INTRODUCTION

1.1  GENERATION OF ANTIBODIES

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Mammalian B lymphocytes are formed first in the liver during fetal life but the bone marrow becomes the site of lymphopoeisis after birth and remains so throughout adult life (Rolink and Melchers, 1991; Kincade, 1987). Genes expressed in B lymphocytes encode the immunoglobulin (Ig) molecule. The monomeric immunoglobulin is composed of two identical heavy (H) and light (L) polypeptide chains. The amino-terminal domain of each polypeptide chain consists of a variable region whilst the carboxy-terminus contains the constant domain(s). Pairing together of a variable light and heavy chain region form the antigen-combining site responsible for antigen-antibody interactions. Each separate variable domain has three areas of high amino acid sequence variability (hypervariable regions) known as complementarity-determining regions (CDR) interspersed by regions of relatively constant amino acid sequence termed framework regions (FR) (Kabat et. al., 1991). CDR3 is the region of greatest variability.

The immunoglobulin heavy chain variable region segment is encoded by a variable (VH), diversity (D), and a joining (JH) gene segments while the light chain variable region is encoded by a variable (VL), and joining (JL) segment. A site-specific recombination reaction known as VH(D)JH recombination brings gene segments present in the germline together to form a functional B cell receptor gene that encodes surface immunoglobulin in a 'virgin' B cell. Virgin B cells in the primary immune repertoire expressing germline encoded antibodies of the IgM isotype migrate from the primary lymphoid organs (fetal liver, fetal and adult bone marrow) into secondary lymphoid organs - spleen, lymph nodes - where contact with antigen, further differentiation into plasma cells may result in the secretion of either IgG, IgA, or IgE isotypes. The significance of isotypes of antibodies is demonstrated in the generation of IgG antibodies which are usually somatically mutated in the variable region and have higher affinities for antigen than IgM (Potter and Capra, 1995).

1.2 VH(D)JH RECOMBINATION AND THE HUMAN IMMUNOGLOBULIN LOCI

Assembly of immunoglobulin genes by the VH(D)JH recombination process machinery under the influence of recombination activating genes (RAGs) brings different gene elements of the V region together to create considerable diversity (termed combinatorial diversity) in the antibody molecule. Additional diversity can occur by random insertion or deletion of a few nucleotides at the junctions of the segments. This is termed junctional diversity. Studies involving the development of the primary immune repetoire expressed in normal individuals, fetal B lymphocytes, and autoantibodies have led to the identification of immunoglobulin gene families. Sequence analysis of individual gene seqments, and mapping of their chromosomal locations have helped to determine which immunoglobulin gene segments are used in the development of the normal immunoglobulin repetoire (Matsuda et. al., 1998; Cook and Tomlinson, 1995).

1.2.1 The human antibody IgH locus

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The human antibody variable heavy chain locus (VH, D and JH genes) is located on chromosome 14q32.3 (Cox et. al., 1982). Sequence analysis, pulse-field gel electrophoresis mapping (PFGE), deletion mapping and cloning suggest that the human VH locus contains 51 functional VH gene segments (Cook and Tomlinson, 1995; Tomlinson, 1997). Another study reported the existence of 44 functional VH genes (Matsuda et. al., 1998). The difference in the number of functional VH genes in these two independent studies is attributed to an insertion/deletion polymorphic region which when present results in the increase of some functional genes. The human VH genes are grouped into 7 families (van Dijk et. al., 1993). Belonging to a particular Ig (VH) family means sharing at least 80% nucleotide sequence homology while genes with less than 70% homology are members of a different family. However, a unique set of VH segments which share high homology (72 - 82%) with VH1 but differ at a clustered region between framework 2 (FR2) and FR3 has been placed into a different family, the VH7 family (Schroeder et. al., 1990). Among the 51 known functional VH segments, VH3 is the largest with 22 members, VH4 and VH1 has 11 each, VH2 has 3 members, VH5 has 2, and VH6 and VH7 has 1 member each (Tomlinson, 1997). There are several family-specific conserved regions within the human germline VH segments. These family-specific sequences were found in codons 9 - 30 in FR1 and 60 - 85 of FR3 (Kabat et. al., 1991; Tomlinson et. al., 1992 Matsuda et. al., 1993). The conservation of sequences is not limited to only the framework regions. Complementarity determining region 2 (CDR2), one of the regions showing hypervariability, also displays some amount of family-specific conservation. Codons 60 - 65 in the 3' portion of CDR2 are conserved in a family-specific way (Honjo and Matsuda, 1995). 25 functional human D gene segments are grouped into 7 families whilst 6 JH human gene segments are known (Tomlinson, 1997).

