I (". •,

June 7982(2!/j 1 r Vo/umr: 3, No . 6

immunology today

Receptors, antibodies and disease from john Newsom-Davis and Angela Vincent

Biochemi.sts d · 1 · · , en ocnno ogists, Immu-nologists a nd neurolog ists sha re a n In te res t in ce ll surface receptors a s was ev ident a t a rece nt symposium held by the C iba Founda tion * When these receptors are targets for a uto­ant ibod ie·s 1 · 1 . 1 . , enc ocn ne or neu ro og ica di sea se ca n resu lt. Attent ion foc ussed pa rti cu lar ly o n receptors (R ) for thyroid stimul a ting horm one (TSH) , Insulin , ace tylcho line (AC h), pro lac­tin and ~2-adrenerg i c ago nists .

The 1· · 1 · . . C 111I C<l Importance o f a utO-fnt ibod ies to receptors, judged pure­y In numerica l terms, varies con-

Siderab l I 1. . . (' . Y · nsu In-res istant diabetes Insuiin-R a ntibody ), for exa mple, is

ex tremely rare - less th a n 50 cases

• Recepto .. A ·b d ' . . , ' s "nt1 o 1es a nd Disease (C1ba ~mposium no. 90) was organized by Dr

<IV Jd Evered a nd cha ired by Professor N. !\: Mitchi son . The proceedings wi ll be published by Pitman Medica l Ltd .

1. AChR

have been reported wo rld wide, to date. On the other ha nd Graves ' di sease (TSI-1-R ant ibody) a nd myas­thenia grav is (ACh-R a nt ibody) are modera tely prevalent wh ile as thma a nd ot her atopic di so rders ( ~2 -a dre n­ergic-R ant ibody) a re very common. Direct imp li ca tion of the re leva nt anti­bodies in the di sease process seems we ll es tab li shed for most of the di sor-ders di scussed, a lthough som e uncer­ta inty ex ists a bout the rol e of the rece ntly identifi ed ~2 -a drenerg ic - R ant ibody.

In as thma a red uction in lung t issue ~2 adrenergic receptors, as meas ured by agonist binding or ~ 2 responses, ha s been es ta b li shed for some time a nd a ntibod ies spec ifi c for the ~ 2 receptor can be identifi ed either by inhibition o f ago ni s t binding, by immunoprecipitation of so lubilized

mntintu:t!onji. !50

2. Insulin -A

4. 8 2-adrenergic-R 5.TSH-R

Fig 1 o· · d b . · · Iagrammatic representation (roughly to sca le) of propose su umt structures f h · · · d · d · · J • d b f hi . o t e prmc1pal receptors d1scusse , m 1catmg t 1e s1ze an num er o

ndmgs ite(s) for the natural ligand (hatched). Receptors a re ranked ( 1-5) acco rding to the ll1formation ava il ab le. Numbers denote the mol. wt.

Transplantation

Applied wisdom from Elizabeth Simpson

The tradition of' an annu al 'Round Table Symposium on Applied Immunology ' was ca rried into its 13th year in Axams, Austria , on 25-27 January.

On these occas ions small numbers of clinicia ns and sc ientists mee t to discuss new developments in resea rc h that have mutual interest.

In the opening sess ion on ce ll­surface antigens H. Bainer (R ij svij k) di scus sed the in -11ivo effec t of mono­clona l a ntibodies (MAbs) directed aga inst T-ce ll subsets in Rhesu s monkeys grafted with a ll ogeneic skin . The MAbs defined huma n lympho­cy te subsets that crossreact with the equiva lent rhesus monkey ce lls . Anti ­bodies aga inst the helper/ inducer subset (OKT4 eq uiva lent) pro longed skin a llograft survival whereas those directed against the cy totoxic/ sup­pressor subset (OKT8 equ iva lent) not only fa iled to prolong survival but m ay have shortened it. T hese results are in line with recent reports that in mice and rats the T ce ll s invo lved in initi­ating graft rej ec tion are of the he lper (Ly J+ ) phenotype a nd not the cyto­toxic/suppressor (LyJ+ 2+) pheno­type . D. Arndt-J ovin (Gottingen) pre­se nted her use of sophisti ca ted phys i­ca l method s (Auoresce nce e nergy transfer, rotat iona l diffusion and tra nslational diffusion) to assess the density, proximity and mobility of mouse H-2 (Kk) antigens detected .with monoclonal antibodies. The basic info rmation obta in ed from these studies should a ll ow direct exa min­at ion of how H-2 a ntigens 'associate ' with such ext rinsic antigens as viruses and thus the necessa ry const ra ints for considering the a lte red se lf versus dual receptor hypothes is of T-ce ll recep­tors. P. Peterson (Uppsala) presente d

