EDUCATION & DEBATE

MonoclonalAntibodies in Medicine

Discovery ofantibodies

Meirion B Llewelyn, Robert E Hawkins, Stephen J Russell

Passive immunisation has been used in clinicalpractice since the end of last century, mainlyfor prophylaxis. Success of early treatments wasmarred by anaphylactic reactions and serum sick-ness because antibodies or antitoxins were notraised in humans. Recombination of gene segmentsduring antibody synthesis means that specific anti-bodies for numerous antigens can be producedfrom a limited gene pool. Killer lymphocytes, phago-cytes, and complement then bind to the constantregion of the antibody facilitating elimination of thepathogen. Development of a method of obtaininglarge quantities of antibodies against a specificantigen (monoclonal antibodies) offers the possi-bility of initiating host defence mechanisms againstany unwanted antigen, though some problems stillremain in preventing the body. from attacking themonoclonal antibody.

History ofpassive immunisationThe first use of passive immunisation is thought by

some to have been on Christmas night 1891.' Therecipient is reported to have been a young boy inBerlin with diphtheria who was cured by injection ofdiphtheria antitoxin, although this cannot be provedtoday.2 Passive immunisation is based on work carriedout by Emil Behring and Shibasabura Kitasato fromRobert Koch's Hygiene Institute in Berlin. On 4December 1890 Behring and Kitasato published alandmark article in which they showed that serumfrom an animal actively immunised against diphtheriatoxin could be used to neutralise even a fatal dose of thetoxin in another animal.3 The same results wereobtained with tetanus toxin, and the effects of thesesera were shown to be specific in that tetanus antitoxindid not protect against a challenge with diphtheriatoxin.The potential for treatment in humans was

immediately apparent. At that time diphtheria was acommon and often fatal disease and Behring set towork trying to produce large amounts of antitoxin thatwould be effective against diphtheria in humans. Thepharmaceutical company Farberwerke Hoechst scaledup production by immunising sheep, and the first largetrials started in 1893 with good results.4 Immunisationof horses was then introduced and by 1894 the casemortality of diphtheria in Paris had already fallen from52% to 25%.5 The first British patients were treated inthe summer of 1894 with favourable results.6 Behringthen suggested the combination of diphtheria toxinand antitoxin serum as a safe method of producingactive immunity in humans. This was introduced in1913 but superseded when inactivated diphtheria"toxoid" was found to induce immunity.

A major problem with passive serotherapy was itstoxicity. Anaphylactic reactions often occurred aftergiving horse serum and serum sickness (fever, rashes,and joint pains) was common. Behring believed thatthe antitoxin effect resided in the protein fraction of theserum and went on to show that ammonium sulphateprecipitated a fraction (now known as gammaglobulin)that retained antitoxin activity. Precipitation reducedside effects but did not completely overcome theproblem of serum sickness, which is now known toresult from the deposition of circulating immunecomplexes. Cleavage of gammaglobulin by pepsinfurther reduced the incidence of serum sickness.

Large scale production of antiserum to diphtheriatoxin was made possible through the work of thebacteriologist Paul Ehrlich,'7 who moved to Koch'sinstitute in 1889. He took part in early work challeng-ing rabbits with bacterial toxins and subsequentlydeveloped guinea pig assays to test the potency ofdifferent batches of antisera. Ehrlich proposed the sidechain theory of toxicity. He suggested that toxinsmediated their effects on cells through preformedprotein side chains and that immunity arose becauseof overproduction of these side chains. The sidechains are reminiscent of what we now know asantibodies, although Ehrlich assumed (incorrectly)that their primary function was nothing to do withtoxin neutralisation.The introduction of human immunoglobulin pre-

parations in 1944 as treatment for and protectionagainst measles finally overcame the problem of serumsickness8 and today pooled human immunoglobulinsare still used.9 Agammaglobulinaemic patients havebenefited from the availability of relatively pure immu-noglobulin preparations, which are given regularly toprevent infection. There are currently two typesof preparation: normal immunoglobulin and specificimmunoglobulin. Normal immunoglobulin is pre-pared from pools of at least 1000 random donations ofhuman plasma and contains antibodies to hepatitis A,measles, mumps, and other viruses. It producesimmediate protection that lasts four to six weeks.Specific immunoglobulins are prepared by pooling theblood of patients convalescing from specific diseases.Preparations are available against several pathogens,including hepatitis B virus, Clostridium tetani, andvaricella-zoster virus. Other specific immunoglobulinsare available or under development4 for example,antirhesus D antibodies raised by deliberate challengeof normal male volunteers with the rhesus D antigenare used routinely to prevent sensitisation of mothersnegative for anti-D antibodies after delivery of anantibody positive baby. There is also some interest inpassive immunisation for AIDS.'0 Some antisera canstill not be produced safely in humans and thus, for

