results of several preliminary studies using molgramostim in conjunction with

hepatitis B (Hess, et al. 1996; 1996b) and tetravalent influenzae virus vaccine

(Taglietti, et al.1994) suggest that rHuGM-CSF has potential as an antiviral

vaccine adjuvant; however, further evaluation is needed in these settings.

2.5.4 Use in anti tumor therapy

In vitro, rHuGM-CSF has been shown to slightly enhance the cytotoxic activity of

peripheral blood monocytes and lymphocytes and markedly increase antibody

dependent cellular cytotoxicity (Masucci, et al. 1989) it also enhances monocyte

cytotoxicity against a malignant melanoma cell line (Grabstein, et al. 1986).

rHuGM-CSF has also been shown to augment the cytotoxic activity of peripheral

blood monocytes in antibody-dependent cellular cytotoxicity against numerous

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human tumor cells in the presence of various monoclonal antibodies

(Ragnhammar, et al. 1994) and to enhance IL-2-mediated LAK cell function

(Baxevanis, et al. 1995; Epling-Burnette, et al. 1993). In tumor-infiltrating

macrophages, it has also been shown to increase secretion of matrix

metalloelastase with subsequent production of angiostatin, which inhibits

angiogenesis and suppresses the growth of lung metastases (Dong, et al. 1997).

rHuGM-CSF may also enhance the immunogenicity of tumor cells through

facilitation of tumor antigen presentation (Fischer, et al. 1988).

Thus, rHuGM-CSF might enhance functions of cells critical for immune activation

against tumor cells, alone or with other cytokines or monoclonal antibodies,

making it potentially useful in the therapy of malignant diseases. In a phase I

study in patients with cancer, administration of sargramostim has been shown to

enhance monocyte count, antibody-dependent cellular cytotoxicity and increased

secretion of both TNFa and interferons (Wing, et al. 1989).

Based on the increasing variety of biologic effects being attributed to

endogenous GM-CSF, as we just discussed, additional clinical uses for

sargramostim and molgramostim are under investigation. Because rHuGM-CSF

has been shown to stimulate the migration and proliferation of endothelial cells

and local application of rHuGM-CSF in animal studies has shown faster wound

healing times, clinical trials have evaluated rHuGM-CSF in patients susceptible to

mucosal damage, such as mucositis, stomatitis, and diarrhea, and those with

nonhealing wounds and ulcers . It is likely that we will see application of rHuGM­

CSF in a variety of settings beyond those classically associated with

myelosuppression.

2.5.5 Differential expression of GM-CSF in disease conditions

Apart from being an established therapeutic agent, GM-CSF, according to

medical research has been associated with some diseased conditions. Serum

concentrations of GM-CSF were found significantly elevated in patients with

infectious and non infectious SIRS (systemic inflammatory response syndrome)

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(Torre, et al.2003). GMCSF is found increased in normal pregnancy whereas it is

found in very low concentrations during pregnancy in RAS (recurrent

spontaneous abortion) (Perricone, et al. 2003). According to another study GM­

CSF levels were found increased in chronic aplastic anemia patients and

significantly decreased in acute and chronic leukemia patients (Wang, et al.

2003).

2.6 Expresson Systems for Recombinant Antibody

Molecules

Recombinant antibody fragments have been produced in various expression

systems, such as bacterial (Better et al., 1988; Skerra and Pluckthun, 1988;

Huston et al., 1988; Bird et al., 1988) , mammalian (Jost et al., 1994; Dorai et al.,

1994) insect (Bei et al., 1995) , yeast (Davis et al., 1991; Ridder et al., 1995b),

plant (Whitelam et al., 1994) and in vitro translation systems (Nicholls et . al.,

1993). Every protein poses unique problems in its expression because of its

unique amino acid sequence. Although general conclusions can be drawn from

the study of one protein, expression has to be optimised for every new protein.