1.2.2 The human variable light chain locus

The human antibody variable light chain loci (VL and JL genes) are derived from either the kappa (κ) locus on chromosome 2p11-12 (Malcolm et. al., 1982) or the lambda (λ) locus on chromosome 22q11.2 (de la Chapelle et. al., 1983). In humans, 60% of immunoglobulin light chains are kappa while 40% are lambda. A total of 40 human Vκ gene segments have been divided into 7 families. The human Vλ locus is divided into 10 families with a total of 38 functional genes (Williams et. al., 1996; Tomlinson 1997).

At the murine light chain locus, λ gene rearrangement always occurs after κ genes fail to rearrange (Retter and Nemazee, 1998). Thus if an appropriate κ gene is not assembled, rearrangement at the λ locus often follows.

1.3 PHAGE DISPLAY TECHNOLOGY

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Nearly eighty years after the discovery of antibodies by Emil Behring in 1894, Köhler and Milstein (1975) first described the production of monoclonal antibodies by hybridoma technology which has been of immense use as therapeutic immunoreagents, and in bio-medical research (Verhoeyen and Windust, 1996).

Though monoclonal antibody production offers an unlimited supply of specific rodent antibodies, the hybridoma technique has been much less successful for human monoclonal antibodies (James and Bell, 1987). A major drawback in the use of rodent antibodies in therapy of human antibodies is the problem of human antimouse antibody response which limits their use. A solution to the problem of immunogenicity of rodent monoclonal antibodies in humans would be to produce human antibodies directly from humans since it is technically much more difficult to immortalize human B cells to human tissue antigens than making (many) rodent antibodies, and also unethical to immunize human donors in human monoclonal antibody production. Recent advances in molecular biology and protein engineering has opened new vistas in biological research resulting in recombinant antibody technology that offers new immunotherapeutic opportunities. The new technology involves cloning of the human immunoglobulin gene repertoire comprising heavy chain and light chain genes, recombining them randomly to give a combinatorial antibody library, and expressing the antibodies on the surface of microorganisms.

1.3.1 Antibody production by combinatorial libraries

The display of peptides on the surface of filamentous phages (Smith, 1985), provided a significant beginning to phage antibody display technology. The first development toward combinatorial antibody libraries involved cloning the repertoire of VH genes from an immunized mouse spleen into bacteriophage lambda (Sastry et. al., 1989). Shortly afterwards, Huse et. al. (1989), produced the first combinatorial antibody library by producing murine catalytic antibodies in lambda phage. PCR sequences of heavy (Fd; VH + CH1) and light chains from mouse RNA were cloned into separate lambda vectors. Vector DNA immediately downstream of the cloned mouse heavy chain DNA was digested away and similarly, vector sequences upstream of the mouse light chain DNA were also cleaved. The two vector arms were digested at a common central restriction site using a common restriction enzyme and religated, such that a random pair of heavy and light chain was combined irrespective of their original pairing in the spleen cells, to give the first random combinatorial antibody library. Screening of the library for clones expressing functional Fabs (VH1-CH1 + VL-CL) were done by the adsorption of Fabs onto nitrocellulose filters and incubated with labeled antigen for detection of specific clones. Seperate heavy and light chain libraries were negative.

