IMMUNOLOGY AND MEDICAL MICROBIOLOGY

Antibodies Ajit Singh

Associate Professor I/c Immunology Section Department of Veterinary Microbiology (ICAR Centre of Advanced Studies) College of Veterinary Sciences CCS Haryana Agricultural University Hisar - 125 004 (Haryana). 23- Jan-2006 (Revised 03- Jul-2006) CONTENTS What are Antibodies? B lymphocyte clones differentiate into plasma cells that secrete Abs Abs are heterogeneous glycoproteins that share many common physico-chemical and structural features Shapes of antigen-binding site/ paratope Dissecting the structure of Abs in order to understand their functions Antibodies vis-a-vis enzymes Different Ab classes and subclasses are for diverse effector functions Structural variants of Abs are genetically determined and antigenically distinguishable Biological functions of Abs Abs as biocatalysts Forces involved in antibody-antigen interactions Affinity, avidity and specificity of antibodies Ig genes and generation of antibody diversity Major genetic mechanisms that diversify Ab specificities and effector functions Theories of antibody production - A brief account Immunotechnology for production of antibodies and the antigen-binder fragments Origin and Evolution of Igs Immunoglobulin superfamily (IgSF)

Keywords
Antibody structure, functions, classes, diversity, Ig superfamily, immunotechnology

What are Antibodies? When a vertebrate animal, including human is injected with or naturally exposed to certain foreign substances, called antigens, it produces specific humoral factors. These appear in peripheral blood after a few days in order to bind and eliminate the antigens from the body. These specific humoral (fluid-borne) factors that provide humoral immunity to the host against the offending antigens are collectively known as antibodies (Abs). Abs were first detected in the sera of animals injected with bacterial exotoxins, such as tetanus toxin and diphtheria toxin, by Emil von Behring and Shibasaburo Kitasato in the year 1886 A.D. Very vast information has accumulated since then on the biochemical nature, physico-chemical properties, biological functions, and diverse uses of Abs. This chapter aims at discussion of all such features, functions, and uses of Abs, the magic bullets as Paul Ehrlich called them at the dawn of the 19th century. How studies on Abs progressed can be seen in distinct eras in the history of immunology (Table 1). Table 1: Eras in the progress of studies on Antibodies (Immunoglobulins) Era
Early

Time span
1886-1910s

Immunochemistry

1920-1950s

Immunobiology

1960-1970s

Modern

1975to date

Important events (the most prominent scientists involved) Discovery of antitoxins in serum (E.A. von Behring & S. Kitasato, 1986) Abs in serodiagnosis & serotherapy Side-chain theory of Ab formation (P. Ehrlich, 1900) Specificity of Ab defined (K. Landsteiner, 1917-1937) Ab chemistry & quantitative precipitation reaction (M. Heidelberger, 1920s) Physico-chemical characteristics of Abs (Marrack, 1930s) Ag template theory of Ab production (Horowitz, Alexander & Pauling, 1930-35) Isotypic, allotypic and idiotypic variants of Abs found (J. Oudin, 1956-1969) Structure of Ab molecule elucidated (Edelman, Porter, Nisonoff & Kunkel, 1958-60) Clonal selection theory of Ab production (FM Burnet, 1960) Ab producing B cells detected (Pernis, 1969) Network theory of immune system (NK Jerne, 1974) Modern immunotechnology begins with introduction of hybridoma technique (G Kohler & C Milstein, 1975) Ig genes and genetic basis of Ab diversity elucidated (S Tonegawa, 1980) rDNA based techniques for production of Abs and their Ag-binding fragments in various formats in E. coli, yeast, insect cells, mammalian cells, plants, transgenic animals and phage display system; newer roles of Abs being explored such as in catalysis, proteomics, biosensors, etc.

B lymphocyte clones differentiate into plasma cells that secrete Abs Abs are products of antigen-specific B lymphocyte clones that are responsible for adaptive humoral immunity in vertebrate hosts. They are produced mainly in secondary lymphoid organs and tissues, such as lymph nodes, spleen, mucosa-associated lymphoid tissues, etc. After stimulation by the antigen, specific B cell clones undergo multiple cycles of cell division and terminally differentiate into plasma cells that secrete specific Abs in large amounts in lymphoid tissues. From lymphoid tissues, Abs are disseminated via peripheral blood circulation to entire body, tissue fluids and secretions. Box 1: Antibodies as antigen-specific defence molecules Antibodies (Abs): Are humoral (fluid-borne) factors produced in vertebrates in response to foreign substances, known as antigens (Ags) Were discovered by E. von Behring & S. Kitasato in 1886 A.D. Are produced by Ag-specific B cell clones & secreted by Ag-specific plasma cells Are glycoproteins made of two identical light (L) chains (25-30 kDa) and two identical heavy (H) chains (50- 70 kDa) Have the monomer structure: L-H-H-L, and the multimeric forms (L2H2)2-6 also exist Exist as classes & subclasses with diverse functions In addition to isotypic (classes & subclasses), have allotypic and idiotypic variants Have five different classes in humans: IgM, IgG, IgA, IgE and IgD Human IgG has four subclasses: IgG1, IgG2, IgG3 & IgG4

Abs are heterogeneous glycoproteins that share many common physico-chemical and structural features Abs are glycoproteins of heterogeneous size, charge, amino acid composition and antigenic nature. Each Ab is however basically made up of four polypeptide chains: two identical smaller polypeptide chains of about 25-30 kDa size and two identical bigger polypeptide chains of 50-70 kDa size. The smaller polypeptide chain is designated as L (light) chain and the bigger as H (heavy) chain. L chain is composed of about 220 amino acids (AAs), whereas H chains have about 440 or 550 AAs in them. Only the H chains are glycosylated. In the basic monomeric form of Ab, the polypeptide chains are joined together by interchain disulphide bonds between cysteine residues in a manner that a structure that may be written as [L-H-H-L] is obtained (Fig. 1). Certain Abs also exist as multimers of the basic monomeric form i.e., as (L2H2)n. The value of n may vary from 2 to 6. Ab molecules migrate to the electrophoretic region of - and - globulins of serum. As most Abs exhibit - electrophoretic mobility, they are called -globulins. Macroglobulins, i.e, the largest sized Abs (IgM) however, migrate to region. Being involved in immune responses, - and 3

globulins collectively are called immunoglobulins (Igs). Monomeric Ab molecules have sedimentation coefficient of 7S- 8S, whereas di- and pentameric molecules have 11S and 19S, respectively. The comparison of amino acid sequences of Abs produced by different myelomas (cancer cells of B cell lineages producing homogeneous L or H chains or whole Ab molecules) or monoclonal Abs reveals that both L and H chains vary in their NH2- terminal (N-terminal) parts. The Nterminal half of L chain, which is about 110 AAs long and shows variations among L chains from different B cell clones is therefore called VL and the remaining COOH-terminal (Cterminal) part being almost invariable or constant among L chains from different B clones is called CL. Similarly, N-terminal one-fourth or one-fifth part of H chain, about 110 AAs, that shows variations among H chains from different B cell clones is called VH. The remaining Cterminal part, about 330 or 440 AAs being almost invariable or constant among H chains from different B clones is called CH (Fig. 1). However, two types of CL and at least eight types of CH exist in mice and humans, based on sequence differences. Physico-chemical properties of human Igs are presented in Table 2.

Table 2: Physico-chemical properties of human immunoglobulins Feature IgM Size (kDa) 970 Ig class IgGs IgAs (IgA1, (IgG1 to IgG4) IgA2), sIgA 146 (IgG1, 160 (IgAs) IgG2, IgG4) 385 (sIgA) 170 (IgG3) 7S 7S (IgAs) 11S (sIgA) L2H2 (L2H2) 2 (L2H2) 2-JSC (sIgA) / / 2-3% 10% 2 2, 4 (sIgA) IgE 190 IgD 180

Sedimentation rate Structural formula

19S (L2H2)5-J

8S L2H2

7S L2H2

L chain type H chain type Carbohydrate content Electrophoretic mobility Valency Amino acid composition: L chain: H chain: J chain: SC chain: Ig domains: L chain: or H chain: Serum concentration

/ 12% 10

/ 10-12% 2

/ 12% 2

220 550 135 1VL+1CL 1V+4C 0.5-2.5 mg/ml

220 440 1VL+1CL 1V+3C 10-20 mg/ml

220 440 135 (sIgA) 550 (sIgA) 1VL+1CL 1V+3C 0.5-2.0 mg/ml (IgA1, IgA2)

220 550 -

220 440 -

1VL+1CL 1VL+1CL 1V+4C 1V+3C 50 mg/ml 30 g/ml

Closer look into the sequences of VL and VH segments of several different Ab molecules reveals that each segment shows three hypervariable regions, also called complementarity determining regions (CDR1 through CDR3), interspersed within four less-variable framework regions (FR1 through FR4) (Fig. 2). VL and VH CDR1-3 are on average 7, 10, and 7 amino acids in size respectively, and more or less centered on AA 33, 55 and 95 respectively. The six CDRs, three each from VL and VH segments, collectively make up the antigen binding site or paratope in a folded Ab molecule.

Both L and H chains of Ig molecule are folded into distinct domain structures, each of about 110 AAs, called Ig domain (Fig. 3). L chain is folded into two Ig domains, i.e., one variable (VL) and one constant (CL), whereas H chain into 4 or 5 Ig domains, i.e., one variable (VH) and three or four constant (CH1- CH3 or CH4). Each Ig domain contains a loop (Ig fold) of about 60-70 AAs enclosed by intrachain S-S- bonds. Each Ig fold has two intrachain disulphide linked sheets of antiparallel -strands. In C domains, one sheet of three-strands is exposed to the exterior, while the other of four-strands makes contact with the C domain of the other H or L chain. V domain Ig fold is somewhat different in the sense that the three-stranded sheet (or fivestranded expended sheet) makes contact with the sheet from the other V domain, while the fourstranded sheet lies on the exterior. The three CDRs from V domain of each chain (six CDRs in all) that make up one paratope are located on the outer edges of the five-stranded sheets. The four-stranded sheets of both the CL-CH1 and CH3-CH3 dimers make close contacts, but oligosaccharides covalently bound to CH2 domains prevent tight packing of CH2-CH2.

Ag-responsive mature B lymphocytes express IgM and IgD as Ag receptor (B cell receptor, BCR) on their surface. But surface IgM is monomeric, unlike pentameric secreted form. Other Igs, i.e, IgG, IgA or IgE expressing B cells are generated during immune response following contact with the Ags. The surface Igs differ from their secreted forms in C-terminal region. The membrane bound Igs have additional spacer, transmembrane and cytoplasmic regions ending in the C-terminus. Surface IgM and IgD have the hydrophobic spacer region of 12 amino acids followed by hydrophobic transmembrane region of 25 amino acids and ending in three hydrophilic amino acids in the cytoplasmic tail (Fig. 4). The surface IgGs have 25 additional amino acids in their cytoplasmic tail, whereas surface IgA has a unique 14 amino acid cytoplasmic tail. The BCR is associated with the signaling components, Ig- (CD79a) and Ig- (CD79b) on the B cell membrane for initiating the signal transduction pathways associated with Ag-driven B cell activation.