2.6.1 Yeast expression system

Yeast expression systems are unique as they offer the advantages of both being

a microorganism and a eukaryote. Unlike E. coli, yeast provide advanced protein

folding pathways for heterologous proteins and with yeast signal sequences

secrete the correctly folded and processed proteins. Therefore functional and

fully folded heterologous proteins can be secreted into culture media. Unlike

mammalian expression systems, yeast can be rapidly grown on simple growth

media. For the expression of clinically and industrially important proteins, yeast is

an attractive option as industrial scale fermentation technology is now widely

used. Whole antibodies and antibody fragments have been expressed using this

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system (Wood et al.,1985; Horwitz et al., 1988). The binding activity of whole

antibody and Fab secreted from yeast were found to be similar to that of their

counterparts derived from lymphoid cells (Horwitz et al., 1988). Single-chain

antibodies have also been successfully expressed in yeast systems, for example,

an anti-fluorescein scFv has been produced in Schizosaccharomyces pombe

(Davis et al., 1991) and anti-recombinant human leukaemia inhibitory factor scFv

has been expressed in Pichia pastoris (Ridder et al., 1995b). scFv proteins which

are produced as insoluble inclusion bodies in E. coli are often soluble when

expressed in yeast (Ridder et al., 995b). In addition, the degradation of

heterologous proteins, often a problem in E. coli, is usually reduced in yeast. Two

types of vector systems are used for the expression of cloned genes in the yeast:

(a) episomal vectors which propagate extrachromosomally and (b) integrating

vectors where chromosomal integration is achieved by homologous

recombination. As heterologous promoters do not function in yeast, only yeast

promoters are used for the expression of the cloned genes. The most commonly

used promoters in yeast are GAL 1, GAL?, GAL5, which are repressed by

glucose and induced by galactose. A variety of selectable markers are used for

the isolation and selection of transformants e.g. LEU2, TRP1, HIS3 and URA3,

used in strains auxotrophic for leucine, tryptophan, histidine and uracil,

respectively. The termination process in yeast is similar to higher eukaryotes

involving termination of transcription, endonucleolytic processing and

polyadenylation. In yeast systems, initiation of translation is inhibited by

secondary structures and high G contents in the 5' untranslated region, therefore,

all non-coding sequences in the 5' end are eliminated. The initiation codon ATG

is usually preceded by an A-rich sequence, such as AAAAAAATG for efficient

initiation of translation. In yeast heterologous leader sequences also do not

function therefore for secretion to occur yeast derived signal peptides are used.

Yeast systems are capable of glycosylation of proteins at Asn-X-Ser/Thr motifs.

However, this glycosylation is not the same as seen in hybridomas and

myelomas, here carbohydrates are not modified beyond the mannose addition

(Kukuruzinska et al., 1987).

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Higher production levels of secreted recombinant antibody fragments have been

achieved in yeast expression systems. Two single-chain antibodies, anti-CD7

and anti-DMI, were expressed at 0.25 mg/1 in E. coli, but when the same

fragments were expressed in Pichia pastoris their yields were increased to 60

mg/1 and 100-250 mg/1, respectively (Eldin et al., 1997). Similarly, the yield of

functional rabbit anti-recombinant human leukemia inhibitory sFv was 1 00-fold

more in Pichia pastoris 1 00 mg/1 than in E. coli (Ridder et al., 1995b ).

2.6.2 Insect cell expression system

Insect cell expression systems have emerged in the last few years as attractive

choices for the expression of recombinant molecules. Baculovirus expression

systems are the most popular of the insect cell expression systems and are

known to produce large amounts of active proteins. The baculovirus system has

been used to express functionally active antibody molecules (Hasemann and

Capra, 1990; Zu Putlitz et al., 1990). Insect cells are able to perform most of the

post-translational alterations which are used by higher eukaryotes, giving them a

significant advantage over the bacterial system (Kang, 1988; Luckow and

Summers, 1988; Maeda, 1989; Miller, 1988). Baculoviruses belong to a large

group of circular double stranded DNA viruses which infect only invertebrates,

usually insects (Granados and Federici, 1986). Their genome ranges from 80 kb

to 200 kb. In a typical baculovirus vector, the foreign gene is placed under the

control of a strong polyhedrin promoter, that enables the gene to be transcribed

at a high level, allowing simple selection of recombinant viruses and causing the

recombinant protein to be secreted in the insect cell culture in large amounts

(Miller, 1988; Maeda, 1989). The polyhedrin promoter is considerably stronger

than most eukaryotic promoters. The most commonly used baculoviruses are the

Autographa californica nuclear polyhedrosis virus (AcNPV) and the Bombyx mori

nuclear polyhedrosis virus (BmNPV) (Adams and McClintock, 1991; Bilimoria,

1991; Kool and Vlak, 1993). The insect cell line Sf9 which derives from

Spodoptera frugiperda is the most widely used host (Summers and Smith, 1987).

