Magic Bullet For Rheumatiod Arithritis Biology Essay

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Introduction -

At the beginning of 20th century the renowed German immunologist and nobel prize winner Paul Ehrlich gave the idea of magic bullet against disease.

Paul postulated that-if a compound could be made that selectivity targeted a disease causing organism then a toxin for that organism could be delivered along the target of selectivity (1).

In the 1970 the B-cell cancer multiple myleoma was known and it was found that these cancerous B-cell all produce a single type of antibodies i.e. a paraprotein.This was used to study the structure of antibodies .Self tolerance was originally described by Ehrlich (1901) as 'horror autotoxicus'. He observed that after immunisation of a goat with erythrocyte of a other goats, antibodies were produced against erythrocytes of other goats but not against their own.

Rheumatoid Arithritis(RA)is a chronic progressive inflammatory autoimmune disease.RA is a systemic disorder where inflammatory changes does not only affect synovial joint but also many other sites including the heart , blood leve land skin.

RA is two or three times more common in females than males and can affect all ages,including children although it is usually develop between the age of 35 and 55.

RA is a systemic chronic disease affecting about 0.5-1% of the population worldwide.(2)

The prevalence is most common across the globe regardless of geographic location and race.(3)

The primary changes that may be reversible include hypertrophy and hyperplasia of synovial cells and fibrinous inflammatory effusion into the joints. if a disease progress there are further secondry changes which may be reversible including -

1. Erosion of articular cartilage and the growth of granulation tissue (pannus) that seprates the bone and distort the shape of joint.

2. Fibrosis of pannus which causes adhesion between the bones , limiting movement.

3 .Ossificatin of the fibrosed pannus , further restricting joints movement.

4 .Spread of granulation tissues to tendons.

5 .Weakening and atrophy of muscles possibly due to limiting exercise.

6 .Development of rheumatoid nodules (subcutaneous collagen nodules) outside the joints. e.g. in pressure area such as elbow ,over the knuckles and in the lungs,pleura ,heart and eyes.

7 .Enlarement of lymph nodes and spleen (lymphadenopathy and spleenomegaly).

8 .Antigen-antibodies(antigammaglobulin)are formed in blood and synovial fluid.

Genetics of rheumatic disease

RA has a heritable risk with a complex genetic basis .therefore to understand basic pathogenesis and clinical characteristics of the genotype to the expression of disease studied. Recent technological advances in genotyping and statistical analysis and international collaborations assembling large cohorts of patients have led to a wealth of new data.

In particular evidence from genome wide association(GWA) studies which provide support for the existence of a common genetic basis to disease.(4).The concept of systemic GWA become practical with the catalogue of libraries of common polymorphisms .Currently over 20 million single nucleotide polymorphism (SNPs) have been identified (5) and plateforms are available to type upto 1 million of these in a single reaction. Although not all SNPs are currently genotyped,as the human is arranged into haplotype blocks in linkage disequilibrium, it is called tag SNPs , which identify these areas of limited variability(6) to achieve good representation of the total amount of genetic variation Most typed SNPs are relatively common (minor allele frequency of >5%) and if association with disease are likely, to have only modest pathogenic effects(odds ratios ORs usually between 1.2-2) as otherwise they would become depleted in a population due to natural selection .

Commom-disease common variant (CD-CV) which assume as accumulation of risk caused of multiple deleterious alleles, to explain current experimental findings.(7)

The genome is subjected to variation at more than the SNP level , and individuals also differ in the copy number of sections of DNA of greater than several kilobases in size ,so called copy number variation (CNV), in fact accounts for more total nucleotide difference between individuals than SNPs. CNV can affect gene expression levels [8] and has been linked to autoimmune disease [9,10].Association studies based on CNV are therefore in their relative infancy. Finally, the genome is subject to modification without a change in DNA sequence; epigenetic mechanisms can have profound effects on gene expression. These include DNA methylation and changes in chromatin structure[11].

The HLA complex

Histocompatibility antigens mean cell surface antigen that evoke immune response to an incompatable host resulting in allograft rejection. These alloantigens are present on the surface of leucocyte antigens (HLA) and the set of genes coding for them is named the HLA complex. The HLA complex of genes is located on short arm of chromosome 6 .The nomenculture of the HLA-system is regulated by official committee of WHO. They have officially recognised alleles and their corresponding antigens by the locus and a number . an A locus is position where a particular gene is located on the chromosome. HLA loci aree multiallelic i.e the gene present on the loci can be anyone of the sevsral alternate forms (alleles). Each allele determines a distinct antigen. There are 24 alleles at HLA-A locus and 50 at HLA-B. HLA system is very pleomorphic. Every individual inherits one set of HLA- genes from each parent.