Shapes of antigen-binding site/ paratope Paratope is a three-dimensional shape formed by six CDRs (three each on VL & VH) brought closer by folding of VL and VH domains together (Fig. 5). Each bivalent Ab has two identical paratopes that can bind two epitopes on multivalent antigens or two haptens. Ig fold of each domain has two layers (interior & exterior) of anti-parallel -pleated sheets connected by random coils or loops. CDRs lie on the tips of these loops connecting the anti-parallel -strands.

Abs produced by different B lymphocyte clones have paratopes of different shapes and complementarity to the antigenic determinants or epitopes so as to achieve the best fit between a paratope and an epitope. Paratope shapes can be as varied as concavities (for accommodating convex or bulging epitopes on protein antigens), long grooves (for epitopes on carbohydrate and nucleic acid antigens), or small pockets (for haptens and monosaccharide epitopes). Polyspecific Abs have however been found that can accommodate more than one type of epitopes within their large polyfunctional binding sites.

Dissecting the structure of Abs in order to understand their functions Abs being proteins are amenable to degradation by proteolytic enzymes (proteases). IgG is considered as prototype Ig for studies on structural details. Two proteases, namely papain and pepsin, cleave the human IgG1 to yield fragments of various sizes. Limited digestion with papain cleaves IgG1 in its proximal hinge region to yield: i) two identical N-terminal fragments of about 50 kDa size, each being composed of [VL+CL+VH+CH1] domains, and ii) the C-terminal fragment of about 50 kDa size, and composed of [Hinge region + 2(CH2+ CH3)]. Each Nterminal fragment is able to bind one hapten molecule or one epitope on a multivalent antigen and therefore designated as Fab (Fragment antigen binding) (Fig. 1). Being monovalent, Fab is not able to agglutinate or precipitate multivalent antigens. These are also not able to bind 8

complement component C1q and are therefore unable to activate the complement by classical pathway. On the other hand, C-terminal fragment, being composed of only constant parts of the H chains still joined by disulphide bonds between cysteine residues in the hinge region, can be crystallized, and therefore designated as Fc (Fragment crystallizable). Fc is able to fix C1q, but does not bind antigen. Pepsin degrades human IgG1 into two different fragments: i) one N-terminal large fragment of about 100 kDa size, composed of [2(VL+CL+VH+CH1) + Hinge region], and ii) several small Cterminal peptide fragments, the largest being of about 110 AAs. The N-terminal fragment is functionally bivalent, i.e., can bind two epitopes and cross-link multivalent antigens so as to agglutinate or precipitate them, and therefore designated as F(ab)2 (Fig. 1). The largest Cterminal fragment is designated as Fc, just to distinguish it from Fc of the papain digested IgG1. In this manner, these early studies were of immense use for elucidating the structure of Ig molecules and continued efforts culminated into determination of the fine structure of Igs of all major classes from various species. Structure of human and mice Igs is known in more details than those of other species. AA sequencing of myeloma proteins and later of monoclonal antibodies in conjunction with the X-ray crystallographic studies for their three-dimensional conformation, have over past four decades revealed several structural features of Abs that could explain their functions. Nucleotide sequencing of Ig gene segments has allowed faster progress of this field of knowledge. The evolutionarily conserved Ig fold having two layers of pleated sheets as described in the preceding paragraphs is clearly elucidated in the structures of Ig prototype and other members of a large family of Ig-like proteins, called Ig superfamily (IgSF). A region lying between CH1 and CH2 domains of IgG molecules is rich in proline residues that allow rotational and translational flexibility to Fab arms and therefore called hinge region. IgG can adopt Y to T shape due to the mobility in its Fab arms that gives advantage of reaching of the two paratopes to two epitopes of multivalent antigens at varying space. T shape is however more commonly of IgA1 than that of other Igs. Hinge region also has cysteine residues that are involved in disulphide bonding between the two H chains. Hinge region is of variable length in different Ab molecules, even different IgG subclasses, but is lacking in IgM and IgE isotypes. In human IgG1, it has 15 AAs and is divided into proximal, core and distal parts. The core has CysPro-Pro-Cys-Pro sequence. A more elongated Cys- rich hinge region occurs in human IgG3 and account for its functional differences from other IgG subclasses. Human IgA1 has a hinge region of 13 amino acids in length, which is rich in O-linked glycosylation sites and is susceptible to bacterial proteases. Human IgA2 lacks hinge region and is not susceptible to bacterial proteases. A full-sized domain in IgM and IgE each i.e., CH2 replaces a typical hinge region (these isotypes have four CH domains instead of three in other isotypes). However, overall structures of these Ab molecules have compensations for flexibility. Ab fragments smaller than F(ab)2, Fab or Fc, to which some function can be assigned, have now been constructed by the application of recombinant DNA methodologies. One such fragment, composed of [1VH+1VL] domains, designated as Fv (Fragment variable), harbours one paratope and thus may also be called a single epitope- binder. Still smaller or rather the smallest epitope-binder composed of a single VH domain have recently been constructed and have vast potential for use in therapy, biosensors, proteomics, etc. Such fragments can be expressed in E. 9

coli or by phage display system in correctly-folded structure that performs the function of specific antigen binding. Precise information is now available on the binding of different types of antigenic determinants to different paratopes, and also on that of several types of effector molecules (C1q and cell surface Fc receptors) and other ligands to Ab molecules outside their paratopes (Tables 3-5). Fc receptors for IgG, IgA, IgM and IgE have been found to be present on various leukocytes (Table 3). Some proteins of bacterial and viral origin have also been found to act as Fc receptors. Notable among them are: staphylococcal protein A, streptococcal protein G and herpes simplex virus proteins gE and gI (Table 4). In addition, lectins of plant and animal origin, fibronectin, rheumatoid factor (IgM or IgG autoantibody) can bind to Igs in their Fc regions (Table 5). Binding sites of Igs that lie outside the paratope region for several different molecules are precisely known at present. In particular, CH2-CH3 cleft of IgG is promiscuous, i.e., acts as binding site for several different ligands and therefore has control over several functions. Table 3: Various soluble and cell surface receptors which bind in the Fc regions of Igs for effector functions Ig binding effector Present on/ in molecule I. FcRs in effector functions FcRIa,b,c (CD64) Most leukocytes FcRIIa,b (CD32) Most leukocytes FcRIIIa,b (CD16) Most leukocytes, including NK cells FcRI (CD89) Monocytes, neutrophils, eosinophils Fc/R Mature B lymphocytes & monocytes FcRI Basophils, mast cells and some other cells FcRII (CD23) Most leukocytes II. Complement binding C1q serum C3b & C4b (thioester serum bonds) Binding to

Lower hinge-C2 Lower hinge-C2 Lower hinge-C2 C2- C3 interface ? C3 C3 C3 & C2 Covalent binding at multiple sites of IgG

10

Table 4: Proteins of bacterial and viral origin (microbial FcRs) binding to Igs in their Fc regions Microbial FcR Staphylococcal protein A (SpA) Streptococcal protein G (SpG) gE & gI Bacteria/ virus Staphylococcus aureus Streptococcus pyogenes strains C & G Herpes simplex virus (HSV-1) proteins Binding to Ig region Mammalian C2-C3 interface & also Fab Mammalian C2- C3 cleft & also Fab Human C2- C3 cleft

Table 5: Lectins and other ligands binding to Igs in their Fc regions Ligand Lectins Fibronectin Rheumatoid factor (IgM/ IgG autoantibodies) Source Plants & animals Body fluids & extracellular matrix Serum & synovial fluid Binding to Ig region Ig glycans Fc of aggregated human Igs C2- C3 cleft

Antibodies vis-a-vis enzymes Basic plan of Ab structure and function is same for all Ab molecules: they are glycoproteins of two identical L chains and two identical H chains or their multimers to bind the antigen specifically and effect its disposal/ destruction. Enzymes are structurally diverse (proteins of different shapes and sizes, ribonucleic acids) but all carry out one basic function of biocatalysis, after binding specifically with their respective substrates. Some Abs have however been found to act as enzymes and are termed as abzymes or catalytic antibodies. Similarities and differences between Abs and enzymes have been summarized in Table 6.

Different Ab classes and subclasses are for diverse effector functions Differences in the CL and CH sequences are also responsible for differences in their biological behaviour. Due to differences in CL of L chains, there exist two classes of L chains, designated as and in nearly all mammalian species. But in any one Ab molecule, only a pair of either or is found in association with a pair of H chains. The ratio of - bearing Igs to - bearing Igs varies in different species: : ratio in mice & rats is 95:5, and in humans 60:40. Differences in CH sequences generate different classes and subclasses of H chains and hence of Abs. In mammals including humans, due to five different H chains, designated as , , , , and , there are respectively five different classes: IgM, IgG, IgA, IgE and IgD. Four subclasses of human IgG, designated as IgG1 (1), IgG2 (2), IgG3 (3) and IgG4 (4), and two of IgA, 11

designated as IgA1 (1) and IgA2 (2) are found in all healthy individuals. In mice, four subclasses of IgG, designated as IgG1 (1), IgG2a (2a), IgG2b (2b) and IgG3 (3) are found in all healthy individuals (Table 7). Table 6: Similarities and Differences between Enzymes and Antibodies Feature Function Chemical nature Size Structure Binding sites Biocatalysis Most are proteins, including some Abs (Abzymes); some RNA (ribozymes); Wide range (a few kDa to MDa) Different for different enzyme Substrate binding site & catalytic site are closely placed to make the active site i) By binding essentially to cofactor/ co-enzyme ii) By conversion of zymogen to active enzyme Non-covalent and covalent bonds with substrate or transition state Enzymes Antibodies Antigen recognition & disposal Glycoproteins

Gain of activity

150-180 kDa (monomers) and 2-6X monomers (multimers) (L-H-H-L)1-6 Ag binding site & effector binding site are distantly placed Fully active in secreted form; Effector binding to C or FcR follows Ag binding Non-covalent bonds with Ag as well as effector molecules

Type of chemical bonds involved Binding

Specific to small molecules or small regions of large molecules Constitutive or induced Different enzymes produced by many different types of cells

Expression Producer cells

Specific to small molecules (haptens) or small regions of large molecules Induced by contact of B cell clone with cognate Ag Ag-specific B cell clones

Table 7: Classes and subclasses of immunoglobulins of human and mice Species Mice Human Immunoglobulin classes & subclasses IgG1, IgG2a, IgG2b, IgA IgM IgE IgG3 IgG1, IgG2, IgG3, IgG4 IgA1, IgA2 IgM IgE IgD IgD