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A baculovirus expression vector is constructed by insertion of the foreign gene

into a specific transfer vector which is usually formed by a bacterial plasmid -

frequently pUC-derived - containing 5' and 3' sequences, a multiple cloning site

with a choice of restriction enzymes for the insertion of the gene of interest and

the polyhedrin promoter.

The gene of interest is inserted into the baculovirus genome by co-transfection of

the insect cells with the transfer vector plasmid DNA and wild type viral DNA. By

using an occlusion-negative/positive plaque assay the transformants that contain

foreign gene sequences are identified under the microscope by their occlusion­

negative phenotype (Hink and Vail, 1973; Volkman and Summers, 1975). The

main advantage of the baculovirus expression system is that it can provide high

levels of the desired protein, as per now 1-500 mg of recombinant protein per

litre of infected insect cells has been reported (Luckow and Summers, 1988). The

success of a foreign gene expression depends on a number of factors. Very

good quality growth media and careful culturing is required. For optimal results,

highly viable insect cells, in the log phase of growth are a pre-requisite. The

insect cells have a considerably higher demand for oxygen which makes

sparging a necessary but risky process because of the stress exerted to the

highly sensitive cells (Weiss et al., 1982). An anti-pancarcinoma antigen (tumour

associated glycoprotein, TAG-72) scFv and its IL-2 fusion protein has been

produced using this system. The sFv-CLCH1 construct has been expressed at 9

~g/ml concentration in culture supernatants, whereas an IL-2 fusion protein of the

same construct was produced at 3 ~g/ml levels (Bei et al., 1995). The main

disadvantage with baculovirus expression system is that the expression of the

foreign protein is controlled by a very late viral promoter and peaks when the

cells start dying from the viral infection. An alternative has been developed which

is based on the stable transformation of the gene, under the control of an

appropriate promoter into insect cells. Host cells for these are derived from

dipteran insects, including the fruitfly and mosquito. These include the Schneider

2 (Schneider, 1972; Sang, 1981) and Kc (Echalier and Ohanessian, 1970) from

Drosophila melanogaster, and C7 (Sarver and Stollar, 1977) derived from Aedes

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albopictus. The Drosophila metallothionein promoter has been used to control

expression and found to be tightly regulated, directing high levels of transcription

when induced by heavy metals, such as cadmium Cd or copper Cu (Maroni et

al., 1986; Otto et al., 1987). Antibiotic resistance genes such as neomycin and

hygromycin are used as selectable markers and can be co-transfected together

with the heterologous genes (Van der Straten et al., 1989). An anti-E selectin

scFv has been expressed using this system at 0.2-0.4 mg/1 levels in culture

supernatants, whereas when the same sFv was expressed in a bacterial

expression system no protein was detected in either the culture supernatant or

the soluble periplasmic extract. However, after refolding the periplasmic protein,

went up to 0.1-0.4 mg/1 of monomeric scFv which was recovered by (Mahiouz et

al., 1998).

2.6.3 Mammalian cells expression system

In mammalian cell expression, signals for synthesis, processing and secretion of

eukaryotic proteins are properly and efficiently recognised by the mammalian

cells though inter- species' differences do exist.

Two general methods exist for the introduction of foreign DNA into mammalian

cells. One is mediated by virus infection and the other by direct transfer of DNA

into the cells employing chemical methods (liposomes, calcium phosphate,

DEAE-dextran and polybrene) and physical methods (electroporation and

microinjection). Though efficiency of transcriptional control elements such as

promoters and enhancers vary considerably in different cell lines, one critical

feature present in all promoters is that they contain two types of recognition

sequences, mRNA cap sites where the mRNA transcript starts and a TATA box,

located 25-30 bp upstream of the transcription initiation site. The TATA box is

known to be involved in directing RNA polymerase II to begin RNA synthesis at

the correct site. Other upstream promoter elements are located up to 100-200 bp

upstream of the TATA box and determine the rate at which transcription is

initiated.

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All expressible cloned genes in mammalian cells include a ribosome-binding site

and an initiation codon. Eukaryotic ribosomes initiate translation by recognising

the consensus sequence GCCGCCArGCCAUGG +4 (Kozak, 1987, 1989). The

position of nucleotides from position 1 to 6 and the presence of a G at position +4

is important for the efficient translation of the cloned genes (Kozak, 1987).