HLA is grouped in three classes-

Class I - HLA-A,HLA-B and HLA-C

Class II - HLA -DR, HLA-DQ and HLA -DP (all of these are present within HLA-D region HLA complex ).

Class III - Complement loci encode for C2,C4 and factor B of complement system and tumour necrosis factors (TNF) alpha and beta.

In RA ,the MHC accounts for around a third of the genetic liability (13).Alleles at HLA-DRB1 contribute much of this risk .

GWA studies confirm the strong association with MHC variants; risk alleles confirm an OR of around 2 to 3 in homozygotes(14) with very high stastical significance(p<10-100) .Additional loci contributing to risk of RA identified by high density genotyping include HLA-DP in patients with anticyclic citrullinated peptide antibodies(15).

Risk variants in the class III cluster which encodes genes such as TNF and complement component C2, C4A, C4B. C4 is crucial in the classical and mannose binding lectin pathways of complement activation and complete deficiency of C4 or indeed other components of the classical pathway are rare.

Interferon signalling: IRF5

Haplotypes of IRF5 are implicated in RA.

TNF associated signalling pathway : TNFAIP3 and TRAF1-C5

TNF-associated signalling pathway genes play a prominent role in the risk for RA and associations with variants in TNFAIP3 (TNFα-induced protein-3) and the TRAF1-C5 (TNF receptor associated factor) locus have been identified (16,17). TNF induced protein-3 known as A20.It is a ubiquitin editing enzyme that acts as a negative regulator of NFκB (nuclear factor). A20 can disassemble LYS63 linked poly-ubiquitin chains from targets such as TRAF6 and RLP1. A second region of A20 catalyses LYS48 linked ubiquitination that target the molecule for degradation by de-proteasome (18). A20 modifies key mediators in the downstream signalling of TLRs (Toll like receptors) that use MyD88, TNF receptor, the IL1 receptor family and nucleotide olygomerization domain protein-2 (NOD2(19)). Tnfaip3 knockout mice develop severe multi-organ inflammatory disease and the phenotype is lethal (20). The SNPrs10499194 in TNFAIP3 carries an OR of 1.33 for RA.

On chromosome 9, the region containing TRFA1 ( TNF receptor associated factor-1) and C5 (complement component 5) genes is associated with significant risk for RA.

T-cell receptor signalling: PTPN22

Outside the HLA region, the first reproducible genetic association for RA came with the implication of PTN22 from a candidate gene approach (21) based on linkage analysis identification of a susceptibility locus at 1P13 (22). It has remained the strongest and most consistent association mapped by GWA studies in RA. The OR for the risk allele is around 1.75 in RA.

Polarisation toward TH1 and TH17 phenotypes: STAT4 and IL23R

STAT4 encodes (signal transducer and activation of transcription factor-4), responsible for signalling by IL-12, IL-23, and type 1 IFNs (23). STAT4 polarises T cells towards TH1 and TH17 phenotypes which has the potential to promote auto-immunity (24). In RA the OR for the risk allele of SNP rs7574865 is 1.32 in one case controlled study (25) with a less strong disease association at rs11893432 in a meta-analysis of GWA studies. ( OR 1.14) (14).

B cell -

B cells undergoes blast transformation to become plasmablast, intermediate transitional cells and then plasma cells. A single plasma cell can synthesize a single antibody of single specificity, of a single immuno-glubulin class.

B cells are depleted in RA. Despite the importance of B cells in the pathogenesis of RA, none of the gene effects identified in the current generation of GWA studies.

Post Translational modification: PADI4

Peptidydyl arginine deeminase-4 (PADI4) is a member of the enzyme family, responsible for the post-translational citrullination of argenine residue in RA synovium, subsequently recognized by anti-cyclic citrullinated protein antibodies. In Japanese (26) and Korean (27) patients, case-control association studies have identified functional haplotypes of PADI4 conferring risk of RA.

ANTIBODIES STRUCTURE,FUNCTION AND MOLECULAR GENETICS-

Antibodies (also called as immunoglobulin) are glyproteins,Which specifically recognise the foreign molecules as a antigens.When antigen invade human or animals an immunological response is triggerd-which involve the production of antibodies by b lymphocytes.