1.3.2 Production of antibody fragments by Phage Display

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The screening procedure of the lambda phage system imposed limitations to the effectiveness of the technique. Screening a library of say 5 x 108 antibodies would require a minimum of 10,000 filter lifts (at 50,000 plaques per filter lift) making the screening extremely laborious. Besides, the antigen must be available in large quantities and be readily amenable to radioactive labeling, and should also not significantly stick to filters in the absence of antibody (Burton and Barbas, 1994). This imposes restrictions on for example, membrane-bound antigens, and hampers the effectiveness of the lambda phage system.

A new technique which was based on Smith’s display of peptides on the surface of filamentous phages was utilized. Foreign DNA fragments were ligated to the minor coat protein III gene of filamentous phages, creating a `fusion phage´. The peptides were shown to bind immobilized antibodies, and led to easy screening of libraries for binding to ligands and antibodies. McCafferty et. al. (1990), displayed single chain variable domain fragment (scFv) antibodies on the surface of filamentous phages. ScFv consists of a heavy chain and light chain variable domains connected on the same polypeptide chain by a flexible oligopeptide linker. The use of filamentous phage genome as a cloning vehicle has some limitations. Since large inserts are known to adversely effect the infectivity of the phages, the risk of the library being populated by rapidly growing deletion mutants was high (Little et. al. 1997). Besides, it is difficult to prepare the double-stranded DNA necessary for cloning, hence phagemid systems were introduced as an attractive alternative to cloning directly into the phage genome (Burton and Barbas, 1994). The display of peptides on phages was applied to proteins. (Kang et. al., 1991a; Breitling et. al. 1991; Chang et. al., 1991; Duchosal et. al., 1992) described the use of phagemid vectors in combination with helper phage rescue for the construction and rapid analysis of combinatorial antibody libraries.

Phagemids are plasmids that contain filamentous bacteriophage origin of replicaton but none of the genes required for replication, DNA packaging, and assembly of phage particles. They are double-stranded, making cloning easier, and allow 100-fold higher efficiencies of transformation compared to phage vectors (Verhoeyen and Windust, 1996; Little et. al., 1995). Phagemids can be packaged into phage particles by 'rescue' with helper phages such as VCSM13 (Stratagene, La Jolla, Ca., USA) that provides all the phage proteins necessary for replication, DNA packaging and assembly of phage particles. The helper phage itself is unable to package its own genome due to a defective origin of replication (Burton and Barbas, 1994). Therefore, cells transformed with a phagemid are infected with a helper phage to provide proteins required for replication and packaging single-stranded phagemid DNA into the filamentous phage particle. The mature phage with the potential to bind antigen displays statistically one copy of Fab-cpIII fusion protein and 3-4 copies of the native cpIII protein which mediates phage infectivity (Fig. 1.0). In a most recent publication, Rondot et. al. (2001) reports a considerable increase in the fraction of phage particles displaying antibody fragments by the use of a newly-developed helper phage (hyperphage), and a mutated E. coli strain.

1.3.3 Phagemid pComb3H

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The phagemid pComb3H (Fig. 1.1) used in this study is a derivative of the original pComb3 phagemid vector (Barbas et. al., 1991). The main features of pComb3 are that it contains both the origin of replication of the multicopy plasmid ColE1 and the origin of replication of the filamentous bacteriophage f1. The phagemid also contains the enzyme beta-lactamase gene that confers ampicillin (carbenicillin) resistance (Apr or Cbr) to bacteria that harbor pComb3 DNA. All other genes required for replication and assembly of phage particles are lacking. Consequently, pComb3 needs a helper phage to provide the phage proteins necessary for replication and packaging. The helper phage VCSM13 which contains a gene coding for kanamycin resistance (Kmr) is used to superinfect bacteria that has been transformed with pComb3 DNA. VCSM13 is a male-specific phage i.e. host bacterial cells must contain an F factor that encodes the proteins forming the pili which is necessary for infectivity of the male-specific phage (Cold Springs Harbor Laboratory Course Manual, 1993).