Enhancers are also used to increase expression. Many enhancers are cell type­

restricted (Voss et al., 1986; Maniatis et al., 1987) and therefore the choice of

promoter and enhancer elements in an expression vector is determined by the

cell type(s) in which the gene of interest is to be expressed. However, many

promoters in common usage such as the SV40, human cytomegalovirus (Boshart

et al., 1985) and the long terminal repeat of the Rous sarcoma virus (Gorman et

al., 1982) are active in many cell types with some quantitative differences.

Isolation of cell lines that express the transfected gene is achieved by

introduction of a second gene encoding for a selectable marker. The gene of

interest and gene encoding the selectable marker can either be included on a

single vector or co-transfected as separate vectors. The selection of stable cell

lines is a time consuming task, as an alternative transient expression systems

give an early indication whether a genetically engineered construct is correctly

prepared. This is particularly important in the case of scFvs, Fvs and humanised

antibodies, where the loss of affinity during the engineering phase is common

and is required to be identified and corrected at an early stage. Transient

expression of engineered chimeric antibodies is commonly achieved in COS cells

(Whittle et al., 1987; Daugherty et al., 1991; Kettleborough et al., 1991 ). Virally

transfected CHO-K1 cell line transfected with the adenovirus E 1 A transactivator

is used for the transient expression of the antibodies (Cockett et al., 1991 ). By

the use of heterologous promoters, enhancers and amplifiable genetic markers,

the yields of antibody and antibody fragments is increased. High levels (well

above the levels seen from parental hybridomas) of chimeric antibodies and

recombinant antibody fragments have been achieved from low copy number cell

lines (Colcher et al., 1989; King et al., 1992). Antibody fragments, a wide range

of scFv, scFv-fusion proteins and similar molecules have been expressed in

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Review of Literature

mammalian cells (Dorai et al., 1994) till date. An anti-c-erb 8-2 (741F8) scFv was

secreted at 10 mg/1 in culture medium with Sp2/0 cells, whereas up to 4-50 mg/1

of the same scFv was recovered after refolding the material from E. coli (Dorai et

al., 1994). Similarly, 1 mg/1 of MOPC 315 scFv was recovered from Sp2/0

expression as opposed to 4 mg/1 in E. coli after refolding the protein (Dorai et al.,

1994 ). The Fv fragment of the 872.3 antibody were expressed in E. coli culture

medium, up to 40 mg/1 in shake flask cultures and up to 450 mg/1 in

fermentations. The yield of 872.3 Fv was 4 mg/1 in CHO cells (King et al., 1993).

2.6.4 Gene expression using Escherichia coli cells

Immunoglobulin fragments are commonly expressed in E. coli. One advantage of

this system is the ability to produce recombinant protein in very large quantities.

Kipriyanov, et al (1997) investigated the effect of growth and induction conditions

on the production of soluble single-chain Fv antibody fragments in Escherichia

coli under the control of wt lac promoter and reported a yield of 16.5 mg/1 in

shake-flask cultures. Martinneau, et al (1998) reported mutated scFv production

yields of 0.5g/l in shake flask and 3.1 g/1 in fermentor. A single-chain Fv (scFv)

antibody fragment against the hepatitis 8 surface antigen (H8sAg) was

expressed in Escherichia coli in the form of two independent fusion proteins,

(fused to human interleukin-2). This strategy of expressing scFv as fusions gave

yields around 30.3 and 27.3 mg r1, of active scFvs respectively (Sa'nchez, et al.

1999). In another study Robin et al (2003) expressed a catalytic single-chain Fv

(scFv482) fragment in different expression systems, Escherichia coli and two

yeasts species to compare their production levels. The scFv482 secreted as an

active form in the culture medium of Pichia pastoris and Kluyveromyces lactis,

gave 4 and 1.3 mg/1 yields after purification. In E. coli, scfv was expressed as

inclusion bodies (12 mg/1) and after refolding its catalytic activity was measured

and found to be comparable to that of the whole lgG. The same observation was

repeated in a study done by Miller, et al (2005). They aimed at heterologous

protein expression in Saccharomyces cerevisiae, Pichia pastoris, and

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Escherichia coli to evaluate their ability to rapidly, efficaciously, and consistently

produce scFv antibodies for use in downstream proteomic applications. scFv

antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3,

and 0.63-16.4mgl-1 culture, respectively. E. coli grow at a very fast rate in

comparison to mammalian cells, giving the opportunity to purify, analyse and use

the expressed protein in a much shorter time. In addition, transformation of E.

coli cells with the foreign DNA is easy and requires minimal amounts of DNA.