Normally an animals immune system recognise its own tissues antigens as ' self ' and therefore does not produce antibodies against these self antigen.Autoimmunity a condition in which when the body produces autoantibodies and immunological competent T-lymphocytes aganists its own tissue.This lead to structural or fuctional damage of tissues.Autoimmunity is literally means 'protection against self' while it lead to development of 'injury to self'.

Structure of Antibodies-

An antibodies comprises four covalently linked polypeptides chains: two identical heavy chains and two identical light chains.

The heavy chains usually contain four and light chain two distinct domins(domain in a discreat folded ,fuctional unit) .

The first domain is each chain is variable (V) domain,VH and Vl on heavy and light chains,respectively. The rest of the heavy chain comprises (four for IgE) constant domains (CH1 to CH3) while the light chains have one constant domain CL,there is a flexible peptide segment (hinge region ) between the CH1 and CH2 domains.

The antibodies V region is composed of the VH and VL domains .The C region is composed of the CH1, CH2, CH3 and CL domains. On digestion of antibodies molecule with papain enzyme releases a single Fc (fragment crystallisable) fragment corresponding to the CH2 and CH3 domains. The two Fab (fragment antigen binding ) fragments are also generated, Corresponding to the antibodies arms.

Within each VH and VL domain, three short polypeptide segments form the hypervariable or complementary determining regions(CDRs) .the remainder of the V domain is much less variable and forms a scaffold that supports the CDRs.CDR3 is the most prominent variable of the CDRs and play a dominant role in antibodies specificity.

STRUCTURE

The Antibody C region determines the class and subclass of the antibodies.Five different classes namely IgG,IgM,IgA,Igd and IgE are desinated on presence of heavy chain.

Class of Immunoglobuli

Heavy Chain

IgG

Gamma(γ)

IgM

Mu (μ)

IgD

Delta (δ)

IgE

Epsilon(ε)

IgA

Alpha(α)

The light chain L are present in two forms Kappa(κ) and lambda(λ). The light are named as kappa and lambanda after the investigaters Korngold and Lapari. Kappa and Lambda chains are present in a ratio of 2:1 in human sera.

On pepsin Digestion ,pepsin cleaves immunoglobulin molecule and give rise to Fc portion and the two Fab fragment is bivalent and two Fab fragment held together in position .This Fab fragment is bivalent and can still preeciptate with antigen .It is called F(ab')2. the pepsin also degrades the Fc portion into smaller fragments.

Structure of antibody-----

For a VH domain -

V segments that code for most of the V domain, including CDRs1 and 2 and the first part of CDR3.

D segments that code for the the immediate part of CDR3,and

J segments that code for the terminal part of CDR3.

In humans ,there are about 51 heavy-chain Vsegments,25D segments and 6J segments[28]

During B-cell development, antibodies -encoding DNA undergoes various rearrangements

Types of antibodies

There are two types of antibodies-

Polyclonal antibodies

Monoclonal Antibodies (Mabs) -are produced against only one specific antigen which is of immediate relevance.

Technique for monoclonal production

1 In Animals-

The method for production of monoclonal antibodies against any desired antigen was first described by Georage Kohler and Ceser Milstein in 1975. They were awarded Nobel Prize for medicine in 1984.

Antibody forming spleen cells are fused with myeloma cells to produce hybrid cells (hybridomas). The resultant hybridoma retains the antibody producing capacity of the spleen cells and the ability of myeloma cells to multiply indefinitely. The details of the technique are as follows:

animals (usually mouse) is immunised with the desired antigen and lymphocytes are harvested from the spleen.

Spleen cells (lymphocytes) are then fused with mouse myeloma cells , grown in culture ,myleoma cells are deficient in the enzyme hypoxanthine phosphoribosyl transferase (HPRT). Fusion is done by incubating these cells in the presence of polyethylene glycol (PEG).

The fused cells (hybrid cells)are grown in the basal culture medium containing hypoxanthine , aminopterin and thymidine (HAT medium).

Only hybrid cells have the properties of both the splenic lymphocytes (HPRT +) and myeloma cells (HPRT -)can grow in culture. The enzyme HPRT is necessary for nucleic acid synthesis and is provided by the splenic lymphocytes in hybrid cells . splenic lymphocytes alone (unfused) cannot replicate indefinitely while unfused myeloma cells are killed by aminopterin in HAT medium.

Clones that secrete the desired antibody are selected for continuous cultivation . These hybridomas can be maintained indefinitely and will continue to form monoclonal antibodies. They can also be grown as tumours in the peritoneal cavity of mice by intraperitional inoculation and monoclonal antibodies are obtained by harvesting the ascetic fluid produced. Hybridomas may be frozen for prolonged storage.