cmztinlll'drm fJ . /51

PFIZER EX. 1582 Page 1

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[J (immunology today J Volum e 3, no. 6

June 1982

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news and features

Receplo rs, antibodin and dis·ease John Newsom-IJavis and Ange la Vincent

Tran.lf!lrmlallon: ajijJ/it:d wi.1rlom Elizabeth S im pson

letters

l nterleukim: a rose ts a rose . Steve n G illi s

rostrum

Is .1-perm imllmno.l'1lpjm:.lsive in male lwmo.1·ex1uds and /H/S i?C /I!III.i::_erf 11/CTI:'

Gene M . Shearer and Ursula H urtcnbach

compass

The vulnerabiLity oj'1kin grajis to allo- and xeno-rmtibodies: a rrmwulmm r1'.1olved:'

Les li e Hrent

C:om.jJlenwnt and .\olubili.;;_alirm o/ irmmme comjJiexes· Nevin 1-1 ughes-J ones

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i\11 mwclonal antibodies - tools to dis.1erl lhe nervmo· system Colinj. Barnsta ble

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The lll(llll711ary gland man immunological organ I ,ars A. I-ianso n

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PFIZER EX. 1582 Page 2

160 frmnmwlrJ!',)' Today, vol . .3, No. (), !f)82

The three-dimensional structure of antibodies

l\!Iarkus Marquart and Johann Deisenhofer Max-Planck Institut fi.ir Biochemic, A.btcilung Strukturforsclntng II, D-8033 Martimriccl, F.R.C.

Antibody molecules arc glycoproteins which occur in vertebrate species. They recognize and bind an enor­mous variety of foreign substances (antigens) and sub­sequently trigger further defense mechanisms at the molecular or cellular level. Specific recognition requires surface structures complementary to the antigen and hence a huge variety of antibody molecules. In contrast the- effector functions need identical interactiun sites in all antibody molecules.

The determination of the primary structure of immunoglobulins 1- 1 and the X-ray crystallographic studies of several antibody molecules and frag­ments1·'.7·111·12-1' led to an advanced understanding of the way in which antibodies meet these opposing requirements.

Fig. 1 Schematic representation of an lgG 1 immunoglobulin molecule. The arms of the Y-shapecl molecule arc formed by the Fab parts, the stern is maclc up by the Fe 'part. The light chains arc linked to the heavy chains IJy a clisulphiclc bridge close to the C-tcrrninus. The two heavy chains are conncctecl via two disulphide linkages in the hinge region.

Fig. I is a schematic drawing of an antibody molecule of class I gG I. It is composed oft wo idcnt ical heavy chains and two identical light chains with mol. wts of 50,000 and 25,000, respectively. Both types of polypeptide chain arc folded into domains: the four domains of the heavy chain arc VH, CIII, CII2, ;mel CH3; the light chain consists of the two domains VL and CL. All domains except CH2 arc arranged in pairs which arc held together by non-covalent f'orccs. Inter-chain disulfide bridges provide further stability.

Among antibody molecules of a given class and species, the V-dornains differ considerably in amino acid sequence, whereas the C-domains have identical sequences. The V-domains are composed of about 110 amino acid residues at the N-terminal end of heavy and light chains. The VH-VL pair together forms the antigen binding site; different antibody specificities arc the result of different amino acid sequences of the V-domains. The sequence variability in V-domains is most pronounced in a f'cw hypcrvariablc rc!jions. On the other hand the framework residues arc well con­served. The constant domains CH2 and CI 13 arc involved in effector functions such as complement activation and binding to receptors on certain cell types. There is significant homology between the amino acid sequences of all C-domains, and of the framework residues of V -domains.