Antibody engineering using E. coli tends to be inexpensive. These reasons

explain the popularity of bacterial systems. E. coli however are not capable of

glycosylating proteins. Therefore if whole antibody molecules are required, which

are glycosylated in the C 2 domain, other expression systems are preferred

whereas it's an ideal expression system for antibody fragments that do not

require glycosylation.

Essential components of Escherichia coli expression system (Fig.2.2)

A

+ r-nas---, p-

-~ 1 f'rGACA(NhrlAiAAf

STAP:lc:'xk#'l

n!ONh S' UMOGAOG IN!-a A& (!H'\,j H1G tRNA 3' 1 tofo.UlJCClJ('.C GOO t.1J"!~~

UlJ<'l {1"!;.)

Figure 2.2: Schematic presentation of the salient features and sequence elements of a prokaryotic expression vector. Shown as an example is the hybrid tac promoter (P) consisting of the -35 and -10 sequences, which are separated by a 17-base spacer. The arrow indicates the direction of transcription. The RBS consists of the SD sequence followed by an A+ T -rich translational spacer that has an optimal length of approximately 8 bases. The SD sequence interacts with the 3' end of the 16S rRNA during translational initiation, as shown. The three start codons are shown, along with the frequency of their usage in E. coli. Among the three stop codons, UAA followed by lJ is the most efficient translational termination sequence in E. coli. The repressor is encoded by a regulatory gene (R), which may be present on the vector itself or may be integrated in the host chromosome, and it modulates the activity of the promoter. The transcription terminator (TT) serves to stabilize the mRNA and the vector, as explained in the text. In addition, an antibiotic resistance gene, e.g., for tetracycline, facilitates phenotypic selection of the vector, and the origin of replication (Ori) determines the vector copy number. The various features are not drawn to scale.

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Review of Literature

Promoters: In E. coli expression systems in order to control expression of the

proteins inducible promoters are normally used. This prevents loss or mutation of

the gene in situations where its production might be toxic to the bacteria. Among

commonly used promoters are lac promoter, the trp promoter and their hybrid,

the tac promoter that is regulated by the lac repressor is induced by isopropyl­

~-galactosidase (IPTG} (Amann et al., 1983; de Boer et al., 1983). Another

popular promoter is the A.PL promoter, responsible for the transcription of the

A. DNA molecule; it is regulated by a temperature sensitive repressor. The T7

RNA promoter is also generally used to obtain tightly controlled, high level,

expression (Tabor and Richardson, 1985; Studier and Moffatt, 1986).

mRNA: In Escherichia coli translation initiation from the translation initiation

region (TIR) of the transcribed messenger RNA uses a ribosomal binding site

(RBS) including the Shine-Dalgarno (SD) sequence and a translation initiation

codon (S0rensen et al., 2002). The Shine-Dalgarno sequence is located 7±2

nucleotides upstream from the initiation codon, which is the canonical AUG in

efficient recombinant expression systems (Ringquist et al., 1992). Optimal

translation initiation is obtained from mRNAs with the SD sequence

UAAGGAGG. The RBS secondary structure is highly important for translation

initiation and efficiency is improved by high contents of adenine and thymine

(Laursen et al., 2002). Translation initiation efficiency is in particular influenced

by the codon following the initiation codon and adenine is abundant in highly

expressed genes (Stenstrom et al, 2001 ). A transcription terminator placed

downstream from the sequence encoding the target gene, serves to enhance

plasmid stability by preventing transcription read through. Transcription

terminators stabilize the mRNA by forming a stem loop at the 3' end (Newbury et

al., 1987). Translation termination is preferably mediated by the stop codon UAA

in E. coli. Increased efficiency of translation termination is achieved by insertion

of consecutive stop codons or the prolonged UAAU stop codon (Poole, et al.,

1995).