12

Different classes and subclasses have evolved to perform diverse effector functions of the immune system (Table 8). Abs have basically two distinct regions that perform two distinct functions: a) antigen binding region made up of VL and VH domains, and b) effector binding region, made up of C-terminal 3-4 Ig domains in the H chains that bind effector molecules and cellular receptors. Table 8: Biological properties of human immunoglobulins Feature IgM Half-life (days) 7-10 Ig class IgGs IgAs (IgA1, IgE (IgG1 to IgG4) IgA2), sIgA 2 21- 23 (IgG1, 6 (monomer) IgG2, IgG4) ? (dimeric sIgA) 7 days (IgG3) Serum & tissue Serum & mucosal Basophils & fluids secretions mast cells in tissues IgG3 (+++), (Only alternate IgG1 (++), IgG2 pathway by IgA1) (+), IgG4 () IgG2 & IgG4 mostly Yes IgD 3

Tissue distribution Complement activation Transplacental transfer High affinity binding to basophils & mast cells Binding to microbial proteins (SpA &SpG) Opsonization ADCC Virus/ toxin neutralization Bacterial agglutination

Mostly in serum ++++

Nave mature B cells

All subclasses but with varying affinity Yes Yes, in mucosal secretions Yes, in mucosal secretions

Yes, Yes, efficiently efficiently Yes Yes Yes Yes, very Yes efficiently

Structural variants of Abs are genetically determined and antigenically distinguishable Isotypes (Ab classes) and subtypes (Ab subclasses) These are structural variants of L and H chains of Igs, encoded by separate genes present in all healthy individuals of the vertebrate species. For example, mouse has two different isotypes of L chains, designated as and , and five isotypes of H chains, designated as , , , , . Four 13

subtypes of exist in mouse and are designated as 1, 2a, 2b, 3. H chain isotypes and subtypes make different Ig classes (IgM, IgG, IgA, IgE & IgD respectively due to , , , , ) and subclasses (IgG1, IgG2a, IgG2b & IgG3 respectively due to 1, 2a, 2b, 3). Human subclasses of IgG are: IgG1 (1), IgG2 (2), IgG3 (3) and IgG4 (4). Human IgA, has two subclasses, namely, IgA1 (1) and IgA2 (2). Abs being proteins themselves have all the features of good antigens and induce production of anti-Abs when inoculated in suitable individuals other than the self. The isotype- and subtypespecific antigenic determinants on L and H chains are restricted to their respective C regions. Different classes and subclasses of Igs can thus be serologically discriminated by using classspecific and subclass-specific antisera raised in individuals of a different species or specific monoclonal antibodies. Allotypes Structural variants of L and H chain isotypes and subtypes exist within individuals of the same species, because of the presence of alleles of isotype and subtype genes in populations. Allotypes are therefore shared among some but not all individuals of a species e.g., G1m(1) and G1m(3) are human IgG1 allotypes showing sequence differences in CH1 and CH3 domains respectively. Most of the allelic differences lie in the constant regions of L and H chain genes, thereby restricting the allotype antigenic determinants to these regions. Allotypes can thus be detected serologically by using anti-allotype allo-antisera (antisera produced by injecting allotypic Ig into the individuals who lack that particular allotype) or specific monoclonal antibody. Idiotypes Structural variants of V regions of L and H chains of Igs that can be detected as antigenic determinants are called idiotypes. Igs produced by different B cell clones in an individual exhibit different idiotypes. So Ig molecules produced by each B clone has its characteristic idiotype. An idiotype is a set of antigenic determinants, called idiotopes, on V regions of L and H chains. Some of the idiotopes are conformational and others are linear. Some of them may also be shared with those on other Igs within the same individual or species or even across the species. Antiidiotype Abs are normally produced in every individual that result in creation of idiotype-antiidiotype networks for regulation of the immune responses. Anti-Id Abs can be produced by immunization of individuals with the Ab of a particular Id or by hybridoma technique. Biological functions of Abs Classical functions of Abs Abs have two major functions i.e., i) antigen recognition by specific binding of Ag in the Agcombining site (paratope), and ii) antigen disposal following Ab binding to various sorts of effector molecules in the regions outside paratope, mostly in their constant domains. Following Ag-Ab complex formation, three major types of effector molecules can bind to Ab leading to Ag disposal by different mechanisms. They are: i) C1q binding to IgM and aggregated IgG molecules leading to activation of the classical pathway of complement, ii) binding to various FcRs on macrophages and other leukocytes for opsonization of the particulate Ags, and also for release of the inflammatory mediators from leukocytes, and iii) binding to FcRIII on natural killer cells for Ab-dependent cell-mediated cytotoxicity (ADCC). The effector molecules binding 14

to Fc regions of different Ig isotypes are presented in table 3. It must however be remembered that Ab-mediated neutralization of toxins and viruses occurs merely by their binding on Ab paratopes. These are examples of Fc region-independent effector function of Abs. In addition, Igs can bind in an Ag- independent manner on the cell surface receptors on epithelial and endothelial cells. Neonatal Fc receptor (FcRn) is responsible for transcytosis of IgG across placenta in humans and rodents, gut epithelium of the colostral IgG in neonates, and in mammary glands secretion of the lactating mammals. FcRn also regulates the levels of IgG in serum. Polymeric Ig receptor (pIgR) present on abluminal side of mucosal epithelia allows binding of polymeric Ig isotypes, namely dimeric IgA and pentameric IgM, and transports these molecules on the luminal side. Then it is cleaved to yield a secretory component (SC) that remains associated with the secreted form of the polymeric Ig. SC protects the secretory Ig from proteolytic degradation on the mucosal surfaces. Igs also interact with several different proteins of bacterial and viral origin (Table 5). Protein A of certain strains of Staphylococcus aureus (SpA) and protein G of Streptococcus pyogenes (SpG) are fully characterized for their binding to most subclasses of mammalian IgG in their Fc regions. These so-called microbial FcRs are structurally different from leukocyte FcRs, the latter being members of IgSF. SpA also binds in Fab region of mammalian IgGs and acts as a B cell superantigen. Proteins from other bacteria and viruses have also been found to bind on Igs. Microbes use the Ig-binding ability to their advantage, thereby affecting the course of the diseases caused by them. Purification of IgG is often done using SpA- and SpG- affinity chromatography. Lectins of plant and animal origin, and various synthetic ligands have also been found to interact with various parts of Igs outside the paratopes (table 6). Such interactions might have bearing on immunological disorders. Important classical functions of different Ab classes and subclasses IgM IgM is the predominant Ab class in primary immune response and exists mostly as pentamers and some as hexamers. It is very efficient in precipitation, agglutination and opsonization of antigens. It fixes very efficiently complement component C1q after binding with the antigen and triggers C-activation by classical pathway. A single pentameric molecule is able to trigger C activation. Being a large pentameric molecule, it is abundant in serum and negligible in tissue fluids. Its secretion onto mucosal surfaces is mediated by polymeric Ig receptor (pIgR). IgG IgG is the predominant Ab class in late primary and secondary (booster) humoral immune responses and remains in circulation for longer time than any other Ab class. It is efficient in precipitation, agglutination and opsonization, but less effective than the pentameric decavalent IgM. It fixes complement component C1q after binding with the antigen and triggers classical pathway of C-activation. But aggregation of IgG molecules on the antigen is required for C1q binding. Its concentration is the highest of all isotypes. It occurs in almost equal amounts in both serum and tissue fluids. Unlike humans and rodents, it is secreted in colostrum and milk of ruminants in more amount than that of IgA. Different subclasses of IgG show variation in their effector functions (Table 8). 15

IgA IgA is abundant in mucosal secretions and the primary mediator of mucosal immunity. Colostrum is rich in IgA. It is present in more amounts than other Ab classes in milk in rodents and humans. It is mainly synthesized in gut and respiratory tract mucosa-associated lymphoid tissues (MALT). In the mucosal secretions, it mainly exists as a dimer joined by a J chain and held together by a secretory component (SC), and designated as SIgA (secretory IgA) (Fig. 6). Human IgA1 subclass is more abundant than IgA2 in serum and secretions, except in colon where IgA2 has more concentration. SIgA protect mucosal surfaces from pathogens by excluding their entry, agglutinating them, interfering with flagellar motility and neutralizing microbial toxins at these sites. Systemic IgA mediates opsonization of microbial Ags by engagement of FcRI on phagocytes. It also induces respiratory burst activity, and release of cytokines and proinflammatory mediators from phagocytes.

IgE This antibody class is involved in allergies (type I hypersensitivity) and parasitic immunity. Unlike other Ab classes, it is heat-labile and inactivated at 56C in 30 min. It binds via high affinity FcRI on basophils and mast cells in tissues, where it remains in bound form, and is therefore also called cytophilic antibody. Allergen binding by these cytophilic IgE Abs on mast cells causes release of the granular contents that are the inflammatory mediators of type I hypersensitivity. It is also called reaginic antibody for its involvement in allergies. It binds to other leukocytes via low affinity FcRII for its role in immune response generation.

16

IgD IgD is cell surface-associated BCR, in addition to IgM monomer on mature B lymphocytes, and is of the same specificity for Ag as that of IgM. Its role in immunity is not clearly understood, but is possibly involved in increasing the efficiency of B cell activation following Ag binding onto the B cell. Non-classical functions of Abs Recently, several V-domain mediated functions of Abs, in addition to Ag binding, have been found. These are: catalysis, nucleotide binding, self-binding and superantigen binding. The conserved segments of the V domains mediate most of these functions, but their biological significance is not clearly understood at present. Different subsets of Abs show these nonclassical functions and a term superantibody has been proposed in order to distinguish them from the conventional Ag-binding function of Abs. Superantibody activities may be involved in autoimmune disease and protection against infections. For example, levels of certain catalytic Abs are increased in autoimmune diseases. These novel activities may also find applications in biotechnology and medicine. Box 2: In vivo biological roles of antibodies Ab neutralizes viruses and toxins by binding with them specifically and blocking virus entry in the cells or toxin binding on cellular receptors Abs complexed with Ags activate classical pathway of complement leading to lysis of the membrane-bound Ags such as bacteria, enveloped viruses and other pathogens Abs act as opsonins to enhance phagocytosis of the Ab-coated Ags Abs link cell surface Ags to NK cells for Ab-dependent cellular cytotoxicity Abs can act as positive and negative regulators of inflammation and cell-mediated immunity Abs are involved in mediating hypersensitivities (IgE in type I, IgG in type II and type III reactions) Abs are involved in autoimmunity Abs are involved in removal of effete cells & debris during normal wear and tear in the body Abs exert direct anti-microbial effects by binding on surface proteins and causing interference with microbial physiology, thereby acting somewhat as antibiotics