BMJ VOLUME 305 21 NOVEMBER 1992

This is the first ofthree articleson the development andclinical applications ofmonoclonal antibodies

Medical Research CouncilCentre and Addenbrooke'sHospital, CambridgeCB22QHMeirion B Llewelyn,MRC trainingfellowRobert E Hawkins, CRCsenior clinical research fellowStephen J Russell, MRCclinician scientist

Correspondence to:Dr Russell.

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example, horse and rabbit antilymphocyte globulinsare still used to treat severe aplastic anaemia and tosuppress rejection of renal allograft.

Antibody structureThe active components of gammaglobulin that

protect against the effects of diphtheria and tetanustoxins are now known to be immunoglobulins, alsoknown as antibodies. IgG, the prototype antibody, isa glycoprotein with a molecular weight of 150000Dalton. The molecule consists of two identical heavychain-light chain heterodimers linked by a disulphidebridge to form a Y shaped structure (fig 1). Each heavychain comprises one variable (V) and three constantimmunoglobulin (Ig) domains, whereas light chainsconsist of a single variable and a single constant Igdomain. Each domain comprises about 110 aminoacids.

Antigen binding sites

Disulphide bonds

CH3 CH3 Fc regioninitiates hostdefence systems

FIG 1-Structure of antibodies. Antigen binding sites comprise twovariable regions (V) and two constant regions (C), one each from theheavy and light chains. Fc region comprises two constant regions fromthe heavy chain

From a functional perspective, the importantfeatures are the antigen binding site (Fab) and the Fcregion (fragment crystallisable), which is responsiblefor initiating host defence mechanisms. The Fc regionis the stem of the Y structure and the two antigenbinding sites are at the tip of the outstretched arms.The hinge region of the antibody is flexible so theantigen binding sites are widely and variably separated,permitting the linking of two identical antigens by thesame antibody; many invading microorganisms such asbacteria and viruses have repeating subunits and thiseffect allows much more effective binding (the avidityeffect). The multiple binding sites also allow aggre-gation and rapid clearance of antigens.The antigen binding site also crystallises quite easily

and this has allowed x ray diffraction analysis ofantibody structure." Several crystal structures are nowknown and they show important conserved features.The antigen binding site is formed by the apposition oftwo globular variable domains, one derived from theheavy chain (VH) and the other from the light chain(VL). The folds of each of the V domains form threeshort loops of amino acids-the hypervariable loops orcomplementarity determining regions (CDRs). At theantigen binding site all six loops (three each fromthe heavy and light chains) come together. Theextraordinary specificity of antibodies to the vastnumbers of possible antigens is possible because of thevariability in both the length and the amino acidcomposition of these loops. Their configurations are,however, not entirely random and can be categorised

into a limited number of recurring canonical forms.'2Alterations in residues of the underlying ,B sheetframework adjust the orientation of individual loopsand also play some part in the structure of the bindingsite. Besides providing insights into the basis ofantibody diversity, detailed analysis of the structuralfeatures of antigen combining sites provides a basis fornew approaches to antibody construction.Although an antibody may have a direct neutralising

effect on a virus or toxin, Fc mediated defence systemsare needed to eliminate the source of the antigen.Antibodies can sensitise a target cell for attack by killer(K) lymphocytes by coating (opsonising) the cell withIgG. K lymphocytes recognise coated target cellsbecause they have receptors for the Fc region of IgG ontheir surface. This mode of killing is known asantibody dependent cellular cytotoxicity. Similarly,phagocytes (including neutrophils and macrophages)target an opsonised antigen through Fc receptors. Thecombination of antibody and antigen can activatethe classical complement pathway and deposition ofcomplement on the target cell can produce lysis orfurther opsonisation.