Proteolytic cleavage at the hinge re!jion yields stable and functional fragments: the antigen-binding frag­ment Fab, and the Fe fragment (Fe was the first anti­body fragment obtained in crystalline form) 1

'.

A 8 F E

H G c D X ARRMGEI1EIH OF STflANDS IN IMI1UNOGLOBULII< DOI·IAII<S

X N-TERMINUS UP •• C-TEfU11NU3 UP

Fig. 2 Schematic drawing of the strand topology in a V­domain viewed parallel to the strands. (x) and (e) indicate N-and C-tenninal ends of the strands point­in~ towards the ob'ierver.

~ El'il'\'i<"r Bionwdi~.t! p,~-;-; J!JH_? II I r, 7-1'! I 'ljH2flllll>ll-lllllllljS2. 7.1 This materia I was copied

at the NLM and may be

~ubject US Copyright Laws

PFIZER EX. 1582 Page 3

!rmmmology Today, z•ol. 3, .. Vo. 6, TU82

Besides IgGl, several other classes (IgM, IgA, IgD, IgE) and subclasses of immunoglobulins have been identified; the diflcrences between these arc located in the constant region of the heavy chain. The two types of light chain (kappa, lambda) can combine with heavy chains of any class.

Domain folding The general folding pattern in all immunoglobulin

domains is very similar. It is shown schematically in Fig. 2 for a V -domain. The folding is characterized by two pleated sheets connected by an internal eli­sulphide bridge linking strands B and C. The two sheets cover a large number of hydrophobic amino acid side chains.

Despite that gross similarity there exist substantial differences when one compares V- and C-domains: C-domains lack strand X, strand D is very short (2-3 amino acids) and connected to strand E. In addition the length of the loop regions inC-domains is different from V -domains, thus changing the overall shape con­siderably.

VI-I and VL, on the other hand, show only minor differences when comparee! with each other (except in the hypcrvariablc regions) as do CL, CHI and CII3.

CH2 represents yet a third type of domain, differentiated from the other C-domains mainly by the branched carbohydrate chain linked to it. It will be discussed in more detail below.

Domain-domain interaction Two kinds of domain interactions occur in irnmuno­

globulins: lateral (or trans) interactions and longi­tudinal (or cis) interactions.

In lateral interactions immunoglobulin domains other than CI-12 strongly associate to form modules VL-VI-I, CL-CH I, CH3-CH3. In V modules VI I may be replaced by VL to form light chain V dimers as seen in the Bcnce-Joncs protein fragments Rei or Auu1

. In Hence-Jones proteins, which arc light chain dimers, one of the light chains simulates the Fab parts of the heavy chain, as described for Mcg 111 •

V modules associate in a different way than C modules do. In V modules HGCD faces (sec Fig. 2) of the domains get into contact, in C modules the ABFE faces arc involved.

A considerable loss of accessible surface area 11 is connected with contact formation of the immuno­globulin domains. It amounts to 1760 A2 , 1923 A2 and 2180 A2 for VL-VH, CL-CH I modules of IgG Kol 12 ·13

and the CH3-CH3 module of an human Fe fragmcnt 11 •1" respectively. In VL-VH association both framework residues and amino acids from hyper­variable segments arc involved. A comparison of V­domain amino acid sequences of different animal species shows that the contacting framework residues arc highly conserved. Also the constant domain residues participating in lateral contact arc either invariant or rcplclcccl by homologous residues in

161

different immunoglobulin chains. This low degree of sequence variability for the residues important for lateral contact formation provides an explanation for the fact that different L-chains can associate with different H-chains to give intact immunoglobulins.

In addition to the extensive Van dcr Waals contacts, there exist a few trans hydrogen bonds, in which mainly polar side chain groups are involved. There are two salt linkages in Kol CL-CI-1 I contact: Glu 125 light chain- Lys 214 heavy chain, Glu 126 light chain - Lys 148 heavy chain, which have their analgon in Cl-13- Cl-13 pairing: Glu 356- Lys 439, Glu 357- Lys 370.