Abs can act as positive and negative regulators of inflammation and cell-mediated immunity. Complement (C) activation by Ag-Ab complexes produces proinflammatory C components. Cross-linking of FcR by immune complexes promotes phagocytosis by macrophages. Specific Abs can influence inflammatory response by removal of microbial antigens that have proinflammatory or anti-inflammatory effect in the host. IgM are often proinflammatory due to their pronounced C activating ability. IgGs can either be pro- or anti-inflammatory, depending on their subclass, concentration, and interactions with stimulatory or inhibitory FcRs. Abs have also been shown to exert direct antimicrobial effects. By binding on surface proteins, Abs cause interference with microbial physiology and exert anti-microbial effects. In this manner, they act somewhat as antibiotics. For example, anti- E. coli lipopolysaccharide (LPS) 17

antibodies interfere with enterochelin release, thereby preventing iron acquisition by bacteria leading to bacteriostatic effect. Abs, when given as Ab-Ag complexes, regulate the immune response to the Ag. IgM-Ag complexes often augment the immune response against the antigen. These immune complexes activate C resulting in binding of C3b component on the Ag. This complex would then be trapped by complement receptors (CRs) on the surface of follicular dendritic cells (FDCs) in the germinal centers. Thus specific B cell clone coming to the site makes contact with the Ag trapped on the FDCs. IgG-Ag complexes are often found to have inhibitory effect on immune response development. It could be that IgG acts as blocking Ab (Ag masking Ab) or inhibits immune response in an FcR-mediated inhibition of B cell activation. Abs as biocatalysts An antibody that can act as a catalyst for a chemical reaction, in addition to its specific binding to the antigen, is termed as catalytic antibody or abzyme. More than 100 different monoclonal antibodies having catalytic activity have been reported that carry out as diverse reactions as pericyclic processes, elimination reactions, hydrolyses, bond-forming reactions and redox reactions. Bence-Jones proteins from multiple myelomas and L chains of certain autoAbs can express peptidase activities. VIPases, DNases, esterases and thyroglobulinase activities have been described in abzymes. Haptens and peptide Ags can induce catalytic Ab synthesis. The transition state analogues can induce the production of antibodies. These Abs in their active sites bind to the transition state more strongly than the substrate. Further genetic manipulation introduces catalytic residues in their active sites. Like enzymes, Abzymes process their substrates through a Michaelis complex in which the chemical transformation occurs followed by product dissociation. The steady-state kinetics for all abzymes obey the Michaelis-Menten rate expression. The value of kcat/ kuncat for abzymes is generally in the range of 101 to 106. It is very difficult to demonstrate catalytic activity of antibodies in antisera, because of their heterogeneity. Monoclonal antibodies and recently phage display library antibody fragments are therefore used for searching catalytic activities in them. Cat ELISA is a test used for screening of abzymes, in which the product formed during catalysis is captured by another product-specific antibody that is then detected by ELISA amplification system. Abzymes are still in infancy, but are promising in the sense that they can carry out certain chemical reactions more efficiently than the classical enzymes do. Forces involved in antibody-antigen interactions Antibody binds to the antigen specifically in its paratope, involving various types of noncovalent bonds. They are listed below: 1. Ionic bonds or electrostatic interactions typically between COO and NH3+ groups on ionized amino acids of interacting paratope and epitope. These bonds are most influenced by pH and ionic strength of the medium. Bonding energy is in the range of 5-8 kcals/ mole. 2. Hydrogen bonds between OH and NH2 groups of interacting paratope and epitope. Bonding energy is 1-2 kcals/ mole.

18

3. van der Waals forces, very weak forces due to electron clouds of atoms and molecules that have come very close to each other. Bonding energy is ~1 kcal/ mole. 4. Hydrophobic interactions due to exclusion of water or polar solvent molecules from the two non-polar moieties approaching each other. Bonding energy is ~5 kcals/ mole. About 50% of the binding strength comes from these interactions. Box 3. Forces involved in Ag-Ab interaction Only non-covalent reversible bonds are formed between Ags & Abs, which are: Ionic interactions (5-8 kcal/ mole) Hydrophobic interactions (5 kcal/ mole) Hydrogen bonds (1-2 kcal/ mole) van der Waals forces (1 kcal/ mole) Although covalent bonds are not involved in Ab-Ag interactions, multiple non-covalent bonds have good amount of bonding energy for providing overall stability to Ab-Ag complexes. Unlike covalent bonds, non-covalent binding of paratope and epitope is reversible and the net outcome of difference between total attractive and total repulsive forces. Therefore, Ab-Ag interactions are explained by using the law of mass action. Ab-Ag interactions are usually exothermic at room temperature or lower temperatures. Obligatory long-range attraction between paratope and epitope also play their role in Ab-Ag interactions. Biopolymers repel each other and no two molecules can come nearer than 3-5 nm to each other. Paratope and epitope can however penetrate the repulsion fields of each other and come within 3 nm of each other due to their shape complementarities, opposite electrostatic charges and hydrophobicity of the paratope. Affinity, avidity and specificity of antibodies Antibody affinity Affinity is a thermodynamic parameter to quantify the strength of binding of two interacting molecules in solution. Antibody affinity is thus the strength of binding of a single paratope with a single epitope in appropriate solution. Ab affinity is also called intrinsic affinity and depends on structural complementarity of paratope and epitope. Small changes in the structure of paratope affect the Ab affinity. Affinities of different Abs range between 105/ M (low affinity) to 1011/ M (high affinity). Ab affinity matures or improves in the developing immune response against the inducing antigen by the process of somatic mutations in CDRs of the Ig V gene segments in B lymphoblasts. B lymphocyte dividing by mitosis following antigen recognition is called B lymphoblast. Ab affinity can be measured experimentally (by equilibrium dialysis, radio-immunoassay, competitive ELISA, etc.), wherein a single species of Ab molecules and a monovalent epitope or hapten are used. According to the law of mass action, KA= [Ab.Ag]/ [Ab][Ag] Where, KA is the affinity constant, [Ab.Ag] is the conc. of Ab-Ag complex, 19

[Ab] is the conc. of free Ab, and [Ag] is the conc. of free Ag Ab affinity is measured using Scatchard equation or other suitable equations. Scatchard equation: K= r / (n-r) (c) Where, r = Conc. of bound Ag (ligand)/ Conc. of total Ab molecules in the system n = Ab valency (= 2 for IgG), c = Conc. of free or unbound ligand. Monoclonal antibodies are homogeneous i.e. each and every monoclonal Ab molecule in a solution is of the same affinity for the antigen. Whereas, Abs against a particular epitope present in antisera are heterogeneous i.e., Abs produced by the B lymphocyte clones in a developing immune response have slight variations in their affinities against the same epitope. Such variations in Ab affinities are introduced by somatic mutations in CDRs of Ig V gene segments in the mitotically dividing B lymphoblasts. Practical importance of Ab affinity In vivo reactions carried out by high affinity Abs are functionally better than those by low affinity Abs, since latter are difficult to be removed from the body and may lead to pathological conditions. High affinity Abs are more useful in serological tests in vitro as they ensure more specificity. Moderate affinity Abs are however more useful in Ab affinity- based purification techniques. Antibody avidity/ functional affinity Ab avidity is the total strength of binding of a multivalent Ab with a multivalent antigen. Ab avidity is governed by the intrinsic affinity of Ab, valencies of Ab and Ag, and stereic hindrance of the interacting molecules. Ab avidity is of more practical consequences than intrinsic affinity. Antibody specificity and cross-reactivity Ab specificity is the ability of Ab molecules to discriminate between the binding of the inducing Ag and that of the antigens chemically related to the inducing one. Ab specificity is governed by the structural (geometrical shape) and chemical (bonding) complementarities between the Ab paratope and the Ag determinant. The binding of Ab to the inducing Ag generally represents the best fit between Ab paratope and the epitope on the inducing Ag. In other words, the Ag that induces the production of specific Ab binds to this Ab with the highest affinity in the tightest possible manner. The epitope on the inducing Ag is called the homologous Ag determinant and the determinants chemically related to the homologous one are called the heterologous or cross-reactive determinants. Some exceptions to this rule exist in that certain heterlogous Ags can bind with higher affinity to an Ab induced by homologous Ag. Such Abs are called heteroclitic Abs. Ag determinants that are chemically different but bind with the homologous Ab are known as mimotopes e.g. peptides mimicking the carbohydrate epitopes for binding to the anti-carbohydrate Ab. Abs display exquisite specificity for the antigens i.e., they can discriminate between stereoisomers, optical isomers and charge differences due to variations in the primary structure of the closely related Ags. 20

Box 4: Ab affinity, avidity and specificity Antibody affinity is the strength of binding of a single paratope with a single epitope Ab affinities range between 105/ M (low) and 1011/ M (high) Ab affinity is measured by using Scatchard equation: K= r/ (n-r).c Ab avidity is the total strength of binding of a multivalent Ab with a multivalent antigen Ab specificity is the ability of Ab molecules to discriminate between the binding of the inducing Ag and that of the antigens chemically related to the inducing one Antisera represent complex mixtures of antibody molecules of polyclonal origins Chemically related epitopes and antigens can bind to homologous Ab, but with lower affinities than that of homologous Ag:Ab pair Epitopic cross-reactivity with homologous Ab results from reduced structural and chemical complementarity of heterologous epitopes for homologous Ab paratope Cross-reactivity of multivalent antigens (antigenic cross-reactivity) with homologous antisera may result from both epitopic cross-reactivity and epitope sharing between homologous and heterologous Ags

Antisera represent complex mixtures of antibody molecules of polyclonal origins. They show many different specificities which arise in two different ways: i) Two or more different Ags given simultaneously in the same animal will result in production of Abs against all of them and therefore the antiserum would react against all of them, ii) Most Ags existing in Nature are multivalent and therefore different Abs are produced against different epitopes of the multivalent Ag. These Abs originate from different B cell clones and therefore antisera contain a mixture of polyclonal Abs. The cross-reactive Ags share some of the epitopes with the homologous Ags are therefore able to bind with Ag-specific Ab in the antisera. Chemically related epitopes and antigens can thus bind to homologous Ab, but with lower affinities than that of homologous Ag-Ab pair. In other words, we can say that epitopic crossreactivity with homologous Ab results from reduced structural and chemical complementarity of heterologous epitopes for homologous Ab paratope. On the other hand, cross-reactivity of multivalent antigens (antigenic cross-reactivity) with homologous antisera containing homologous Abs against different epitopes on the homologous Ag may result from both epitopic cross-reactivity and epitope sharing between homologous and heterologous Ags. Ig genes and generation of antibody diversity The extent of antibody diversity No two immunoglobulin molecules produced by two different B lymphocyte clones are alike. A huge number of different B lymphocyte clones exist in healthy individuals of all vertebrate species that bear immune system. The clones are different for they bear different antigen-specific B cell receptors (BCRs) on their surface. The number of B cell clones in different species is 21