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Review of Literature

Current expression systems: A wealth of expression systems designed for

various applications and compatibilities are now available. Approximately 80% of

the proteins, submitted to the protein data bank (PDB) in 2003 were prepared in

an E. coli expression system. The T7 based pET expression system

(commercialized by Novagen) is by far the most used in recombinant protein

preparation. Systems using the A-PL promoter/cl repressor (e.g., Invitrogen

pLEX), Trc promoter (e.g., Amersham Biosciences pTrc), Tac promoter (e.g.,

Amersham Biosciences pGEX) and hybrid /ac/T5 (e.g., Qiagen pQE) promoters

are also common (Hannig and Makrides, 1998). An interesting system is based

on the araBAD promoter (e.g., Invitrogen pBAD).

The pET expression system: Studier and colleagues first described the pET

expression system, which has been developed for a variety of expression

applications (Dubendorff and Studier, 1991; Studier et al., 1990). More than 40

different pET plasmids are now commercially available. The system includes

hybrid promoters, multiple cloning sites for the incorporation of different fusion

partners and protease cleavage sites, along with a high number of genetic

backgrounds modified for various expression purposes. Expression requires a

host strain lysogenized by a DE3 phage fragment, encoding the T? RNA

polymerase (bacteriophage T? gene 1 ), under the control of the IPTG inducible

/acUV5 promoter. Lac I represses the lacUV5 promoter and the T? /lac hybrid

promoter encoded by the expression plasmid. A copy of the fact gene is present

on the E. coli genome and on the plasmid in a number of pET configurations.

Lac I is a weakly expressed gene and a 1 0-fold enhancement of the repression is

achieved when the overexpressing promoter mutant Laclq is employed (Calos,

1978). T? RNA polymerase is transcribed when IPTG binds and triggers the

release of tetrameric Lacl from the lac operator. Transcription of the target gene

from the T7/lac hybrid promoter (repressed by Lacl as well) is subsequently

initiated by T7 RNA polymerase. The T? promoter is a 20-nucleotide sequence

not recognized by the E. coli RNA polymerase. T? RNA polymerase transcribes

maximally 230 nucleotides per second and is five times faster than E. coli RNA

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Review of Literature

polymerase (50 nucleotides per second). Background expression from pET

expression plasmids is diminished by the presence of T? lysozyme

(bacteriophage T? gene 3.5 amidase), which is a natural inhibitor of T? RNA

polymerase. Co-expression of T? lysozyme is achieved by either plasmid plysS

or plysE. These plasmids harbour the T? lysozyme gene in silent (plysS) and

expressed (plysE) orientations, with respect to the cognate tetracycline

responsive (Tc) promoter (Studier, 1991 ).

E. coli host strains: The strain or genetic background for recombinant

expression is highly important. Expression strains should be deficient in the most

harmful natural proteases, maintain the expression plasmid stably and confer the

genetic elements relevant to the expression system (e.g., DE3). E. coli BL21 is

the most common host and has proven outstanding in standard recombinant

expression applications. BL21 is a robust E. coli B strain, grows vigorously in

minimal media but is non-pathogenic and unlikely to survive in host tissues and

cause disease (Chart et al., 2000). BL21 is deficient in ompT and lon, two

proteases that may interfere with isolation of intact recombinant protein.

Derivatives of BL21 include recA negative strains for the stabilization of target

plasmids containing repetitive sequences (Novagen BLR strain), trxB!gor

negative mutants for the enhancement of cytoplasmic disulfide bond formation

(Novagen Origami and AD494 strains), lacY mutants enabling adjustable levels

of protein expression (Novagen Tuner series) and mutants for the soluble

expression of inclusion body prone and membrane proteins (Avid is C41 (DE3)

and C43(DE3) strains).

Stability of the messenger RNA: Gene expression is also controlled by the

decay of mRNA. The average half life of mRNA in E. coli at 37°C ranges from

seconds to maximally 20 min and the expression rate depends directly on the

inherent mRNA stability (Rauhut and Klug, 1999; Regnier and Arraiano, 2000).

Control of mRNA stability in recombinant expression systems is desirable.

Efficient translation initiation and consequent immediate ribosomal protection

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from degradation stabilizes the mRNA and is achieved by selection of ribosomal

binding sites lacking inhibitory secondary structure elements. Stable hybrid

mRNAs are constructed by implementation of efficient five prime and three prime

stabilizing sequences as a barrier against exonucleases.