Mouse monoclonal antibodies, however, proved unsuitable for human therapeutic use because of strong antimouse immune response. Human monoclonal antibodies have been developed by modification of the original technique. Genes for particular antibody fragments have been fused to bacteriophage genes. Large quantities of the desired antibody can be obtained by infecting bacteria with the appropriate bacteriophage. Such antibodies hold great promise for immunotherapy.

Diagram of production of monoclonal antibodies by hybridoma technology.

2) Production in cell-culture

This technique requires some expertise, special media and can be expensive and time-consuming. There has been considerable research on in vitro methods for growing hybridomas and these newer methods are less expensive, faster, and produce antibodies in higher concentration than has been the case in the past[1]

Evolution of MAbs

The significance of MAbs lies in their specificity and immortality. Whereas hybridoma development of murine MAbs was the requisite for the development of MAbs as drugs.

The Mabs are classified into generation as per their evolution and immunogenicity as follows:

First Generation Mabs- majority of earlier Mabs available were murine,rabbit or rat proteins purified following immunonization of the animal with an antigen preparation.These are labelled as first generation antibodies.

Second Generation Mabs-These are referred to as chimeric ,humanized, primatized or pure human Mabs.

Applications of Mnoclonal antibodies-

Example FDA approved therapeutic monoclonal antibodies

Antibody  

Brand name  

Approval date  

Type  

Target  

Approved treatment(s)  

Gemtuzumab

Mylotarg

2000

humanized

CD33

Acute myelogenous leukemia (with calicheamicin)

Trastuzumab

Herceptin

1998

humanized

ErbB2

Breast cancer

Abciximab

ReoPro

1994

chimeric

inhibition of glycoprotein IIb/IIIa

Cardiovascular disease

Alemtuzumab

Campath

2001

humanized

CD52

Chronic lymphocytic leukemia

Bevacizumab

Avastin

2004

humanized

Vascular endothelial growth factor (VEGF)

Colorectal cancer

Panitumumab

Vectibix

2006

human

epidermal growth factor receptor

Colorectal cancer

Cetuximab

Erbitux

2004

chimeric

epidermal growth factor receptor

Colorectal cancer, Head and neck cancer

Certolizumab pegol

Cimzia

2008

humanized

inhibition of TNF-α signaling

Crohn's disease

Ranibizumab

Lucentis

2006

humanized

Vascular endothelial growth factor A (VEGF-A)

Macular degeneration

Omalizumab

Xolair

2004

humanized

immunoglobulin E (IgE)

mainly allergy-related asthma

Natalizumab

Tysabri

2006

humanized

alpha-4 (α4) integrin,

Multiple sclerosis and Crohn's disease

Rituximab

Rituxan, Mabthera

1997

chimeric

CD20

Non-Hodgkin lymphoma

Tositumomab

Bexxar

2003

murine

CD20

Non-Hodgkin lymphoma

Ibritumomab tiuxetan

Zevalin

2002

murine

CD20

Non-Hodgkin lymphoma (with yttrium-90 or indium-111)

Eculizumab

Soliris

2007

humanized

Complement system protein C5

Paroxysmal nocturnal hemoglobinuria

Efalizumab

Raptiva

2002

humanized

CD11a

Psoriasis

Palivizumab

Synagis

1998

humanized

an epitope of the RSV F protein

Respiratory Syncytial Virus

Adalimumab

Humira

2002

human

inhibition of TNF-α signaling

Several auto-immune disorders

Infliximab

Remicade

1998

chimeric

inhibition of TNF-α signaling

Several autoimmune disorders

Muromonab-CD3

Orthoclone OKT3

1986

murine

T cell CD3 Receptor

Transplant rejection

Basiliximab

Simulect

1998

chimeric

IL-2Rα receptor (CD25)

Transplant rejection

Daclizumab

Zenapax

1997

humanized

IL-2Rα receptor (CD25)

Transplant rejection

Treatment of rheumatoid arithritis-

In the last years, advances in our understanding of the immune system, as well as the advent of the era of biotechnology, have triggered great interest in the development of new therapies for autoimmune rheumatic diseases. Our better understanding of these disorders has also shifted treatment strategy from a more conservative approach to a much more aggressive one, especially in rheumatoid arthritis (RA).[29]

1. cytokine blockade,

Interleukin (IL)-1 receptor antagonist, IL-18 binding protein, soluble TNF receptor,

antibodies to TNF-α, IL-6, IL-15, IL-17, and BLyS.