Fig. 1.0 Overview of the Phage display system with pComb3H. Amplified light and heavy chains permits the production of phages expressing antibodies whose genetic information are carried within the phage coat. Desired antibodies are selected by successive rounds of biopanning, and subsequently ELISA on the antigen of interest. Soluble Fabs are produced upon restriction of phage coat protein gene III from the phage genome.

Phage display however suffers from potential drawbacks. Non-productive aberrant chains are often very well expressed and tend to be non-toxic to the bacterial host, whereas cells expressing functional Fab-gene III fusions may have a growth disadvantage and are selected against (Fischer et. al., 1999). The Fab-gene III fusion protein can also cause vector instability, creating deletions in the antibody fusion genes (Courtney et. al., 1995). The combinatorial construction of Fabs in the library makes it difficult to discriminate original VH - VL pairings from 'de novo' formed pairs, and original pairing may be lost (Fischer, 1998). The panning process which by itself is very difficult could lead to over-amplification of plastic binding clones which are inherently present in the libraries.

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Fig. 1.1 pComb3H is a modified derivative from the original pComb3 phagemid. The lac Z promoter drives the synthesis of the light chain and Fd/gene III transcript. The two ribosome binding sites initiate the translation of the two separate polypeptide chains; a complete light chain (VL - CL), and the Fd fragment of a heavy chain (VH - CH1) fused to the carboxy-terminal domain of the gene III protein. Random combination of light chain and Fd fragment PCR products which have been independently amplified are cloned into the plasmid to generate a combinatorial antibody library. The leader peptides omp A and pel B target both polypeptides to the bacterial periplasm where the soluble light chain fragment and the membrane-bound Fd fragments associate via a disulfide bond. The Sac I and Xba I restriction enzyme sites are provided for cloning light chain fragments whilst Fd fragments are inserted at Xho I and Spe I restriction sites. Cleavage of pComb3H by the restriction enzyme Spe I (A|CTAGT) and Nhe I (G|CTAGT) which have identical cohesive ends that can be re-ligated results in the removal of the gene III product of M13 producing soluble Fabs. The wild type vector is provided in the form pComb3H-SS where the SS designation is for stuffer fragments. The stuffer fragments have not been well characterized in detail but they lack the restriction sites found in the vector. (After Rader and Barbas, 1997).

Phage antibodies are either produced as scFv or Fab fragments in E. coli, devoid of the Fc portion. For therapeutic use the Fc portion is required to yield the month-long half-life typical of antibodies (Barbas, 1995). The immobilization of antigen in plastic wells during panning results in masking or inaccessibilty of some epitopes on the antigen, compromising the conformational integrity of the antigen.

1.3.4 Recombinant antibody gene expression in E. coli

The wealth of knowledge accumulated from E. coli genetics makes it an attractive cloning vehicle for the expression of recombinant antibody fragments. Besides, growth of E. coli is inexpensive, cell growth is fast, and DNA transformation as well as transfection (infection) with phage is extremely efficient (Cold Spring Harbor Laboratory Course Manual, 1993). A disadvantage with using E. coli is that a whole antibody molecule including the Fc portion which is important for effector functions, and glycosylation which influence the complete molecule cannot be expressed in E. coli, since E. coli cannot glycosylate proteins (Verhoeyen and Windust, 1996).

1.4 SYSTEMIC LUPUS ERYTHEMATOSUS

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Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology involving multiple organs with diverse and variable clinical manifestations. The disease is characterized by production of anti-nuclear antibodies, generation of circulating immune complexes, activation of the complement system, initiating and causing inflammation of the joints, kidney, brain, lung, skin, etc. The American Rheumatological Association have established criteria to guide medical practioners in diagnosing SLE cases (Table 1.1).