Codon usage: Codons rare in E. coli are often abundant in heterologous genes

from sources such as eukaryotes, archaeabacteria and other distantly related

organisms with different codon frequency preferencies (Kane, 1995). Expression

of genes containing rare codons lead to ribosomal stalling at positions requiring

incorporation of amino acids coupled to minor codon tRNAs (McNulty et al.,

2003). Two alternative strategies are utilized to remedy codon bias. site-directed

mutagenesis of the target sequence for the generation of codons reflecting the

tRNA pool in the host system is beneficial for increasing expression levels and

for alleviation of mistranslation (Calderone et al., 1996; Kane et al., 1992). But

this strategy is generally not foolproof (Wu et al., 2004 ). Whereas co­

transformation (second approach) of the host with a plasmid harbouring a gene

encoding the tRNA cognate to the problematic codons is a less time consuming

method (Dieci et al., 2000). Hence by increasing the copy number of the limiting

tRNA species, E. coli matches the codon usage frequency in heterologous

genes.

Expression of antibody fragments: The expression of recombinant antibody

fragments in the reducing environment of the cytoplasm leads to the formation of

insoluble inclusion bodies, which contain unfolded protein. A number of refolding

strategies are being employed, but they need to be optimised for each molecule

as each antibody molecule harbors a unique protein sequence. Most strategies

imply isolation of inclusion bodies, the solubilisation of the recombinant proteins,

and their renaturation in an environment that promotes the correct disulphide

bond formation and adoption of the appropriate three-dimensional shape.

Solubilisation of the inactive proteins is achieved by using denaturing agents,

such as guanidine HCI or urea. Mild detergents, which do not bind too strongly

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to the protein (Tanford, 1968), are also used (Lacks and Springhorn, 1980;

Kurucz et al., 1995). Reducing agents, such as P-mercaptoethanol or

dithiothreitol OTT are used to reduce inter and intra-chain disulphide bonds that

might have formed during the lysis of the bacteria and solubilisation of the

protein. The formation of disulphide bonds is performed by simple air oxidation

(Anfinsen et al., 1961 ), in some cases promoted by the presence of metal ions

(Saxena and Wetlaufer, 1970). 'Shuffling' (breaking and reforming) of the

disulphide bonds to increase the chance of obtaining the correct configuration is

promoted by use of disulphide isomerases (Carmichael et al., 1977) or by the

inclusion of a redox couple, made by a mixture of reduced and oxidised thiol

groups (Saxena and Wetlaufer, 1970), for example, as is provided by

glutathiones. Every antibody or antibody fragment is unique. Hence, theoretically,

there is no one universal refolding protocol. Therefore to optimize the a particular

refolding protocol there are many parameters that are handled. These include

factors for example temperature of refolding, the time, the concentration of

protein, the presence of a co-solvent or redox couple and the pH of the reaction.

This optimization for a particular antibody or fragment seems time consuming but

the yields are worth it. Using fermented cultures up to 100-130 mg/1 of active

scFv or scFv fusion proteins have been prepared (Huston et al., 1995). Instead of

the cytoplasmic exoression, recombinant proteins by the use of leader sequence

can be directed to periplasmic space (Skerra and Pluckthun, 1988). Periplasmic

space lies between the inner and outer membrane of Gram negative bacteria,

and has oxidising environment. There exist a number of chaperonin-like

molecules and disulphide isomerases which help in refolding of the recombinant

antibody. This approach was first used in the expression of Fv (Skerra and

Pluckthun, 1988). A number of leader sequences are now being used, including

the peiB leader from the pectate lyase gene of Erwinia carotovora (Lei et al.,

1987) and the leader sequence derived from alkaline phosphatase gene. These

sequences get cleaved by the signal peptidases inside the periplasm (Ferenci

and Silhavy, 1987). In some cases the recombinant antibody material in the

periplasm has been found to 'leak' through the outer membrane into the culture

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medium (Ward et al., 1989). The extent to which this occurs is depends on the

bacterial strain, the induction conditions and, most importantly, on the individual

amino acid sequence of the antibody (Knappik and Pluckthun, 1995), rather than

on the signal sequence (Suominen et al., 1987). This provides a number of

advantages; it permits rapid screening for antibody secretion and, when the yield

is high, it allow for direct purification of material from the supernatant. However,

not all antibodies are secreted in this way. Periplasmic extract is frequently

obtained by osmotic lysis. This endows us with an advantage that the protein is

often present at high concentrations, in a reasonably pure form (Skerra, 1994 ).

Bacterial expression therefore has an important role to play in the production of

recombinant antibody-based molecules, in particular for the fragments that do not

require glycosylation.

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