however different, depending on the size of the species and blood composition. For example, an adult mouse has about 107 B cells and therefore, a possible maximum of 107 B cell clones producing 107 different Abs that bind specifically with an equal number of different Ags. However, the number of antigens in the whole universe is enormous, and therefore 107 different Abs make a relatively limited number for the species to cope up with the vastness of the antigenic world. Nevertheless, molecular biology studies during past three decades have clearly suggested that using genetic information contained in segmented genes for L and H chains, molecular mechanisms can potentially produce diverse Abs of the order of 1016. Then, how can a mouse produce antibody against any imaginable Ag introduced into it? One possible answer lies in the fact that BCR can bind specifically with one Ag and the same can also bind with cross-reactive Ags initially giving sufficient threshold signal to trigger the immune response. Subsequent to this initial event, the developing immune response improves Ab affinity against the inducing Ag by somatic hypermutation. This may bring the figure to 109 different Abs at the most. Recently discovered polyspecific antibodies may further solve this puzzle to some extent. But we also remember at the same time that any individual encounters only a limited number of Ags in its surroundings during its lifetime and this limit can be of the order of millions. Which antigens will be encountered at what time in an individual is however not predetermined to a large extent and the individuals are not pre-cognizant of the Ags in the changing environment! Ig genes and their genomic organization Ig is composed of two identical IgH chains and two identical IgL chains derived from Ig or Ig gene segments. The murine IgH locus has upto a thousand V gene segments in about 1 Mb region that begins about 100 kb upstream of C on chromosome 12. A cluster of 4 J gene segments lie about 7.5 kb upstream of C. Between VH and JH are present 13 D gene segments. VH gene segments at their 3- ends and JH gene segments at their 5-ends are flanked by recombination signal sequences (RSS) containing 23-bp spacer sequence. D gene segments are flanked on both sides by RSS with 12-bp spacer sequence. Eight CH genes are located downstream of the VDJ gene segments in a 200 kb region on chromosome 12. The order of murine CH genes starting downstream of JH is 5C-C-C3-C1-C2b-C2a-C-C- 3. As C is the first to recombine with a functionally rearranged VH(D)JH exon, IgM is the first isotype expressed on the B cell surface. Alternative RNA splicing of the V(D)JH exon to the C exons leads to the expression of IgD. Thus, both IgM and IgD are expressed on nave mature B cells. All other isotypes are produced by class switch mechanism described elsewhere. Switch regions are present upstream of every C gene except C. Each C region gene has a separate exon for each CH domain and hinge region and an additional M exon for cell membrane-bound form of Igs. Ig locus is about 3 Mb size on murine chromosome 6 and has 140 V, and 4 functional and one non-functional J gene segments just upstream of a single C gene. Unlike VH and Ig, V gene segments are in both transcriptional orientations and are rearranged by both deletions and inversion of intervening sequences. V gene segments are flanked by 12-bp RSS and J by 23-bp RSS.

22

Ig locus is about 200 kb on murine chromosome 16 and has 3 functional V flanking 23-bp RSS. Upstream of 3 functional and one non-functional C are J gene segments flanked by 12bp RSS. Genomic organization of mouse IgH, Ig and Ig loci are shown in Figure 7.

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Human IgH, Ig, Ig gene segments are present on chromosome 14, 2 and 22, respectively (Table 9). The number of different gene segments is shown in Table 10. Genomic organization of human Ig genes is similar to that of mouse. The order of human CH genes starting downstream of JH is 5- CM-CD-CG3-CG1-CA1-CG2-CG4-CE1-CA2- 3 encoding respectively , , 3, 1, 1, 2, 4, , 2 regions. Like murine CH gene, each human CH gene has a separate exon for each CH domain and hinge region and an M exon for cell membrane-bound form of Igs. Table 9: Chromosomal location of Ig genes in mouse and man Ig gene encoding polypeptide Ig gene location on chromosome number chain Mouse Man IgH IgL () IgL () 12 6 16 14 2 22

Table 10: Approximate number of Ig gene segments (excluding pseudogenes) in mouse and human Gene segment VH DH JH V J V J Approximate no. of gene segments Mouse 1000 13 4 25 4 3 3 Man 50 25 6 40 5 30 4

Molecular mechanisms that underlie Ab diversity Several molecular mechanisms as listed below generate antibodies of huge diversity: (i) Existence of multiple V & J gene segments for L chains and V, D & J gene segments for H chain in the germline DNA (in undifferentiated hematopoeitic stem cells). However, germ line DNA is not transcribed into any functional mRNA for L and H chains. It has to be rearranged by genetic recombination in developing B cells in primary lymphoid organ (bone marrow) to bring together distantly placed gene segments. Out of many possible combinations and permutations, only one set of the recombined V-J gene segments of L chains and one set of the recombined V-D-J gene segments of H chain are present in transcribable form in any developing B cell clone. 24

(ii) (iii)

(iv) (v) (vi)

Imprecision in V-J and V-(D)-J recombination initiated by recombination activating gene (RAG) proteins introduce further changes in the VL and VH gene sequences. N- nucleotide additions 3- to VH and D gene segments (non-template dependent addition of a few nucleotides) by terminal deoxynucleotidyl transferase (TdT) in pro-B cells are responsible for VH gene sequence changes. Pro-B cells refer to those cells committed to B lineage. Reassortment of H and L (/) polypeptide chains contributes significantly to the diversity of Abs. Somatic (hyper)mutations in the rearranged VL and VH gene segments of mitotically dividing B lymphoblasts following contact with antigen. This mechanism ensures production of high affinity specific antibodies. Gene conversion by means of pseudogene insertion into very few functional V gene segments also generates diverse Ab specificities in some species such as poultry and rabbits. Box 5: Molecular mechanisms that generate Ab diversity

Molecular mechanisms are capable of generating Ab diversity of the order of 1016 There are several different molecular mechanisms for generating Ab diversity i. Presence of multiple V, J gene segments for L chains & V, D, J gene segments for H chains ii. Imprecision in recombination of different V(D)J gene segments iii. N- nucleotide additions to H gene by terminal deoxynucleotidyl transferase iv. Random reassortment of H & L chains v. Gene conversion in some species vi. Somatic hypermutation in activated B cells The first five mechanisms are Ag-independent occurring in developing B cells in the primary lymphoid organ & the last one occurs in activated B cells following Ag exposure in the secondary lymphoid organs/ tissues

Major genetic mechanisms that diversify Ab specificities and effector functions V(D)J recombination diversifies Ab specificities before contact with the Ags V(D)J recombination is a process by which the paratope-containing variable regions of receptors on B and T cells (BCR and TCR respectively) are generated in primary lymphoid organs. It involves rearrangement of the gene segments in the developing B and T cells. V(D)J recombination is initiated by recombinase, consisting of recombination activating gene (RAG-1 and RAG-2) products, which cuts DNA precisely at conserved recombination signal sequences (RSS). The RAG-induced double-strand DNA breaks are repaired by non-homologous endjoining (NHEJ) DNA repair pathway. RSS is made up of conserved heptamer non-conserved spacer sequence conserved nonamer, i.e., [5-(CACAGTG)(relatively non-conserved 12-bp or 23-bp intervening spacer sequence) 25

(ACAAAAACC)-3]. RAGs can put together RSS having a 12-bp spacer sequence with only that having a 23-bp spacer sequence and this requirement is called the 12/23 rule. The 12/23 rule prohibits direct VH to JH joining and ensures the usage of D gene segments during normal V(D)J recombination. RAGs form a synaptic complex (SC) by associating with 12-bp RSS, 23-bp RSS and their flanking coding gene segments. Following SC formation, RAGs introduce a single-strand nick in the DNA between the heptamer and the gene-coding segment. The 3-OH on one DNA strand of the gene coding segment attacks the opposite DNA strand forming a covalently sealed hairpin coding end (CE) and a blunt 5-phosphorylated signal end (SE). Hairpin CEs are nick-opened to create 3 overhangs of 4-5 nucleotides, which are filled-in via DNA polymerases to generate palindromic sequences, called P nucleotides. This serves as a mechanism to diversify Ab. To further diversify junctions, lymphoid-specific terminal deoxynucleotidyl transferase (TdT) adds random non-template nucleotides (N- nucleotides) to the 3-coding ends. TdT is not required for V(D)J recombination or lymphocyte development, but affects diversification of overall repertoire. NHEJ repairs broken DNA ends during G1 phase of the cell cycle. The CEs are modified by both the loss and addition of nucleotides, while the SEs are repaired precisely using the same doublestrand repair factors. Hairpin opening requires additional factors such as DNA-dependent protein kinase and Artemis. Later stages of NHEJ possibly also require XRCC4 and DNA ligase 4. RAG-1 and RAG-2 genes are expressed predominantly in the developing lymphocytes and their deficiency arrests the development of B and T lymphocytes at progenitor stages leading to severe combined immunodeficiency. RAG expression is co-incident with beginning of IgH rearrangement in Pro-B cells and is terminated during the transition from pro-B to pre-B cell stage. Box 6:V(D)J recombination that diversifies Ab specificities V(D)J recombination: Is a mechanism to generate diverse Ag receptors on B & T cells in primary lymphoid organs Is initiated by recombinase, consisting of two gene products, namely, RAG-1 & RAG-2 RAGs cleave DNA at recombination signal sequences (RSS) at 3 to V & J gene segments, and both 5- & 3- to D gene segments RSS is made up of 5-[heptamer- spacer sequence (12/ 23 nt)- nonamer]-3 RAG-induced ds DNA breaks are repaired by non-homologous end-joining DNA repair process Lymphoid-specific terminal deoxynucleotidyl transferase adds non-template (N)nucleotides to 3 end of V and D gene segments Ig gene rearranges prior to Ig gene Either or chain (isotypic exclusion), and only one functional H allele & one L allele (allelic exclusion) are expressed in a B cell clone 26

B cells express the functional products of only one IgH allele and one IgL allele, in a process called allelic exclusion. Second allele is allowed to rearrange only in those B cells in which the first V(D)J rearrangement is non-productive, thereby preventing the assembly of multiple Ag receptors in a single B cell. IgL isotype exclusion also occurs in developing B cells. Ig locus rearranges prior to Ig genes in pre-B cells and only the cells that fail to generate a productive Ig would proceed to rearrange Ig genes. IgH locus rearranges in an ordered fashion with D to JH before subsequent V to DJ rearrangement. Association of IgL with IgH makes mature Ag receptor in the form of IgM expressed on B cell surface, which provides further feedback regulation and allelic exclusion. If a newly generated B cell expresses a productive but self-reactive Ag receptor, the process of receptor editing may replace this receptor with a non-self reactive one. Receptor editing involves extended RAG expression for further rearrangement of IgL chain. Somatic hypermutation in V(D)J gene for maturation of Ab affinity occurs in dividing B cells after contact with the antigen After Ag-induced activation of B cells in the germinal centres, mutations at a high rate (10-310-4 / bp/ generation) are introduced into V region exons of IgH and IgL chains. This process is known as somatic hypermutation (SHM). The mutated V regions create Ag receptors of higher affinity than the original ones, a process called affinity maturation. These higher affinity receptor- bearing B cells are selected by the Ag for further differentiation into plasma cells and memory B cells. IgM production, however, occurs before the initiation of SHM, and therefore does not undergo affinity maturation. Box 7: Somatic hypermutation in Ig V gene segment that improves Ab affinity for the Ag Somatic hypermutation (SHM): Occurs in assembled IgV gene segment in activated B cells following contact with Ag in the germinal centres of secondary lymphoid organs Introduces mutations into V region of IgH & IgL chains at a rate of 10-3 10-4/ bp/ generation Different affinities of the Ag receptors are produced as a result Ag selects the highest affinity receptor bearing B cells to proliferate and differentiate further SHM is initiated by activation-induced deaminase (AID) in activated B cells SHM is initiated by activation-induced deaminase (AID) that deaminates cytidine residues on DNA resulting in dU/dG mismatched DNA base pairs. AID is expressed in activated B cells that are undergoing SHM and class switch recombination.