2. cell depletion,

Antibodies to CD20 on B cells

3. or regulatory cell surface receptor blockade.

Abatacept (CTLA-4 fusion protein), Efalizumab (anti-CD11a antibody), Alefacept

(LFA-3 fusion protein). Compared to conventional treatment, these agents may target the immune system more selectively and therefore have fewer non-specific side effects, although many cytokines are certainly pleiotropic. Biologics are not less potent in their immunosuppressive abilities than conventional immunosuppressive medications and affect general immunocompetence as well as the autoimmune process. Consequentially, their use in an elderly population requires special considerations. This review will focus on recent literature and on the benefits and risks of newer biologic agents, with particular emphasis on diseases that are prevalent in this age group.

Antibody engineering

Progress in biotechnology and greater understanding of pathogenesis of RArendered the production of 'man-made' antibodies . A number of strategies led up to the 'bespoke' process of antibody design that we are now familiar with.[28]

Chimeric antibodies

first therapeutic antibodies were murine proteins produced from murine(concerning rodents-mice,rat) 'hybridomas' by conventional fusion technology [30]. In rheumatology , one of the earliest anti- CD4 mAbs was murine [31]. Limitation to the use such 'foreign' molecules was their immunogenicity. For example, OKT3, a murine mAb against human CD3, was effective for reversing allograft rejection.

Therapy was often ineffective, however, due to neutralising anti-antibody - anti-globulin or human anti-murine (HAMA) - responses [32]. A further potential limitation of using murine mAbs was their interaction with human effector functions.[32]

There are subtle differences in amino acid sequence between murine and human Fc regions and between murine and human FcγR(fragment crystasalliable gamma receptor ). Consequently, the interaction between a murine mAb and human FcγR will be suboptimal, potentially limiting the cytotoxic potential of the antibody in the therapeutic situation.

Neuberger and colleagues [33] first demonstrated the feasibility of linking a murine antibody V-region gene segment to a human C-region gene segment. The resulting gene construct encoded a chimeric, 'half human/half mouse', mAb . The chimeric C region did not interfere with antigen binding but, affect the effector function of the encoded mAb. The production of 'matched sets' of chimeric mAbs confirmed the expected inter-class and inter-subclass variation of effector function, enabling the selection of the appropriate C region for a particular therapeutic task and the birth of 'designer' mAbs [34,35]. Two chimeric mAbs are used in everyday rheumatological practice: infliximab and rituximab . Both possess a human IgG1 C region and these highly effective drugs neutralise tumour necrosis factor-alpha (TNF-α) and kill B cells, respectively. Nonetheless, their murine V regions retain the immunogenicity of a foreign protein. The consequences of immunogenicity vary from anaphylaxis, which fortunately is rare, to lack of efficacy and infusion reactions, which are more common. For example, human anti-chimeric antibodies are a significant cause of secondary inefficacy of infliximab , whereby mAb requirements increase with time and treatment may eventually become ineffective [36].Currently infliximab is used in combination with methotrexate(MTX) which decreases the development of anti-infliximab antibodies. Infusion reactions are also more frequent in the presence of anti-globulins [37]. A number of factors influence immunogenicity, including background immunosuppression, dose, and route of therapy [38].

Humanised antibodies

The Careful examination of the V-region peptide sequence of a mAb allows the identification of the CDRs. In the mid-1980s, it was shown that genetic engineering could be used to 'transplant' the CDRs of a murine antibody onto a human V-region framework, generally without a loss of specificity (CDR grafting) [39].To

optimise the 'fit' and ultimate affinity, the chosen human V gene was generally one that closely resembled that of the parent mouse mAb. The potential advantage of humanisation was a further reduction in immunogenicity, although the selected V-region backbone was not always one that was used commonly by the natural human antibody company or performer (regulary) [40]. The first humanised therapeutic mAb, CAMPATH-1H (alemtuzumab), was shown to be minimally immunogenic in patients with rheumatoid arthritis (RA) [41]. This drug is highly effective at killing

lymphocytes and is now licensed for the treatment of chronic lymphocytic leukaemia(CLL) Tocilizumab, a humanised mAb against the interleukin-6(IL-6) receptor that is currently in phase III development for RA, was also developed by CDR grafting, ocrelizumab, an anti-CD20 mAb that is currently in phase III trials for RA . A number of techniques have subsequently evolved for generating humanised and 'human' mAbs. Because of their murine CDRs, humanised mAbs theoretically retain a degree of immunogenicity (human anti-human, or HAHA, responses) . IgM mAbs in small quantities from unstable cell lines, and immunological tolerance provided a significant barrier to raising human mAbs against human targets.