ANTIBODY CLASSES

There are five classes of immunoglobulin, classifiedaccording to the type of heavy chain they possess13:IgG, IgA, IgM, IgD, and IgE. IgG comprises 70% ofthe immunoglobulin pool and is produced mainly insecondary immune challenges. There are four IgGsubclasses (IgG 1-4) which vary in their capacity toinitiate host defence systems. IgGl and IgG3 mediateantibody dependent cellular toxicity and can bindcomplement whereas IgG2 and IgG4 are relativelydeficient in these functions (IgG2 can bind comple-ment, although less efficiently than IgGl and IgG3).IgG is present in the intravascular and extravascularspaces and, importantly, can cross the placenta toprotect the developing fetus and new born child. IgM,which forms 10% of the immunoglobulin pool, has apentameric structure and is mainly confined to theintravascular space. It is a potent complement fixingantibody but cannot produce antibody dependentcellular cytotoxicity. IgA (secretory immunoglobulin)forms around 20% of the pool. It has a dimericstructure and is present in secretions such as saliva andmilk. IgD forms less than 1% of plasma immuno-globulin and is mainly found on the surface of B cells.Its function is unknown. IgE binds to Fc receptors onthe surface of basophils, eosinophils, and mast cellswhere cross linking by antigen triggers the release ofpharmacological mediators that can cause immediatehypersensitivity reactions.

Genetic basis ofantibody diversityThe finding that each antibody had its own amino

acid sequence in the variable region suggested aseparate gene for each antibody produced. If this wereso, most of the human genome would be needed tocode for the enormous number of antibody moleculesthat could exist. In 1965 Dreyer and Bennett hypothe-sised that each class of constant region is encoded forby one gene but that large numbers of genes exist forthe variable domains (VH and V1).'4 They correctlysuggested that antibody genes would be formed byrecombinations between these genes during B celldevelopment. We now know that assembly of thegenes for variable domains occurs by random joining ofa number of gene segments (V, D, and J for heavychains or V and J for light chains) and that additionaldiversity results from loss or addition of nucleotides atthe junctions between these gene segments (fig 2).1" Inthe case of the heavy chain, there are about 75 VHsegments, around 30 DH segments and six JH segments

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Germline DNA:

VH genes HH segments .H segments

Rearrangement v D J C g

Transcription and RNA editing

Messenger RNA V t) J CH

:- -|T'ranslationFIG 2-Antibody diversity the P iIIIZE iiI .joininig of V, D, J segments ofthe 1 2 -:heavy chain. The genes are Complementrityencoded by a relatively small determining regionsnumber ofgermline genesegments but the random IiEl Nucleotides ad-ded or reimoved during joining-recombination gives rise to largenumbers ofexpressed genes i di-esit

on each chromosome."4 6 The V segment encodes mostof the variable domain including the first and secondcomplementarity determining regions (CDR1 andCDR2) whereas the highly variable CDR3 is encodedby D segments and by nucleotides at the V-D and D-Jjunctions. The VDJ segment is then apposed to theconstant region at the messenger RNA stage, afterediting of the large nuclear RNA transcripts produced.The number of unique immunoglobulin variableregions possible due to random recombination of V, D,and J gene segments is very large. Moreover, thejoining positions of VH, DH, and JH segments are notalways the same and the DH segment may end up inany of its three possible amino acid reading frames.This, along with random loss or addition of nucleotidesat the boundaries between the segments furtherincreases diversity.'5 The x and X light chain loci alsoundergo recombination of VL and Jl coding regions(there is no equivalent of the heavy chain D segment).Since any light chain is thought to be able to combinewith any heavy chain an enormous diversity (estimatedat over 1010 antibodies) can be produced from arelatively small number of DNA segments. Furtherdiversity arises later through the introduction ofscattered point mutations into the DNA of theassembled heavy and the light chains.'7 This is knownas somatic hypermutation and usually occurs duringthe immune response to foreign antigen (see below).

Humoral immune responseDuring a typical infection protective antibodies are

produced against the invading organism. After aninitial lag phase the level of antibody detectable in thebloodstream rises rapidly, reaches a plateau, and thenfalls to low levels once the pathogen has been eradi-cated. IgM is the first class of antibody to appear in thisprimary antibody response, followed by IgG, whichhas a longer lag period. Re-exposure to the samepathogen gives rise to a "memory" or secondaryresponse that is stronger and has a much shorter lagphase; it is dominated by IgG and tends to be of higheraffinity.