Any 4 of the 11 criteria is an indication of SLE. Patients with SLE produce autoantibodies to self-components with antinuclear antibodies being prevalent. Among these are autoantibodies to chromatin, the U1 and Sm small nuclear ribonucleoprotein (snRNP) particles, the Ro/SSA and La/SSB RNP complexes (Kotzin, 1996). U1 is a uridine-rich snRNA complexed with various polypeptides designated as 70-kD, and proteins A - G. Anti-Ro/SSA and anti-La/SSB antibodies have been implicated to have a direct pathogenic role in the development of some manifestations such as skin lesions of subacute cutaneous lupus erythematosus and congenital heart failure (Tomer et. al., 1993). Anti-Ro/SSA and anti-La/SSB antibodies in SLE are 17 - 63 % and 6 - 35 % respectively. Other autoantibodies include anti-phospholipid antibodies (which complexes to β2-glycoprotein 1) that are associated with thrombotic complications in some patients. Another subset of autoantibodies in systemic lupus erythematosus are directed to cell surface molecules. These immunopathological autoantibodies are observed in diseases such as idiopathic thrombocytopenia (platelet destruction) and hemolytic anemia (red blood cells destruction). Anti-dsDNA antibodies which are the hallmark of systemic lupus erythematosus (Stollar, 1989) and which serves as a major diagnostic marker for the disease appear to play a prominent role in the immune complex glomerulonephritis.

Table 1.0 The 1982 revised criteria for SLE (Tan et. al., 1982).

Criterion

Definition

1. Malar rash

Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds

2. Discoid rash

Erythematous-raised patches with adherent keratotic scaling and follicular plugging; athropic scarring may occur in older lesions

3. Photosensitivity

Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation.

4. Oral ulcers

Oral or nasopharyngeal ulceration, usually painless, observed by a physician

5. Arthritis

Nonerosive arthritis involving two or more peripheral joints, characterized by tenderness, swelling, or effusion

6. Serositis

a. Pleuritis - convincing history of pleuritic pain, or rub heard by physician, or evidence of pleural effusion, or
b. Pericarditis - documented by electrocardiogram, rub, or evidence of pericardial effusion

7. Renal disorder

a. Persistent proteinuria greater than 0,5 g per day, or greater than 3+ if quantitation is not performed, or
b. Cellular casts, red cell, hemoglobin, granular, tubular, or mixed.

8. Neurologic disorder

a. Seizures, in the absence of offending drugs or known metabolic derangements, e.g. uremia, ketoacidosis, or electrolytic imbalance, or
b. Psychosis, in the absence of offending

9. Hematologic disorder

a. Hemolytic anemia with reticulocytosis, or
b. Leukopenia, less than 4 x 106/ml two or more occasions,
c. Lymphopenia, less than 1.5 x 106/ml,
d. Thrombocytopenia, less than 100 x 106/ml in the absence of offending drugs.

10. Immunologic disorder

a. Positive LE cell preparation, or
b. Anti-native DNA antibody in abnormal titer, or
c. Anti-Sm present, or
d. False-positive serologic test for syphilis known to be positive for at least 6 months and confirmed by negative Treponema specific test

11. Antinuclear antibody

An abnormal titer of ANA by IF or an equivalent assay at any point in time and in the absence of drugs associated with drug-induced lupus

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Anti-dsDNA antibodies which rarely occurs in other conditions are virtually diagnostic of SLE. The combination of high levels of anti-DNA antibodies with how levels of C3 is virtually 100 % diagnostic of SLE in patients who are suspected to have the disease on clinical grounds. Most anti-dsDNA antibodies recognize the sugar phosphate backbone of DNA, while others are base specific or bind to unique three-dimensional structures on DNA. The pathological consequence of anti-DNA antibodies is their role in kidney damage. Immune complexes of DNA-anti-DNA are deposited in the kidney glomeruli, or sometimes they form in situ in the kidney. An alternate hypothesis have it that anti-dsDNA antibodies are able to cross-react with glomerular structures that are not DNA in origin (Kotzin, 1996). Local complement activation induces glomerulonephritis with a detectable consumption of serum complement (Balow, 1991). It is important however to note that the presence of high levels of abnormal serum anti-dsDNA titer does not correlate with any particular manifestation of SLE except nephritis. Thus, it is not possible to predict from serological results that a given patient will develop renal disease, for example (Tomer et. al., 1993).