CH2 is an exception, as it forms a single unit without lateral domain interactions (see Fig. 3) *. Instead it interacts with bound carbohydrate, which is attached to Asn 297. The CI-12 residues that are involved in carbohydrate contact arc, with a few exceptions, structurally in the same positions as the residues that form the CH3-CH3 contact (face ABFE in Fig. 2). This demonstrates that the carbohydrate in CI-12 provides a substitute for the C-C con­tact and presumably helps to stabilize the Cl-12-domain. The branched carbohydrate forms a few hydrogen bonds with the Cl-12-domain, but the dom­inant interactions are hydrophobic in nature. The carbohydrate covers a hydrophobic patch of the protein made up of, Phe 241, 243, Val 262, 264, Tyr 296, Thr 260, Arg 30 I, which would otherwise be exposed to the solvent. The loss of accessible surface area of one CI-12 domain is 522 A2, which is only about half as much covered surface area as seen in CH3-CH3 contact (I 080 A2). This observation could explain the apparent 'softness' of those parts of the CI-12-domain, as seen in the crystal structurc 11 ·1 ",

which are most remote from the CI-13-CI-12 interface. The functional relevance of carbohydrate in anti­

bodies is unclear. It might be involved in intracellular movements of the glycoprotcins and in secretion u. 1 H. It m'ay well be that the origin of the altered functional properties of carbohydrate-free antibody variants is structural destabilization.

In contrast to the extensive lateral interactions, nonbonded longitudinal interactions along the heavy chain or light chain are much weaker or do not exist at all. However, they arc interesting because con­formational changes in antibodies affect those inter­actions.

Fig. 3, which represents the Fe part of an IgG I molecule shows the CI-I2-CI-13 interaction. With a loss in accessible surface area of 778 A2 this contact has roughly one 'third of the size of CI-13-CI-13 contact. The residues that participate in CI-12-CI-13 contact are highly conserved in all Ig classes, suggesting that this contact is likely to be found in IgG and IgA and as CH3-CI-I4 contact in IgE and IgM. .

*Most readers will need a stereo viewer (commercially available) to sec in three dimensions the structures shown in the paired diagrams on pages 162, 163 and I 66.

This material wascoP"ied at the and may bE ~ubje;:t USCoP"yright Laws

PFIZER EX. 1582 Page 4

Fig. 5 Amino acid comparison of residues 98- 119 (Eu num­bering) of M603, New, Kol and Eu h eavy chain s. T he und erlined res idu es were left out in Fi g. 6c .

End of VH 98

M603 : Cys Ala Arg Asn Tyr Tyr New Cys Ala Arg Asn Leu lie Kol Cys Ala Arg Asp Gly Gly Eu Cys Ala Gly Gly Tyr Gly

Gly Ser Thr Ala Gly Cys His Gly Phe lie Tyr Ser

Fig. 3 Ste r·eo draw ing of a s pa ce fi llin ~ m od e l o f human Fc-frag-!11C lll.

Th·· molt· < uk cs bu ilt from t \\ O cdt· clti<;il pol ypt'ptidc c hain -; l< h ;ti n !. cIt; , in 2J. an d idc·nti cal c;H·i>o lw rlra; ,· L\ ~"""Jl ~. llotlt lt ;ik c; ; 11 ·c rr·l a tc<i ll\ .q>pro xi lll iltt· rl i;, rl s . ,,.cl , l, l;l{'k : (: JJ 2-domains of<hain I <IIll i < lt;,in 2, n· ~ p<T t i v c l ~.

hill <', o ,·;lnt;e : C l ll-dont;Jill'i and< .tr­hnlt\ d rat•· o f c lt;1in I ;md c h;tin 2 !'I 'Sjl< T l i\'t· Jy.