Cellular basis ofthe immune responseAntibodies are produced by B lymphocytes. These

cells develop in fetal liver and subsequently in bonemarrow. It is at these sites that immunoglobulin generearrangements occur and potentially harmful selfreactive B cells are deleted from the repertoire. In itsresting state each B cell displays an antibody of onespecificity on its surface. At any one time there areabout 108 different B cell clones in the body, each

displaying a unique antibody. When antigen enters thebody it binds specifically to only a few of these restingB cells, stimulating them to divide and to mature intoplasma cells that secrete large amounts of the immuno-globulin which was previously displayed on theirsurface. This model of clonal selection of B cellswas originally proposed in the 1950s by McFarlaneBurnett.'8 During antigen driven B cell proliferationthere is a switch in the main class of antibody producedfrom IgM to IgG, IgA, or IgE (class switching), and Bcells that secrete higher affinity antibodies begin toemerge (affinity maturation). The B cell class switch isa complex process which is regulated by externalfactors including local secretion of cytokines byantigen specific helper T cells. Some of the pro-liferating cells do not become secretory but continue todisplay their surface antibody and continue to circulatelong after the infection has cleared. These memory Bcells (mainly derived from cells which have undergoneclass switching) are responsible for the more rapidonset and greater intensity of the secondary immuneresponse.

Monoclonal antibodiesIn 1975 George Kohler and Cesar Milstein of the

Medical Research Council Laboratory of MolecularBiology in Cambridge described an elegant system ofobtaining pure antibodies of known specificities inlarge amounts (fig 3). 9 An astute reporter fromthe BBC World Service immediately recognised thepotential of the discovery of monoclonal antibodies butit was some time before they were widely used. In theoriginal method, a mouse is immunised repeatedlywith the desired antigen and the spleen, which containsproliferating B cells, is removed. B cells normally die inculture, but can be immortalised by fusion with a non-secretory myeloma cell. The resulting hybridoma canthen secrete large amounts of the antibody encoded by

FIG 3-Production of monoclonal antibodies. Mice are immunisedwith antigen and their B lymphocytes are then fused with a non-secretory myeloma cell line. The myeloma cell line has a mutation in thehypoxanthine guanine phosphoribosyl transferase (HGPRT) gene andis therefore killed in hypoxanthine aminopterin thymidine (HA T)medium. During selection in HAT medium, unfused myeloma cells arekilled and unfused mouse B cells die spontaneously. Splenic B cellmyeloma cell hybrids (hybridomas) survive HAT selection since the Bcell supplies a normal HGPRT gene. The antigen specific B cell alsosupplies immunoglobulin genes which encode its unique antibody.Screening of large numbers of hybridomas usually identifies a fewclones which produce antibody of the desired specificity (against theimmunising antigen).

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Immunise with Cell culture-antigen .. myeloma line

Fuse in| polyethylene glycol

Spleen cells .. J Myeloma.cels

HAT medium 'Select hybrid cells

j Assay for antibody

tioanev + Freeze

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z

Emil von Behning andcolleagues immunisingguinea pigs its B cell fusion partner. Supematants from the hybrids

that survive the selection procedure outlined in figure 3are tested for binding to the original antigen.-Alan Williams showed in 1977 that monoclonal

antibodies could be raised against biologically intere-sting MoleCUleS20 and this triggered the developmentof a stream of useful monoclonal based diagnosticprocedures. It soon became apparent, however, thatrodent monoclonal antibodies were unsuitable or in-effective for treating humans because they initiatehuman defence systems poorly and are themselves thetarget of an immune response that can greatly shortentheir circulating half life. One solution is to producehuman monoclonal antibodies, but this is difficult forseveral reasons. Firs'tly, human B cells immortalisedby fusion with a myeloma cell or by infection with

!~~~~~~~~~~S2Epstein Barr virus2 are'unstable and can rapidly losetheir capacity for antibody production. Even whenthey are successfully transformed with Epstein Barrvirus antibody yields tend to be low and the virususually transforms IgM secreting cells that producelower affinity antibodies. Secondly, hyperimmunisa-tion of human subjects is problematic, particularlyagainst self antigens. In vitro immunisation-that is,antigenic stimulation of cultured human lymphocytes

provides a partial solution to this problem, butantibody production is still unstable after immortalisa-tion. The third problem is that the most convenientsource of human lymphocytes is the peripheral blood,a poor source of B cells producing specific high affinity

antibodies. Such cells are more abundant in the spleen,bone marrow, or lymph nodes.The recent arrival of antibody engineering tech-

niques (detailed in the second review of this series)provides several altemative solutions to the productionof human monoclonal antibodies. We are thereforebeginning to see a renaissance of immunotherapy as atreatment after its eclipse by chemotherapy. In hisaddress to the Naturalists Congress in 1895 Behringpredicted, "I have no fear that the thought which formsthe basis of serum therapy will ever disappear out ofmedicine"5 and 100 years later this prediction seems tobe holding well.