Human antibodies

In 1989, Orlandi et al[42] showed that it was possible to use the polymerase chain reaction (PCR) to clone immunoglobulin V domains. As a result 'libraries' of immunoglobulin VH and VL sequences were created within plasmid and phagemid vectors, allowing the expression of a huge diversity of antibodies [43]. Sequence conservation meant that a relatively small number of 'forward' (3′) and 'backward' (5′) primers could be used to amplify a large proportion of the V-domain repertoire from an appropriate source, including peripheral blood. The incorporation of restriction endonuclease recognition (RER) sites into primers facilitated the subsequent in-frame cloning of amplified V-domain sequences. An extension of the technology allowed the mutation of a cloned V domain using a number of methods. For example, in 'spiked PCR', the forward primer is synthesised under conditions that introduce low-frequency random mutations, providing a mixed population of many subtly different primers. Because the forward primer encodes CDR3, the resulting

PCR product encodes a V-domain mixture with subtly variable CDR3s and hence find specificities. In contrast, 'error-prone' PCR (using non-stringent amplification conditions or non-proofreading polymerases) results in sequence variability throughout the amplified V domains. These and similar techniques, when applied to a cloned V domain, generate variants of altered affinity in a manner analogous to affinity maturation. Other techniques include 'chain shuffling', in which a 'fixed' VH or VL domain is allowed to pair with a library of partner domains, biasing the resulting Fvs toward a desired specificity [44]. Properly directed selection enabled the derivation of a human mAb starting from a murine sequence [45]. This technology had several advantages. The ability to rapidly capture and clone a significant proportion of the V-domain repertoire from a biological sample was a major advance.

Hence, the new technology bypassed the need to use animals for mAb generation - libraries could be created from human blood samples, because the VH- and VL domain libraries could be randomly combined and mutated, it became possible to generate specificities absent from the natural repertoire of the source tissue, bypassing immune tolerance mechanisms. To exploit these advances, novel techniques were needed to screen the massive V-domain libraries for desired specificities. Thus, through the use of peripheral blood B cells from a non-immunised individual, PCR amplification might result in 107 VH sequences and a similar number of VL sequences. Random pairing of these would result in a 'library' of 1014 different combinations, each cloned into a plasmid. Transformation of a bacterial culture with this library could result in 109 distinct Fv specificities (limited largely by transformation efficiency). Phage display technology provided a method for screening such libraries. Filamentous bacteriophages are simple viruses that infect bacteria. They comprise a nucleic acid core and a surrounding protein capsid. By cloning V domains in-frame with specific capsid proteins, the encoded Fv could be expressed at the phage surface. In particular, functional scFvs (single chain covalently linked heavy and light chain V domains) could be expressed. These molecules comprise a VH and a VL joined by a short, flexible, peptide linker. In this way, libraries of VH and VL domains could be converted into an antibody fragment phage library, each phage displaying a distinct specificity on its surface [46,47]. Each phage is effectively a 'recombinant genetic display package' expressing an Fv on its surface and containing the encoding DNA within. This physical linking of specificity and DNA provided a major advance. To select phage expressing Fv of desired specificity, it was necessary simply to incubate supernatant from a phage-infected bacterial culture with a solid support (for example, test tube or Petri dish) to which the target antigen was attached, a process called as 'panning'. Unbound phage could be washed away, leaving bound phage, a proportion of which was specific for the target antigen. Bound phage then could be eluted and further enriched by infecting a second bacterial culture and repeating the panning process a number of times . Once an Fv(non covalently linked heavy and light chain domain) of appropriate specificity and affinity was identified, it could be recloned into a vector containing appropriate C domains for further drug development. .

The ability to produce a 'fully human' mAb of any desired specificity was a major advance over earlier technologies. Adalimumab, a 'fully human' anti-TNF mAb, was developed in this way and is licensed for use both in RA and severe Crohn disease. Belimumab is a mAb against B-lymphocyte stimulator (BLyS) which was developed using this technology and is in the early phase of development for a number of rheumatic indications (Table 2). Despite the theoretical advantage of fully human mAbs in terms of immunogenicity, however, CDR3 is not germline-encoded . Therefore, this portion of any immunoglobulin molecule is not subject to conventional immune tolerance mechanisms and may remain immunogenic, particularly on repeated administration.