F il{. -~ II{G I molecul e K o l. T h<· l·;ti> pa ri s ;11H I th\' hint;c Sl'L(IllC'Ilt <1r•: well ordcTnl inth!' h: n l c rys tal s. th e h jl.tlt i-; di soni<T•· d a nd not \' is il >it-l'('rl · \ ' 1.-rlom ;,j ll'i I>L<l' k : I :1.-rlqm; ' i'" J,J", .. \ ' 11 -rl o m;c in -; o r< •ll t; •· : ( :I I I -<I om;, in s ;11 H I hi n !.\<' S<'L( Ill \' 111

0 segment

li e Cys Ser Ser Al a Ser Cys I )C

PFIZER EX. 1582 Page 5

J segment

1 10 Try Tyr Phe Asp Val Try Gly Ala Gly Thr

Asp Val Try Gly Gin Gly Ser Gly Pro Asp Tyr Try Gly Gin Gly Thr

Pro Glu Glu Tyr Asn Gly Gly

Thr Leu Pro Leu

Fig. 6 Antigen binding region of lgGl Kol. (a) The extended third hype r­va ri a ble loop or the heavy c ha in (res idu es d raw n in b lue ;1re pre­suma b ly coded for by the D segme nt 21· ·~'1 ) folds into the puta ­t ive a nti gen binding pocket fo rmed b y VL (bl ack) and VH (reel ) . (b) C a backbone a nd sidec ha ins o f Kol a nti ge n binding pocke t. Co lours have the sa m e m ean ing as in Fi g. 6a. (c) A rtifi c ia l d e le ti o n o f nin e r es idu es in th e third h y p e r­·va ri able segm e nt o f Ko l, whic h m a kes it o f equ a l le ngth w ith lgG I Eu ~". revea ls a deep curved cl eft.

119 Val Thr Val Ser Ser Val Thr Val Ser Ser Val Thr Val Ser Ser Val Thr Val Ser Ser

PFIZER EX. 1582 Page 6

164

The CH2-CH3 orientation is found to be somewhat variable and influenced by external forces. In the Fe fragment crystals the two chemically identical chains arc in a different environment. As a consequence the CI-12-CI-13 orientation varies by about 6°. In Fc-Protein A complex crystals this arrangement differs slightly from that of Fe crystals 1 1

.

More drastic changes are observed in VH-CH I and VL-CL longitudinal contacts, when chcrnically different Fab fragments are compared. These differences in longitudinal arrangement arc most con­veniently described by an elbow angle, which is enclosed by the pseudo diads relating VL to VH and CHI to CL respectively. The elbow angle may vary from more than 170° to 135 o when we compare Kol Fab with McPc Fab 12 ·il.I'J.zo.

In two cases the elbow angles of the same molecule in two different crystal lattices were comparee! and found to differ by 8° and 17° rcspcctivcly 1 '1 ~ 1 • In Fab New, with an elbow angle of approximately 137°, there exist a few longitudinal contacts between VL and CL and VI-I and CH 122 ·2', whereas there arc no non-bonded longitudinal contacts in intact Kol and Fab Kol (sec Fig. 4 ), which arc characterized by an open elbow angle. We interpret these observations to

mean that in Fab Kol the V-C arrangement is flexible in solution. In the crystal the molecule is stablizccl by packing interactions; these will be discussed from a different point of view later.

The antigen-binding area Comparison of amino acid sequences of variable

parts has demonstrated the hypcrvariability of some segments. These were considered to be involved in antigen binding21 . Indeed, crystal structure analyses of I g fragment-hapten complexes show that hap tens bind in a cleft or depression formed by the hyper­variable segments.

The VL dimer of Rcj7·'1 may serve as an illustrative example. The symmetrically arranged hypervariable regions form a deep slit-like pocket around the diad relating the two VL monomers. The walls of the slit are lined by tyrosincs 49, 91, 96, Asn 34 and Gin 89; the bottom of the pocket is formed by Tyr 3(J and Gin 89. A trinitrophenyl group binds to the Rei fra~ment and fills the binding pocket completely.

Anoth-er example of an IgG fragment-hapten complex is Fab New, which is known to bind among other ligands a hydroxy derivative of vitamin K

12".

The hypcrvariable segments of New form a shallow groove with approximate dimensions of 16 x 7 A and a depth of 6 A.