We thank Cesar Milstein, Geoff Hale, Kerry Chester, andGreg Winter for encouragement and critical reading of themanuscript. REH is a Cancer Research Campaign seniorclinical fellow and was formerly a Medical Research Counciltraining fellow. MBL is funded jointly by MRC and Celltech,SJR is an MRC clinician scientist with additional supportfrom Kay Kendall research foundation and Louis Jeantetfoundation.

1 Singer C, Underwood EA. A short history of medicine. 2nd ed. Oxford: OxfordUniversity Press, 1962.

2 Zwister 0, Vor 100 Jahren: die Entdeckung des Diphtherie-Heilserums.Die Gelben Hefte 1990;4:153-9.

3 Behring EA, Kitasato S. Uber das Zustandekommen der Diphtherie-Immunitat und der Tetanus-Immunitat bei Thieren. Dtsch Med Wochenschr1890;49: 1113-4.

4 Gronski P, Seiler SR, Schwick HG. Discovery of antitoxins and developmentof antibody preparations for clinical uses from 1890 to 1990. Mol Immunol199 1;28:1321-32.

5 MacNalty AS. Emil von Behring. BMJ 1954;i:668-70.6 Dale H. Paul Ehrlich. BM_ 1954;i:659-63.7 Browning CH. Emil Behring and Paul Ehrlich. Nature 1955;170:570-5.8 Stokes J Jr, Maris EP, Gellis SS. The use of concentrated normal human serum

gamma globulin (human immune serum globulin) in the prophylaxis andtreatment of measles. J Clin Invest 1944;23:531

9 Department of Health. Immunisation against infectious disease. London:HMSO, 1990.

10 Karpas A, Hill F, Youle M, Cullen V, Gray J, Byron N, et al. Effects of passiveimmunization in patients with the acquired immunodeficiency syndrome-related complex and acquired immunodeficiency syndrome. Proc Nad AcadSci USA 1988;85:9234-7.

11 Poljak RJ, Armzel LM, Avey HP, Chen BL, Phizackerly RP. Saul F. Three-dimensional structure of the Fab fragment of a human immunoglobulin at2 8-A resolution. Proc NatlAcadSci USA 1973;70:3305-10.

12 Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air G, et al.Conformations of immunoglobulin hypervariable regions. Nature 1989;342:842-83.

13 Nisonoff A, Hopper JE, Spring SB. The antibody molecule. New York:Academic Press, 1975.

14 Dreyer WJ, Bennett JC. The molecular basis of antibody formation: aparadox. Proc NatlAcad Sci USA 1965;54:864-9.

15 Tonegawa S. Somatic generation of antibody diversity. Nature 1983;302:573-81.

16 Pascual V, Capra JD. Human immunoglobulin heavy chain variable genes:organisation, polymorphism and expression. Adv Immunol 1991;49:1-74.

17 Berek C, Milstein C. Mutation drift and repertoire shift in the maturation ofthe immune response. Immunol Rev 1987;96:23-41.

18 Bumet FM. The clonal selection theory of acquired immunity. Cambridge:Cambridge University Press, 1959.

19 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody ofpredefined specificity. Nature 1975;256:495-7.

20 Williams AF, Galfre G, Milstein C. Analysis of cell surfaces by xenogenicmyeloma-hybrid antibodies: Differentiation antigens of rat lymphocytes.Cell 1977;12:663-73.

21 Steinitz M, Izak G, Cohen S, Ehrenfeld M, Flechner I. Continuousproduction of monoclonal rheumatoid factor by EBV-transformed lym-phocytes. Nature 1980;287:443-5.

(Accepted 19 October 1992)

ANY QUESTIONS

Is there a risk of upper respiratory tract infections being passedon when children share 2 agonist inhalers?

I am not aware of any published work in relation to thisquestion. Theoretically, however, it is possible that viralinfections could be transmitted in this way under certaincircumstances. Most viruses die as a result of exposurewithin a few hours, but it is likely that viable virusparticles survive on the inhaler and may be transmitted indroplet form or as a contaminant on the hands or on theinhaler. Such transmission would be possible for the

common cold and for other respiratory viruses, such asrhinovirus or respiratory syncytial virus. If two peopleused the same inhaler within a relatively short time-evenan hour or so-these infections could be transmitted.Transmission of bacterial infection is unlikely as aconsequence of shared use, but there may be a theoreticalpossibility of transmitting tuberculosis. Under thecircumstances it seems sensible to continue to enforcethe recommendation that such inhalers should not beshared.-A RICHARD MAW, consultant otolaryngologist,Bristol

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