1.5 IDIOPATHIC THROMBOCYTOPENIC PURPURA

Platelets differentiate from megakaryocytes which are very large polyploid bone marrow cells. Upon migration from the bone marrow space, one-third are sequestered in the spleen, while the remaining two-thirds circulate for 7-10 days. The normal blood platelet count varies between 150,000 - 450,000/μl and since only a small fraction are consumed in the process of hemostasis, most platelets circulate until they become senescent and are removed by phagocytic cells.

The normal function of platelets is their adherence and aggregation to cover damaged areas. Thus when the endothelium is damaged, subendothelial layers come into direct contact with the blood, and platelets adhere and aggregate to cover up the damaged areas (Deckmyn and De Reys, 1995; Macchi et. al., 1997). In the subendothelium, platelets bind directly through their collagen receptors (e.g. gpIa/IIa, gpVI), or indirectly through the adhesion protein - von Willebrand factor (vWF), forming a bridge between the subendothelium and the vWF receptor on the platelets. vWF binds to platelets through contact with glycoprotein Ib (gpIb). The multimeric vWF interacts with gpIb on other platelets resulting in agglutination. Aggregated platelets become activated, produce and secrete or facilitate the production of additional platelet-activating substances such as adenosine diphosphate (ADP), serotonin, thromboxane A2, thrombin, and gpIIb/gpIIIa which help in the recruitment of additional platelets. Platelet to platelet contact is maintained when gpIIb/IIIa on adjacent platelets are bridged by fibrinogen during platelet aggregation. Deficiency of gpIIb/IIIa, or antibodies interfering with their function, particularly antibodies blocking the final step of platelet aggregation i.e. inhibiting fibrinogen to bridge adjacent gpIIb/IIIa, have profound effects in humans; it results in severe bleeding. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease caused by circulating antibodies that react with target antigens (the glycoproteins gp IIb-IIIa, gp Ib-IX) on platelet membranes, with the consequence that the platelets are recognized and eliminated by the host's immune system (Woods et. al., 1984; Kiefel et. al., 1991). The anti-glycoprotein antibodies function as opsonins and accelerate platelet clearance by phagocytic cells. The resultant thrombocytopenia (depletion of platelets) induces a purpura (hemorrhage discharge into skin, mucous membranes, internal organs and other tissues) when the platelet count reaches a critical level, usually < 30,000/μl (Karpatkin, 1997). Platelets interact with Fc parts of antibodies via the FcγII receptor, cross-linking of which can lead to platelet aggregation. Other mechanisms involve Fc independent platelet inactivation and complement-mediated platelet activation. Autoantibodies in idiopathic thrombocytopenic purpura (ITP) can occur in combination with another disease or not, and can be accompanied by thrombocytopenia or without. Anti-platelet antibodies are involved in hematological complications in patients with systemic lupus erythematosus displaying circulating auto-antibodies to platelets, red blood cells, lymphocytes, and nuclear and cytoplasmic cellular components.

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Treatment of idiopathic thrombocytopenic purpura is directed towards the inactivation or removal of a major site of platelet destruction and anti-platelet antibody production namely, the spleen. Administration of corticosteroids prevents sequestration of antibody-coated platelets by the spleen. There arises decreased Fc γ-receptor expression on macrophages thus inhibiting phagocytosis of platelets. In addition, the amount of platelet-associated IgG decreases, thus corticosteroids also impair antibody production and/or binding to platelets. Splenectomy which is only done seldomly, removes the potential site of destruction of damaged platelets as well as the source of anti-platelet antibody production. Since a third of platelet mass is sequestered in the spleen, splenectomy results in increased platelet count of about 30%. However, post-splenectomy thrombosis is a benign self-limited condition that does not require specific therapy. High dose IVIG (1 g/kg in 2 days) is given as emergency treatment in ITP patients with platelet count below 10,000/μl. IVIG induces increased platelet counts and the mechanism of action is believed to be via blockade of the reticuloendothelial system and, the presence of anti-idiotype antibodies in the IVIG preparation (Bussell and Hilgartner, 1986).