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Antibody class switch diversifies Ab effector functions Different classes and subclasses of Igs have arisen by constant (C) gene duplication during evolution in order to diversify biological functions. The V domain determines Ab specificity for Ag and the C domains determine Ab class specificity and effector functions. Recombination of one V gene with various CH genes can produce Ab molecules of the same Ag specificity but of different classes and therefore of different effector functions. Antibody class or isotype switch is a process of change of Ig heavy chain class in a B cell during the development of immune response. The change is only in the CH region, and not in the VH region or the L chain. Ab class switch begins within 4 days after activation of B cells, but prior to somatic mutation in germinal centers (GCs). Thus, IgG, IgA and IgE are made later than IgM during a primary response and represent most of the Abs made during a secondary (memory) response. IgM to IgD class switch occurs by the mechanisms of alternative RNA processing and termination of transcription, but within B cells already expressing IgM BCR of the same Ag specificity. Unlike Agindependent expression of IgD on mature B cells, switch from IgM to all other classes and subclasses i.e., IgG, IgA and IgE occurs only after the contact with antigen. Class switch recombination (CSR) involves looping-out and deletion of intervening DNA between tandemly repeated sequences called switch regions. CSR is influenced by the nature of antigen, T-B interaction, and cytokines in the microenvironment. Cytokines act usually together with B cell activators for switch recombination to occur. They influence switching by regulation of germline transcripts. IL-4 increases germline mouse 1, human 4 transcripts, and germline transcripts in activated B cells, thereby directing switching to IgG1 and IgE in mice, and IgG4 and IgE in humans. TGF- increases germline transcripts, thereby regulating class switching to IgA in both mice and human B cells. Non-T cell synthesized IL-4 (by mast cells), IFN- (by NK cells and macrophages), and TGF- (many types of cells) can act in conjunction with Tind antigens for Ab class switch. T-B contact involving CD40L: CD40 interaction is the most important contact-dependent signal for inducing class switching to most isotypes. Other receptor ligand interactions include: CD58 (LFA-3) on B cells interacting with CD2 on T and NK cells. Before B cells switch to produce a particular Ab class, unrearranged CH gene of that class is transcribed to produce germline transcripts that do not produce any functional protein. Such germline transcripts seem to be indispensable for switching. The primary transcripts are made up of the I exon, S (switch) region and C exon. Mitogens in the presence of cytokines induce or suppress germline transcription of specific CH genes in stimulated B cells that subsequently results in switching to the same isotype. DNA sequences in the intergenic region upstream of CH genes recombine, resulting in deletion of the chromosomal DNA between switch regions. Switch (S) regions are located 5 to each CH gene except C. Unlike VDJ recombination, switch recombination lacks sequence specificity for the site of recombination and can occur at many different sites within S regions. S regions consist of simple tandem repeats of 1-10 kb length. The repeat unit length of S, S, S and S are respectively 20-, 80-, 40-bp and 49/ 52-bp. Unlike V(D)J recombination, switch recombination sites lack consensus DNA sequences. 28

Box 8: Class switch recombination (CSR) that produces diverse Ab classes & subclasses Antibody class or isotype switch is a process of change of Ig heavy chain class in a B cell during the development of immune response in the germinal centres One rearranged VH gene segment joins to different CH regions in the progeny of Agspecific B cell during immune response Mature nave B cell expresses both IgM & IgD on its surface, both specific to only one Ag One B cell clone after Ag contact produces molecules of only one Ab class IgM to IgD switch occurs by alternative RNA processing in the same B cell IgM switches to IgG/ IgA/ IgE by CSR mechanism CSR involve looping-out and deletion of intervening DNA between switch (S) regions Switch (S) regions are located 5 to each CH gene except C. S regions consist of simple tandem repeats of 1-10 kb length The repeat unit length of S, S, S and S are respectively 20-, 80-, 40-bp and 49/ 52-bp Unlike V(D)J recombination, switch recombination sites lack consensus DNA sequences Germline transcript are synthesized prior to the switching process Ag nature, B cell mitogens, cytokines and activated Th cells influence class switch They influence switching by regulation of germline transcripts. AID cleaves DNA in the S region and the cut ends are joined by non-homologous endjoining repair mechanism

AID, the enzyme expressed in activated B cells in the germinal centers is involved in DNA cleavage of the S region. AID can deaminate cytidines on single-stranded DNAs and is important for both somatic hypermutation and CSR. The DNA breaks are repaired by non-homologous end-joining and mismatch repair proteins. Differences among the above three mechanisms are presented in Table 11. Theories of antibody production- A brief account Today we know that antibodies are specific molecules produced in response to antigens by B lymphocytes in vertebrates. Number of antigens that an individual may encounter in its lifetime is enormous. Since an antibody binds to only one antigenic determinant in the fittest manner/ specifically, individuals must produce a huge number of diverse antibody molecules to deal with a very vast number of antigens in Nature. With the inception of the discipline of Immunology in the last quarter of the 19th century, the question of how antibodies of great diversity were produced attracted the attention of immunologists. During the 20th century, various theories were put forward that tried to explain: a) how antibodies are produced, and b) how specific antibodies of huge diversity are produced. Chronologically, at least four important theories have been put forwarded to explain the above questions:

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Table 11. Differences among V(D)J recombination, somatic hypermutation (SHM) and class switch recombination (CSR) Point of difference Recombination between Time and site of recombination Consequence V(D)J recombination V-D-J exons SHM CSR V(D)J gene with one of CH genes Following contact with Ag, B cells in germinal centers Formation of different Ab classes & subclasses AID-mediated d.s. DNA breaks, specific to S regions Base excision, mismatch repair, NHEJ Yes, before CSR Marked influence on class switch Marked influence

DNA cleavage

DNA repair

Germline transcription Influence of Ag Effect of mitogens & cytokines T-B contact

- [only mutations in V(D)J gene] Before Ag contact, B Following contact and T cells in primary with Ag, B cells lymphoid organ in germinal centers Formation of diverse Affinity VH domains & maturation for the paratopes inducing Ag Site-specific d.s. breaks AID-mediated by RAGs at short ssDNA lesions in conserved signal V(D)J gene sequences Non-homologous end- Base excision, joining (NHEJ) DNA mismatch repair, repair pathway NHEJ No No None Selection of high affinity B cell clones ?

None

Does not influence

Affects

1. 2. 3. 4.

Side chain theory (Ehrlich, 1900) Antigen template theory (Alexander & Mudd, Breinl & Horowitz, Pauling, 1930-40) Clonal selection theory (Burnet, 1960) Network theory (Jerne, 1974)

Side chain theory (Ehrlich, 1900) Paul Ehrlich postulated that the white blood cells (WBCs) bear various surface receptors with side-chains. Binding of foreign substances to these side-chains induces the cells to produce multiple copies of the surface receptors. Excess of the receptors are shed into serum to serve as antibodies.

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In this theory, production of increased amounts of antibodies and their interaction with antigens was explained. However, antibody receptors on WBCs being limited in number did not match with vastness of antigens in nature. Therefore, the theory was discarded in favor of a new concept that antigens could somehow direct antibody specificity during their synthesis in the blood cell. This concept led to the proposition of a new theory during 1930-35, popularly known as antigen template theory. Antigen template theory (1930-35) Collectively, the Ag- template theorists proposals were to state that the antigen served as template for the antibody that adopted a shape and chemical affinity complementary to that of the antigen. This theory remained unchallenged up to 1950s- before the dawn of Molecular Biology. Burnet and Fenner, and Jerne during 1940-50 collected evidence that led to rejection of the theory. The theory was rejected because it could not explain the following important immunological observations: exponential production of Abs outnumbering their Ag templates, Ab production even after disappearance of Ag from the body, affinity maturation of antibody molecules, and immunological tolerance. New theories were put forward to explain the observations. The most appealing among them was Burnets Clonal selection theory. Clonal selection theory (Burnet, 1960) F.M. Burnet proposed that a vast number of different lymphocyte clones is present in vertebrate individuals. One lymphocyte and its clones (identical offsprings) can produce just one kind of antibody-cum-receptor. Binding of the antigen with the antibody-cum-receptor molecule triggers the cell to multiply and produce more numbers of the same receptor. In other words, antigen selects the lymphocyte clone that bears specific antibody on its surface and induces it to proliferate and produce Abs in large amounts. Diverse lymphocyte clones are generated at random during fetal development, but those reacting to the self-antigens are deleted. Clonal deletion during fetal life ensures the development of immunological tolerance to the selfantigens. Clonal selection theory has been validated by numerous experimental findings. The theory has revealed a law of immunology, albeit with some refinements over the years: One lymphocyte produces antibody of one specificity. This theory does not explain the interactions of cells and antibody molecules that make up an immune network. Jerne pointed out the existence of immune network in his theory of antibody production in 1974. Network theory (Jerne, 1974) Proposed by Jerne in 1974, the theory explains how a dynamic network is created within every individual by interacting antibody molecules due to the presence of idiotopes and paratopes in V region of their structure. All Ab molecules in an individual are interwoven by paratope-idiotope interactions. Antibodies recognizing the idiotopes, called anti-idiotype Abs, are normally produced in the absence of external Ag stimuli in an individual. Idiotopes and paratopes on antiidiotypic Abs interact to create a large network.