McPc 603, a mouse IgA (K) Fab fragment 2" binds phosphorylcholinc. The site of hapten bindin!j is a large wedge shaped cavity, with dimensions 15 x 20 A and a depth of 12 A. Only five of the six hypcrvariablc regions contribute to the formation of the cavity: L­chain hypcrvariablc regions one and three, and all three H-chain hypervariable regions. The second hypcrvariablc region of L-chain is screened from the

!mTmmolot;v Torl(lv, 1'111. i, Xo. li, /')82

cavity by the first hypervariable loop of L-chain and the third hypervariablc loop of 11-clwin. The deeper cavity in 1\.lcPc(>03, as cornp;u·ed to Fab New, is due to longer hypcrvariable loops. The first hypcrvariable region of L-chain and the third hypcrvariablc region of I !-chain is three residues and the second hyper­variable loop of the 11-ch;tin is two residues longer in McPcMl3 than in New.

PhosphorylcholirH' occupies only a small part of the cavity and interacts vi;t Vander \Va;ds forces, electro­static interactions, ;md hydrogen bonds with the protein.

In contrast to the above examples Ig(; Kol shows no cleft or depression in the antigen-binding region. In Ig(; Kol the hc;tvy chain h;ts a rather long third hyper­variable loop, which contains six residues more than :\!(>03 ;mel <·ight more residues th<m Fall New. The ;nnino acid -;equcnces of the third hypervariahk regions of 1\.i(>OV'·. New"\ Kol 2 ' and Eu 2 ' arc com­pared in Fig . .S. The scquciHT alignment and clas-;ifi­c;ttion in VII, J) ;tnd .J segment 2'• 2'' is somewhat arbitrar·y, especially for the beginning ofthe.J segment as a nucleotide scqtrciH'l' has been determined only f(>r 1\.·160321'. The ;rdclitional residues in Kol with the nearly palindromic amino acid sequence -Gly-Phe­Cys-Ser-Scr-/\la-Scr-Cys-Phc-Giy fold into the puta­tive antigen binding site and fill it completely (sec Fig. Cla,b ). The two cystcins arc disulphidc bridged and form the start ;md endpoints of a short antiparallcl [3-sheet, comprising residues -Cys-Scr-Scr-i\.la-Scr-Cys-. If in <t rnodelhuilding experiment nine residues are cut f'rorn the t hi rei hypcrv;t ria hie region of the Kol heavy chain, thu'> making it of equal length with l!j(; I Eu-''. a deep curved clef'! ;rppc;trs (Fig. (Jc), which easih· could accomrnod;rtc haptcns. \Vith respect to the anti­~en binding ;m~;t lg(; Kol thus looks <ts if it c<HTied its own hapten in forrn of an extended third hyper­variable loop. Another peculi;rrity of Ig(; Kol might be of interest in thilt context. In the Kol crystal lattice the hypervari;tblc p;trls of one molecule touch the hin12;c and spati;rlly <tdjilccnt '>ee;rnents of a symmetrically rcl;ttecl rnolecule. This contact consists or three S;tlt linkages (Arg 4<) light chain-(:( )()I I light chain, ,\sp .)() light chain-Arg 215 hc;tvy chain, Asp 5.1 hc;l\y chain-Lys J3tt heavy chain), a fcvv hydrogen bonds and extensive Van der vVaals interactions. Thus, the lattice contact f(nrnd in Kol crystals might give an instructive model for antibody-antigen interaction, as antigens are usu;rlly rn;tcrornoleculcs which co\'er a much larger part of the <llltibody than haptcns do.

The hinge segment The hinge segment which covalently links Fall and

Fe p<trts, has ;r unique primary and sp;ttial structure. Its central region consists of two parallel disulphiclc­linked poly L-prolinc helices with an ;rmino acid sequence -Cys-Pro-Pro-Cys- 12 1 ;. In the Ig(; I subclass represented by the Kol molecule the poly-proline double helix is short (Fig. 7). llowcvcr, in lg(;3 the hinge sequence is quildnrplic;ttcd '" and model build-

PFIZER EX. 1582 Page 7

lrmmmology Today, vol . .3, No. fi, f1J82

ing suggests that the poly-proline segment of this molecule may be more than I 00 A long.