1.6 INTRAVENOUS IMMUNOGLOBULINS (IVIG)

Intravenous immunoglobulins (IVIG) are pooled normal polyspecific immunoglobulin G (IgG) obtained from serum of several thousand healthy donors. IVIG contains primarily unmodified IgG which has functionally intact Fc-dependent effector functions and only trace amounts of IgA, IgM, soluble CD4, CD8 and HLA molecules (Blasczyk et. al., 1993). Since IVIG is prepared from large donor pools (typically more than 10,000), they represent a broad spectrum of antibodies reflecting the natural exposures and immunizations of the adult populations from which the donors were drawn (Wolf and Eibl, 1996). IVIG therapy is mostly used in patients suffering from primary immunodeficiency syndromes. IVIG is also administered in anti-inflammatory diseases and several autoimmune diseases such as idiopathic thrombocytopenia (Imbach et. al., 1985) autoimmune hemolytic anemia, dermatomyositis, refractory systemic lupus erythematosus, myasthenia gravis, Kawasaki disease (Fischer et. al., 1996; Wolf and Eibl, 1996).

The proposed mechanism of action of IVIG in autoimmunity and systemic inflammatory diseases include: the blockade of Fc-gamma (Fc-γ) receptors on phagocytic cells, interference with activated complement, modulation of cytokine production and release, modulation of T and B lymphocyte functions, suppression of autoantibody production, and selection or shaping of immune repertoires (Mouthon et. al., 1996). IVIG has been observed to block and saturate Fc-gamma receptors on cells of the reticuloendothelial system namely, macrophages and monocytes, as well as neutrophils, and T and B cells. Administration of IVIG to ITP patients resulted in decreased clustering of IgG-coated thrombocytes (Kimberly et. al., 1984) and an increased platelet count (Clarkson et. al., 1986). These observations are attributed to the blockade of Fc-γ receptors-mediated clearance of autoantibody-coated thrombocytes. IVIG has been shown in vivo to interfere with activated complement by binding to the C3b and C4b components and diverting them from interacting with target cells during complement activation (Basta et. al., 1991). Basta and Dalakas (1994) reasoned the observed decrease in complement deposition in small vessels of dermatomyositis patients treated with IVIG was most likely due to formation of complexes between IVIG and activated complement components such as C3b. The anti-inflammatory effects of IVIG in vivo have been observed in the way IVIG interferes with cytokine production in B and T cells. In vitro studies on cultured monocytes have revealed that IVIG behaves as an anti-inflammatory agent as it triggers the production and extracellular release of interleukin-1 (IL-1) receptor antagonist (IL-1ra), a naturally occurring inhibitor of IL-1 activity. IL-1 is a pro-inflammatory cytokine produced by monocytes and macrophages in response to lipopolysaccharide (LPS), or Fc-γ receptor cross-linking by surface-bound IgG, soluble polymeric IgG or IgG-containing immune complexes (Wolf and Eibl, 1996). In vivo administration of IVIG to Kawasaki disease patients led to a decreased production of IL-1 (Leung et. al., 1989) as well as in rabbits injected with LPS mixed with IVIG (Arend et. al., 1991). It is believed the beneficial effect of IVIG in modulating decreased amounts of pro-inflammatory monokines is that it suppresses production of TNF-α and IL-1 by elevating intracellular levels of cyclic adenosine 3', 5'-monophosphate (cAMP) following its interaction with Fc-γ receptors on monocytes (Shimozato et. al., 1991).