Human immunoglobulin transgenic mice

Development of 'fully human' antibodies is the development of mice that are transgenic for the human immunoglobulin locus. These mice have been manipulated such that their endogenous immunoglobulin genes are disrupted and are replaced by their human counterparts [48,49]. In some cases, all human immunoglobulin genes have been inserted, including all heavy-chain classes [50]. When these mice are immunised, they produce 'human' antibodies via physiological processes that include affinity maturation. mAbs then can be developed using conventional fusion technology or even phage display technology. Ofatumumab and golimumab, fully human antibodies against CD20 and TNF-α, respectively, both currently in phase III development for RA, were derived by this process.. Although a number of 'fully human' therapeutic mAbs have been developed by both phage display and transgenic mouse technology, it is too early to say whether one approach has specific advantages over the other. As highlighted in a recent review [51], phage display may provide a more limited potential repertoire than transgenic mice due to restrictions on antibody expression in bacteria. Furthermore, a higher proportion of mAbs derived from phage display require 'lead optimisation' to improve their affinity, presumably due to the lack of in vivo affinity maturation. However, both types of mAb have proven clinical efficacy, suggesting that these are complementary technologies with important roles in future mAb development.

Fusion proteins and non-monoclonal antibody entities-

To treat rheumatological disease are fusion proteins are used, in which the extracellular domain of a cell surface receptor is fused to part of an immunoglobulin C region, generally human IgG1, to create a soluble form of the receptor ( Table 2). Etanercept (a fusion protein of CTLA4) is the best recognised in rheumatological practice, representing a soluble form of the p75 TNF receptor that inhibits TNF-α

activity. The IgG1 C region increases the size and hence the half-life of fusion proteins but potentially also imparts other functions such as complement activation and FcγR binding [52]. Abatacept(CTLA4 immunoglobulin), a fusion protein of CTLA4 and human IgG1, competes with CD28 for binding to CD80 and CD86, thereby interfering with T-cell activation. In the C region has been mutated to reduce complement activation. Atacicept (TACI-Ig) is a soluble form of the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). TACI is a ligand for both BLyS(B- lymphocycte stimulating) and BAFF (B-cell activating factor) and atacicept therefore neutralises both of these B-cell growth factors, distinguishing it from both belimumab and the BLyS receptor fusion protein, BR3-Fc, which neutralise BLyS only [53]. Thus, fusion proteins are generally simple to design

These are single-chain polypeptides that are engineered for full ligand binding and effector function but that are one third to one half the size of a conventional mAb [54]. TRU-015, directed against CD20, comprises an anti-CD20 Fv attached via a linker to an Fc that has been modified to reduce complement activation but to maintain FcγR binding. It is currently undergoing early-phase studies in RA and SLE. The SMIP™ technology(small modular immunopharmaceutical) equally permits the incorporation of receptor fragments in place of an Fv , for example- toxins in place of an Fc. Whereas smaller biological entities may require more frequent dosing, potential advantages include improved tissue penetration that, in RA, might provide greater access to inflamed synovium. The smallest antibody fragment drugs currently under development are single VH or VL domains (nanobodies® and domain antibodies or dAbs™) [55-57]. Aside from their small size, potential advantages include ease of production and greatly enhanced stability, potentially allowing oral administration. If required, the half-life of such antibody fragments can be extended using PEGylation or via fusion to an Fc region. Such an approach was taken for the development of an anti-TNF dAb that is currently being tested in phase II trials in psoriasis [58]. Dual-specificity agents that neutralise two distinct cytokines simultaneously or bring a target and effector cell into apposition can also be created. The latter approach was pioneered many years ago in the form of bispecific antibodies [59].

Fc modifications

Tthe main focus of biotech activity has been, quite reasonably, the mAb V region - developing mAbs with novel specificities or improved affinities. The 'downstream' effects of mAbs and fusion proteins, following ligand binding, rely on the C region/Fc - and not all sequelae are desirable. For example- most CD4 mAbs studied in RA trials were profoundly depleting, whereas non-depleting mAbs were more potent tolerogens in animal models. Similarly, it is thought that complement activation is responsible for some of the infusion-associated adverse effects of mAbs. A profound example of the consequences of FcγR binding was witnessed following the administration of TGN1412 to six health volunteers in a phase I clinical trial in 2006 [60]. Massive cytokine release was triggered when the Fc of the 'agonistic' CD28 mAb bound to human FcγR. The isotype of TGN1412 was human IgG4, which has a lower affinity than IgG1 for FcγR and does not activate complement. The lack of interaction between human IgG4 and monkey FcγR probably explains why the mAb appeared safe in primate studies.