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The network maintains lymphocytes in a state of suppression until the Ag is introduced. Ag introduction removes the suppressive signals and allows stimulatory signals for the Agrecognizing and responding B cell clone to proliferate and produce specific Ab. The steady state equilibrium is restored by various regulatory mechanisms involving idiotope-paratope interactions. Immune response to an antigen is thus only a transition state in the dynamic equilibrium of the immune system. Immunotechnology for production of antibodies and the antigen-binder fragments Immunotechnology is a branch of biotechnology that deals with production of immunologicals by application of traditional and modern methods. Immunologicals are the substances of immunological interest such as Abs, cytokines, antigens, complement, etc., which find uses in disease diagnosis, prevention and treatment, epidemiology, proteomics, biosensors, catalysis, research in life sciences, etc. In this chapter, modern methods for production of Abs and the smaller Ag-binder fragments is discussed briefly. Specific antibodies can be produced by antigen immunization of suitable vertebrate animals, in vitro by hybridoma technique, and rDNA based techniques in bacteria, yeast, insect cells, mammalian cells, and transgenic animals and plants. Antisera produced by animals are a mixture of antibodies originating from different B cell clones i.e., they contain polyclonal antibodies reacting specifically against various epitopes of the multivalent antigens used for immunization. Repeated immunization of an animal with a particular antigen often mixed with adjuvant results in production of very high titres of antibodies in sera, also called hyperimmune sera. Antisera and hyperimmune sera have traditionally been used for serodiagnosis and serotherapy of infectious and certain non-infectious diseases, and research in life sciences. However, difficulties have been encountered in the use of antisera for therapeutic purposes. In vitro production of antibodies has certain obvious advantages: avoidance of large number of animals, defined specificity to single epitopes, scope for further genetic manipulations to produce various Ab formats, etc. Modern methods include hybridoma technology for production of full length monoclonal Ab (mAb) and recombinant DNA technology for production of fragments of Abs. Advent of prokaryotic and eukaryotic systems for Ab expression during 1980s marked the beginning of a new era in Immunotechnology. Subsequently, introduction of phage display system during early 1990s for expression of Ab fragments created a further milestone. These engineering techniques have made possible the construction of mutilated forms of Abs (Ab fragments) that show a great potential not only in the established fields but also in the fields of modern science and medicine such as biosensors, micro-arrays, proteomics, enzyme inhibition and catalysis, novel drug development, in vivo tumour targeting, etc. In fact, antibodies produced by traditional immunization protocols are exceedingly being replaced by Ab fragments (Fab, single-chain Fv, single-domain Ab, etc) produced by rDNA technology. Hybridoma technique for monoclonal antibody production Georges Kohler and Cesar Milstein at Cambridge University, U.K. first developed hybridoma technique for monoclonal Ab production in 1975. The technique involves the fusion between a myeloma (plasmacytoma) cell and a plasma cell to obtain a hybridoma cell that secretes Ab of 32

a defined specificity. Myeloma cells are cancerous cells of B cell origin that can be perpetuated by in vitro culture in suitable media. Myelomas used for hybridoma technique may or may not be secreting antibody or H or L chain of undefined specificity. NS0 and Sp2/0 are examples of nonsecretor myeloma cells of mouse origin most frequently used as fusion partner. YB2/0 is a myeloma of rat origin and GM4672 is a human lymphoblastoid cell line. Plasma cells are obtained from spleen, lymph nodes or bone marrow of Balb/c inbred strain of mice after immunization with the antigen. Myeloma cells are suitable for production of hybridomas only after they have been made deficient in the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK) that incorporate hypoxanthine and thymidine precursors respectively in salvage pathway of DNA synthesis. Therefore, HGPRT myeloma cell can utilize only de novo pathway of DNA synthesis that can be blocked by aminopterin. Thus, being deprived of both de novo and salvage pathways of DNA synthesis, HGPRT myeloma does not survive in media containing hypoxanthine, aminopterin and thymidine (HAT). Plasma cells in vitro have only limited life and do not survive more than 7-10 days. The hybridoma cell obtained from fusion of a myeloma cell with plasma cell acquires the immortality trait from the myeloma and Ab synthesizing capability and HGPRT from the plasma cell partner. HAT medium can thus be used as selective medium for hybridoma that is capable of utilizing salvage pathway of DNA synthesis. Box 9: Immunotechnology for the production of full-length monoclonal Abs Modern immunotechnology for Ab production includes hybridoma technique and recombinant DNA- based techniques Hybridoma technology involves production of hybridoma by fusion of myeloma cells with plasma cells from suitably immunized animal of inbred strain Individual hybridomas are isolated and grown in cell culture or animals for production of monoclonal Abs Monoclonal Abs are products of a single B cell clone that are specific to a single Ag determinant/ epitope Monoclonal Abs have diverse applications in diagnosis, therapeutics, infectious disease investigation, biocatalysis as abzymes, industry, life science research, etc. Mouse-mouse, rat-rat, and human-human homohybridomas, and heterohybridomas between mouse-rat, mouse-human, and rat-human have been obtained. But heterohybridomas are often less stable than the homohybridomas. Major steps in monoclonal antibody production are: 1. Immunization of Balb/c mice with Ag using a suitable immunization schedule. 2. Maintenance of myeloma cells in culture. 3. Isolation of spleen/ lymph node/ bone marrow cells from the immunized mice and fusion with myeloma cells with polyethylene glycol. 4. Culture of cells after fusion in HAT selection medium for 14-21 days. 5. Screening of hybridoma supernatants for Ab secretion using a suitable serological test. 33

6. Isolation of single hybridomas by limiting dilution or colony formation in soft agar for production of monoclonal Ab. 7. Secretion of isolated hybridomas for Ab production. 8. Sub-culturing of hybridomas for stability. 9. Large-scale production of monoclonal Ab in culture flasks (5-50 g/ ml), spinner bottles (100-200 g/ ml), hollow-fibre bioreactors (0.5-10 mg/ml) or in Balb/c mice by producing ascites (2-10 mg/ml). 10. Purification and characterization of monoclonal Abs. 11. Freezing of hybridomas in liquid nitrogen for long-term storage. Applications of monoclonal Abs: In diagnosis of infectious and certain non-infectious diseases. In immunotherapy of infectious diseases and cancer. In epidemiological investigations of infectious diseases. In monitoring of vaccination programmes. In research in life sciences. In catalysis. In industry for purification of biomolecules. Recombinant DNA techniques for expression of Ab fragments Antibodies are increasingly being produced by molecular genetic techniques. These methods are used for either constructing Abs that are difficult to be produced or when some modification is needed. Myeloma cell lines (e.g. NSO) to produce Fab or scFv, transient expression in COS cells and stable expression in CHO cell lines have been tried and all of them give mammalian type glycosylation. Nonetheless, prokaryotes are still the most common expression system for recombinant Abs as scFv or Fab. The Ab fragments can be expressed in periplasmic space or in the cytoplasm. Expression in Pichia pastoris using appropriate vectors for scFv, insect cell lines using baculovirus expression vectors and plant tissue culture have also been attempted. The expression systems that are being used increasingly in recent times are discussed below: i. Creating Ab libraries by phage display system In phage display system, Ab fragments are expressed on filamentous phages as fusion product with phage coat proteins, particularly minor coat protein III. A number of different phagemid vectors have been used for the purpose. Phage display links genotype with the phenotype, so the system is becoming popular. This method circumvents the need to immortalize the B cells as done in hybridoma technology. Numerous libraries of Ab fragments from human and domestic animal species have been prepared using phage display system. In addition, display systems based on yeast, bacteria, viruses and ribosomes, are being currently developed in various formats and exploited for the construction of antibody libraries. The application of these technologies has already provided rich harvests of products and processes for pharmaceutical industry, medicine and research. In early 1990s, Ag-specific Ab fragments were made from phage libraries constructed from spleen cells of immunized mice. This was followed by construction of phage display libraries of human V genes from human B cells immunized against infectious agents, or from autoimmune 34

diseases or cancers. Later, nave Ag-binders libraries have been made from V gene pools from IgM H and L chains obtained from B cells of non-immunized healthy individuals. Synthetic Ab libraries then followed using germline V genes on which diversity was introduced by oligo cloning. Ag-binders selected in vitro are particularly useful for non-immunogenic, toxic and conserved epitopes. Library size ranging from 2x105 to 3x1011 has been achieved by using various library vectors for nave, synthetic and semi-synthetic libraries. ii. Expression of Abs in transgenic animals Large amounts of transgenic proteins have been produced in mammary glands of domestic animals. Goats are more suitable than cattle and sheep for such purpose, because of their short period of attaining maturity, short gestation period, good milk yield, etc. iii. Plantibodies or Abs produced in transgenic plants Ab fragments as scFv have been produced in tobacco. It employs Agrobacterium tumefaciens transformed with scFv cDNA, which transfers DNA to integrate it into the plant cells and express therein. Other plants such as corn, rice and potato tubers can also be potentially used for expression of Ab fragments. Plantibodies could be a cheap and plentiful source of Abs. Box 10: Immunotechnology for the production of Ab libraries and Ab fragments in different formats Ab libraries of 106 specificities can be constructed by using phage display system Ab fragments are expressed on filamentous phages as fusion product with phage coat proteins, particularly minor coat protein III A number of different phagemid vectors have been used for the purpose Phage display links genotype with the phenotype This method circumvents the need to immortalize the B cells as done in hybridoma technology Other strategies include production of repertoires of Ag-binder fragments by transgenic mice and use of SCID mice

iv. Artificial antibodies or plastibodies Synthetic polymers, having paratope mimicks, also called mimotopes, as part of their structure can serve as artificial antibodies to bind with various antigens in vitro. They have been used in so-called molecularly imprinted sorbent assays to detect haptens and may find more uses in future. v. Various formats of antibodies produced by rDNA techniques a. Chimeric Ab and humanized murine monoclonal Abs In vivo use of Abs from sources other than humans suffers from three major problems: i) inherent immunogenicity, ii) poor penetration in tissues and iii) rapid clearance by binding onto FcRs on various leukocytes. Application of rDNA techniques has led to the production of newer formats 35

of Ab fragments to remove or reduce the above-mentioned constraints on their use in therapy of human diseases. For therapeutic uses, IgG subclasses are preferred for they are stable, have long half-life, have different effector functions and can be easily purified and stored. Humanization is modification of murine monoclonal Abs by rDNA techniques for use in human therapy. First of such attempts resulted in chimeric Abs i.e., murine monoclonal Abs in which the entire murine C regions were replaced with human C regions by genetic engineering technique, leaving only V regions of murine origin. The chimeric Abs had reduced problem of immunogenicity in humans. The problem was further reduced to minimal by replacement of entire murine IgH and IgL sequences, except CDRs, by corresponding human sequences. Such Abs are called fully humanized Abs. Currently, alternative strategies are being devised for production of fully human Abs for therapeutic and other in vivo uses. Such strategies include production of repertoires of Ag-binder fragments by phage library system, by transgenic mice, and by reconstitution of SCID mice with human peripheral blood lymphocytes with subsequent immunization with Ags and rescuing immune B cells by fusion with myeloma cells. b. Bispecific Abs and other Ab formats Elegant engineering techniques can now produce fully human Ig molecules with desired characteristics. Abs have been modified in order to increase their therapeutic potential. Bispecific Abs (BsAb) comprises of two different specificities, for example, one for binding to the target and other to the effector cell. In this way, they can improve target selectivity and reduce undesirable reactions resulting from activation of complement. Effector cells could be phagocytes, Ag presenting cells or T lymphocytes. F(ab)2 heterodimer, a prototype BsAb, has been produced by chemical cross-linking of Fab or F(ab) 2 fragments of parental Abs. Quadromas (fusion of two hybridomas) and genetic engineering have also been used for construction of BsAbs in various formats in order to optimize their structure, minimize toxicity, and avoid purification and affinity maturation processes. For more tissue penetrance, BsAb consisting of only two scFv (single-chain variable fragments), also called minibodies, have been produced. Tetravalent BsAbs (Ab-scFv, dimeric minibodies and dimeric diabody-Fc molecules) have also been produced to bind to four epitopes. Immunoconjugates, i.e., Abs conjugated to radio-isotopes, toxins, enzymes or even whole effector cells, can be used to bring the conjugates to the target cells for therapeutic purposes. c. Nanobodies- the smallest Ag-binders` Single-domain antibodies (dAb) have paratope made of one VH domain only and are thus the smallest Ag-binders produced to-date. Interestingly, dAbs derived from heavy chain antibodies (HCAbs) of camelid species (camel and llama) have excellent stability, solubility and high affinity binding. These features are usually lacking in dAb fragments derived from other species. Phage display of dAbs derived from camelid HCAbs gene segments has therefore found much favor for their wider potential in medicine and biology. A considerable fraction of camelid Abs is homodimers of heavy chains only. A number of biotechnological and medical applications of dAbs originating from HCAbs are expected particularly in the fields where their smaller size and targeting of uncommon epitopes such as 36