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Some immunomodulatory effect of IVIG is due to its reactivity with functional molecules on lymphocytes. IVIG reacts with CD5 and CD4 molecules on lymphocytes (Hurez et. al., 1994; Vassilev et. al., 1993). CD5+ (B-1) subpopulation of B cells are believed to be a predominant source of autoantibody producing cells (Casali and Notkins, 1989), thus the presence of anti-CD5 antibodies in IVIG may be of potential relevance for therapeutic modulation of autoimmunity. IVIG has also been shown to react with the CD4 molecule on T and B cells (Lam et. al., 1993). Anti-CD4 antibodies purified from IVIG have been found to inhibit in vitro infection of CD4+ T cells by HIV-1 (Hurez et. al., 1994). IVIG contains antibodies capable of reacting with the V-region of other antibodies (anti-idiotypic antibodies) and B cell antigen receptors. The presence of anti-idiotypic antibodies in IVIG is demonstrated by specific retention of autoantibodies on affinity columns of IVIG coupled to sepharose. These anti-idiotypic antibodies in IVIG have the ability to inhibit the activity of autoantibodies or downmodulate their production (Wolf and Eibl, 1996). IVIG also contains IgG fractions against certain Vβ chains of the T cell receptor lineage (Marchalonis et. al., 1992) and have the capability to regulate the function of pathogenic autoreactive T cells. The suppressive effects of IVIG have been observed in its ability to inhibit the proliferation of in vitro activated B and T lymphocytes (Wolf and Eibl, 1996). IVIG was observed to have suppressed the production of IgM by EBV-transformed B lymphoblastoid cells (Kondo et. al., 1994), and also inhibited B cells stimulated by pokeweed mitogen from producing antibodies (Kondo et. al., 1991). In addition, the presence of other immunologically active proteins (other than immunoglobulins) in IVIG preparations may contribute to its immunomodulatory effects. These include HLA class I and II antigens and soluble CD4 and CD8 molecules. These molecules are thought to play a role in cell-cell interactions during immune response and may contribute to immunomodulation of IVIG (Wolf and Eibl, 1996). Other antibody activities found in IVIG preparations (due to its large antibody repertoire) which could influence its mechanism of action include antibodies to alloantigens such as blood group antigens (Gordon et. al., 1980), HLA antigens, and rheumatoid-factor-like isotype specific antibodies (Quinti et. al., 1987).

1.7 THE IDIOTYPIC-ANTIIDIOTYPIC NETWORK IN AUTOIMMUNITY

Idiotypic networks are thought to regulate humoral and possibly cell-mediated immunity. First proposed by Jerne (1974), the theory hypothesizes that the variable regions of antibodies or B cell receptors (idiotypes) can induce the formation of a second set of antibodies or B cell receptors (anti-idiotypes) with specificities against the first set. Some of the anti-idiotypic antibodies may recognize the antigen-combining site or paratope on the primary antibody in which case they appear as the 'internal image' of the original inducing epitope on the antigen. These antibodies are termed Ab2β. Other anti-idiotypes may recognize epitopes lying outside the paratope on Ab1 (the primary antibody). These subsets of anti-idiotypes are referred to as Ab2α. The network theory predicts that the idiotypes on Ab2 antibodies can also elicit Ab3 or anti-antiidiotopic antibodies whose idiotopes mimic that of Ab1, thereby limiting the network as decreasing levels of antibody are produced in each round of activation (Kuby, 1997). The network theory further predicts that within an individual, the interconnection between complementary idiotypes and anti-idiotypic structures on antibody molecules, as well as B and T lymphocytes leads to an immunoequilibria state (Potter and Capra, 1995). This state could potentially be disrupted through mimicry of self-antigen by auto-antiidiotypic antibody resulting in a regulatory breakdown, the outcome of which could be autoimmune disease. Theories of the idiotypic network controlling autoimmunity are about observations that certain anti-idiotype antibodies administered perinatally can lead to prolonged inhibition of the corresponding (autoimmune) idiotypes later in life, presumably by blocking immunoglobulin receptors on immature B cells which are then deleted (Kearney and Vakil, 1986). However, certain anti-idiotype antibodies given neonatally enhance idiotype-positive B cell responses even in the absence of T cell help (Leffel, 1997).

1.8 AIMS OF THIS STUDY

The objective of this study is the use of the phage display technology to investigate:

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  1. the molecular analysis of Fabs bound by IVIG in a patient with SLE,
  2. compare these Fabs with similar ones from patients with AITP cloned in our laboratory,
  3. understand the mechanism(s) by which IVIG interact with Fabs.


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