Engineering of mAb Fcs is now relatively common, following the identification of key residues that underlie both complement and FcγR binding [61-64]. In general, modification is performed to reduce effector function, although it may also

be enhanced [65]. For example- the CTLA4-Ig Fc is mutated to reduce complement activation, which may reduce the incidence of infusion reactions. Certolizumab pegol (pEGylated Fab fragment)has a unique structure among mAb therapeutics. It comprises the Fab fragment of a humanised TNF-α mAb conjugated to polyethylene glycol. It is acting as a pure TNF-α antagonist. PEGylation increases the half-life of the molecule, which remains smaller than a conventional mAb [66]. It is efficacious in RA and Crohn disease, which attests to the importance of TNF-α neutralisation in their treatment, without an absolute requirement for Fc-mediated effector mechanisms. Several engineered CD3 mAbs are currently in development for indications that include psoriatic arthritis and RA. These have been modified to reduce FcγR binding to harness the efficacy of CD3 blockade with reduced side effects. The original murine CD3 mAb, OKT3, potently reversed allograft rejection but caused a profound cytokine release syndrome on initial dosing, mediated via FcγR binding [67]. Otelixizumab is a humanised rat mAb in which asparagine has

been replaced by alanine at residue 297 of the human IgG1 Fc. This is the o-linked glycosylation site, where carbohydrate is incorporated into the mAb structure. The mutation therefore creates an aglycosyl mAb that in vitro and preclinical data suggest has significantly reduced effector function , and this has been confirmed by clinical studies in allograft recipients and type-1 diabetics [68,69]. Teplizumab is a humanised Fc-mutated version of OKT3. It has been rendered 'non-mitogenic' by the mutation of two key FcγRbinding residues and has demonstrated efficacy in psoriatic arthritis [70]. A third CD3 mAb with similar properties is visilizumab. in this case inflammatory bowel disease trials have demonstrated that its efficacy is accompanied by significant first dose-associated cytokine release [71].Advances in glycobiology have led to an explosion of knowledge around carbohydrate structure-function relationships, which is now being exploited in glyco-engineering. Sugar contributes between 3% and 12% of the mass of an immunoglobulin molecule, the precise Fc sugar content and structure influencing effector function [72,73]. This can be modified either chemically or by producing mAbs in cell lines expressing particular sugar-modifying enzymes. For example- a glyco-engineered form of rituximab that has enhanced ADCC (antibody-dependent cellular cytotoxicity) activity has been created [74]. Even a mAb that potently activates complement and strongly binds FcγR will not necessarily lyse cells expressing its target antigen. Conversely, some targets are particularly attractive for cell lysis. CD52 is one such target and even a human IgG4 CD52 mAb (IgG4-CAMPATH or IgG4-alemtuzumab) induced profound lymphopenia despite absent complement activation and weak FcγR binding . Similarly, mAbs against distinct epitopes of the same antigen can have widely differing cytotoxic characteristics [75]. The critical features of the target antigen have not been fully defined, but close apposition between mAb and target cell membrane is a key parameter, as is the case with alemtuzumab and CD52 [76]. Interestingly, alemtuzumab has a relatively low affinity for CD52, demonstrating that high

affinity is not required for potent cytotoxicity.

Table 2

S.NO

Category

Nomenclature

Example

Brand name

Specificity

1.

Chimeric

-ximab

Infliximab

remicade

TNF-α

Rituximab

Rituximab

CD20

2

Humanised

-Zumab

Aletuzumab

MabCampath

CD52

Tocilizumab

RoActemra

IL-6R

Certolizumzb pegol

Cimzia

TNF-α

Otelixizumab

Aglycosal

CD3

Teplizumab

Fc-mutated

CD3

Visilizumab

Fc-mutated

CD3

3

Fully Human

-mumab

Adalimumab

Humira

TNF-α

Ofatuzumab

Humax-CD20

CD20

Belimumab

Lymphostat-B

BLys

s

Golizumab

TNF- α

4

FFusion protein

-cept

Etanercept

Enbrel

TNF- α

Abatacept

Orencia

CD80/CD86

Atacicept

BLys/BaFF

Conclusion-

Progress in biotechnology and a greater under standing of the pathophysiology of the immune disease, novel therapy may offer patients markedly improved outcomes and perhaps even remission.The growing appreciation of need for more effective therapies has driven the development of novel therapy for Rheumatoid arithritis

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