catalytic sites of enzymes might be beneficial. HCAbs are a unique source of enzyme inhibitory Abs. Several dAbs recognizing Ags of practical importance, such as -lactamases, trypanosome variable surface glycoprotein, carcino-embryonic antigen, cutinase, etc. and against unstable proteins have been produced. Box 11: Immunotechnology for the production of Ab fragments in different formats Newer formats of Ab fragments to remove or reduce the constraints on the therapeutic use of Abs in human diseases Humanization is modification of murine monoclonal Abs by rDNA techniques for use in human therapy Chimeric Abs are murine McAbs in which the entire murine C regions are replaced with human C regions by genetic engineering technique, leaving only V regions of murine origin The chimeric Abs had reduced problem of immunogenicity in humans Replacement of entire murine IgH and IgL sequences, except CDRs, by corresponding human sequences makes fully humanized Abs. Bispecific Abs (BsAb) comprises of two different specificities, for example, one for binding to the target and other to the effector cell For more tissue penetrance, BsAb consisting of only two scFv (single-chain variable fragments), also called minibodies, have been produced Tetravalent BsAbs (Ab-scFv, dimeric minibodies and dimeric diabody-Fc molecules) have also been produced to bind to four epitopes Immunoconjugates are Abs conjugated to radio-isotopes, toxins, enzymes or even whole effector cells used for therapeutic purposes. Single-domain antibodies (dAbs), also known as nanobodies, having paratope made of one VH domain only are the smallest Ag-binders produced to-date Ab fragments have been produced in transgenic plants (plantibodies), transgenic animals and even on synthetic polymer scaffolds (plastibodies)

Phage display construction is much simpler and more efficient with camelid dAbs as compared to Fabs or scFvs. Randomization can be introduced at a much higher %age of CDR positions without exceeding practical library size. Problem of shuffling original VL-VH pairings is also avoided. However, non-human nature of these products limits their usefulness. Synthetic dAb libraries, particularly those based on human VH framework alleviate these problems. Camelised human dAb library has been constructed. vi. Uses of rDNA produced Abs and Ag-binders in modern science and medicine Antibodies have been traditionally used in diagnosis, epidemiology, prevention and treatment of infectious diseases, and forensic medicine, molecular evolution and research in life sciences. In recent times, antibodies produced by modern methods have assumed new roles to play in modern science. Such roles are listed below:

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1. 2. 3. 4. 5. 6. 7.

Abs as biocatalysts Ab microarrays for proteomics Abs in biosensors Novel drug development In vivo tumor imaging Gene therapy Molecular evolution Box 12: Applications of rDNA produced Ab fragments in different formats

Antibodies have been traditionally used in diagnosis, epidemiology, prevention and treatment of infectious diseases, and forensic medicine, molecular evolution and research in life sciences Antibodies produced by modern methods have assumed such new roles to play in modern science as: Abs as biocatalysts Ab microarrays for proteomics Abs in biosensors Novel drug development In vivo tumor imaging Gene therapy Molecular evolution

Origin and Evolution of Igs Evolutionary relationships have been established with the availability of gene and protein sequence data for Igs of all classes of vertebrates derived from the placoderms, the primitive jawed fish, namely, sharks, teleost fish, amphibians, reptiles, birds and mammals. Mammals have the most diverse Ab classes and subclasses. Based on general structural properties, all placoderm-derived vertebrates possess Abs comparable to IgM isotype of mammals. The chains in sharks and mammals are of the same size (70 kDa) and cross-react serologically. There exists strong homology among corresponding chains of lower vertebrates and mammals. C domains of IgM in vertebrates across phyla show 30% identity, whereas framework region (FR) sequence homology is in the range of 40-75%, indicating its evolutionary conservation. In lower vertebrates (amphibians, reptiles and birds), H chain larger than mammalian is found that make IgY, instead of IgG. Other distinct Ig classes are also found, such as IgX in amphibians, IgN in lungfish, reptiles and ducks. Mammalian or L chains occur with varying relative frequencies from species to species: : ratio in human is 60:40, in mouse 95:5, and pigs 5:95. and chains diverged in ancient time when ancestors of sharks and humans diverged. Mammalian and shark L chain show 40% identity in C region, >50% in V region and 75% in FR4. 38

Despite these similarities in Ig genes, their genomic organization varies considerably among various phyla. H and L domains are encoded by V, (D), J and C gene segments that are organized differently in different species. To create Ag receptor diversity by V(D)J recombination, two genes, RAG-1 and RAG2, are required, the homologues of which are expressed in all jawed vertebrates. Available data suggests that Igs comparable to those of higher vertebrates are found in the most primitive living vertebrates, i.e., the cyclostomes (lamprey and hagfish), but not in invertebrates. Protochordates, like tunicates, make molecules of 25-30 kDa size to contain V and C gene segments that are antigenically related to H chain of lampreys and sharks. This molecule is proposed as a missing link or protoimmunoglobulin.

Immunoglobulin superfamily (IgSF) IgSF is a large family of proteins that have immunoglobulin-like structure. Members of IgSF share the basic structural unit of Ig, also called Ig homology unit (Ig domain) of about 110 amino acids. Each domain has an Ig fold of 60-70 amino acids folded into two layers of anti-parallel -pleated sheets (3+4 -strands in C domain, and 5+4 -strands in V domains) linked by an intrachain S-S- bond. A more primordial motif, called H unit, has recently been found that is somewhat different from V and C homology units, and probably originated in early metazoa to carry out cell-surface recognition functions unrelated to vertebrate immunity. Ig fold structure is thus conserved in evolution and can be found in proteins of a wide variety of species including invertebrates and ancient vertebrates. Although structurally similar to Ig, the IgSF members vary in size and are functionally very diverse. Ig fold serves as a scaffold for arrays of various binding sites on it. Self- self and selfnon-self hydrophobic interactions occur among Ig-like domains of the members for carrying out various biological functions. Many members are involved in immune responses, but others have no direct relationship to the immune system. The list of members of IgSF is long and increasing. Important members of IgSF are given in Table 12.

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Table 12: List of the members of Ig superfamily* IgSF member All Igs TCR-1 & TCR-2 Leukocyte FcRs (CD16, CD23, CD32, CD64, CD89, etc.) pIgR Major function Ag recognition & disposal MHC- Ag peptide complex recognition Ig binding via Fc region to leukocytes IgSF member CD158a,b (KIR) CD80, CD84, CD86, CD115 CD121 a-b, CD126, CD130 Major function Killer cell inhibitory receptor on NK cells Macrophage/ monocyte functions Interleukin receptors

Polymeric IgA & IgM transport across epithelia FcRn IgG transport across placenta, gut epithelium, mammary glands & IgG catabolism MHC class I & II Ag presentation molecules 2- microglobulin Associated with MHC class I molecule CD1a-e Glycolipid Ag presentation

CD66a-f

Granulocytes

CD47, CD48, CD50, CD54, CD58, CD100, CD101, CD147, CDw149 N-CAM (CD56)

Most leukocytes

CD19, CD22, CD79a-b, CD28

In B cell function

CD2, CD3, CD4, In T cell function CD7, CD8, CD90, CD96, CD117, CD146, CD152, CD155, CD166 * The list is not exhaustive

Nervous-systemassociated molecules PDGFR Growth factor/kinase receptors oprin Snake venom metalloproteinase inhibitor in Southern opossum Fibrinogen-related Hemolymph of proteins freshwater snail (invertebrate) UNC proteins Present in the Tyr kinase growth invertebrate factor receptor, Caenorhabditis titin, Twitchin, elegans dim-1, axonin

Suggested Reading
1. 2. 3. Immunology 6th edition by Roitt, I. et al.. Mosby, Elsevier Science Ltd., London (2001). Immunology, Immunopathology and Immunity 6th edition by Sell, S. American Society for Microbiology Press, Washington D.C. (2001). Kuby Immunology 4th edition by Goldsby, R.A. et al. W.H. Freeman & Co., New York (2003).

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Immunobiology 6th edition by Janeway, C.A., Jr. et al. Churchill Livingstone- Garland Science Publishing (2005). Introduction to Immunology 3rd edition by Kimball, J.W. MacMillan Publishing Co., New York (1990). Handbook of Experimental Immunology, vol. 4 by Weir, D.M. Blackwell Scientific Publishers, London (1986). Molecular Immunology, edited by Atassi, M.Z. et al. Marcel Dekker Inc., New York (1984). Methods in Molecular Biology, vol. 51 (Antibody engineering protocols) edited by S. Paul. Humana Press Inc., New Jersey (1995) Antibody engineering: A practical guide edited by Borrebaeck, C.A.K. W.H. Freeman & Co., New York (2000). Methods in Molecular Biology, vol. 248 (Antibody Engineering: methods and protocols) edited by B.K.C. Lo. Humana Press Inc., New Jersey (2004) Immunology: The making of a modern science edited by Gallagher, R.B. et al. Academic Press Ltd., London (1995). Encyclopedia of Immunology 2nd edition by Delvis, P.J. Academic Press Inc., London (1998). Immunoglobulin genes 2nd edition by Honjo, T. & Alt, F.W. Academic Press Inc., London (1995). Advances in Immunology, vol. 61 (1996); 62 (1996); 65 (1997); 69 (1998); 72 (1999); 78 (2001); 82 (2004); 86 & 87 (2005). Current Topics in Microbiology and Immunology, vol. 240 (1999); 260 (2001). Immunology Today, vol. 18 (1997); 21 (2000). Trends in Immunology, vol. 22 (2001); 25 (2004). Trends in Biotechnology, vol. 21 (2003); 22 (2004). Scientific American, vol. 293 (2005): 67-71. Infection and Immunity, vol. 72 (2004): 6191- 6196.

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