The poly-proline segment, a relatively rigid struc­ture, is f1anked on both sides by flexible segments: The segment on the· N-tcrminal side is well defined in the crystal lattice of Kol due to crystal packing inter­actions, but it lacks internal interactions, that would provide stability in solution. The C terminal segment is disordered and f1cxible in Kol crystals and in the Fe crystal structurc 1l· 1;. The rigid hinge segment allows independent rnovcrnent of the Fab arms and the Fe part. There is direct evidence for f1cxibility in the crystal lattice of Kol 111 '1 and Zic 11 . This is in contrast to the abnormal IgC protein Dob, which lacks a hinge region'"· The significance of the hinge for Fab-Fc f1exibility is obvious.

Complement binding The binding of the Clq component of the Cl

cornplcx to antigen-antibody complexes is the first step in the classical pathway of complement activationllll. The Clq head pieces bind to the CH2 domains of antibodies li.H .. Protein A, a constituent of the cell wall of .)'1(/jJh]'!ororms rlltrr:ll.l, binds to the Fc­part of antibody mol~cules of certain classes and sub­classes, but docs not interfere with complement binding. The determination of the crystal structure of the complex between FB (one of the four Fc-binding domains of protein A 17 ) and Fc-fragment showed that protein A binds at the CH2-CH3 contact 1 i.lH_ Fig. 8 shows a space-filling model of the FB-Fc complex. The area of CH2 not covered by FB must contain the Clq binding site. In view of the size of the Clq head pieces (mol. wt 50,000) it appears unlikely that they can bind at the inner sides of CII2, i.e. ncar the e<lrbo­hydrate. The most plausible binding site is therefore ncar the tip of CH2 on the outer side of the domain. It is worth mentioning that this region is disordered in crystals of the FB-Fc complex which indicates that this part of the CI 12 domain is f1exible. Possibly, f1cxibility is required for antibody Clq interaction.

Summary and perspectives Investigations of the three-dimensional architecture

of antibodies have elucidated the folding of the polypeptide chains into domains, and the spatial arrangement of the domains. The structural basis for understanding antibody specificity and antibody flexibility was obtained. Segmental flexibility is an important property of antibodies: Flexible segments of the polypeptide chains at the switch and hinge regions allow the Fab fragments to change their shape and their relative orientation. Conformational changes of this kind arc necessary to rneet the geometric require­ments which <lrisc on binding of antibodies to multi­valent antigens.

The unclcrstancling of the effector functions of anti­body molecules is much less complete. One of the central problems is the explanation of the strong enhancement of Clq binding to antigen-antibody

165

complexes as compared to free antibody molecules. Two mechanisms have been considered (for a review see Ref. 39): since Clq is multimeric with at least six antibody binding sites, binding may be enhanced by the formation of antigen-antibody aggregates through crosslinking. Alternatively, antigen binding might induce a conformational change in the Fc-part which enhances affinity for Clq.

There is strong evidence for the importance of aggregation, but a mixed mechanism which involves aggregation and a conformational change cannot be ruled out.

The studies described here were almost cxcl usively carried out with myeloma or Bencc-.J ones proteins because these were the only homogeneous immuno­globulins which could be obtained in sufficient quantity. 1-lowcvcr, in most cases the specificities of such molecules is unknown. Recently, lan.~c amounts of homogeneous antibodies elicited against strepto­coccal or pneumococcal polysaccharides became available from certain rabbit and mouse strains1 11 11. These sources, and the usc of hybrids obtained from myeloma and spleen cells have made it possible to obtain homogeneous antibodies of defined speci­ficity12·11. Structural studies of 'natural' antigen-anti­body complexes can be expected to lead to a rnorc complete understanding of antibody function. Crystallographic work on a specific antibody and of its antigen is already in progress 11 .

Acknowledgements We thank Prof. R. Huber for helpful discussions.

References 1 Edc1rnan, G. M. (1970) Sri . .-11!1. August, H1-87 2 Porter, R. R. (1976) Sri . .-11!1. October, Hl-H7 3 1-Iilschmann, N. ( 1 %9) .NolllnCJI.I.II"I/.IIho/)1'11 56, 195-205 4 Edmundson, /\. B., Ely, K. R. and /\bola, E. E. ( 1978) Cont.

Toji. Mol. !rllllllmol. 7, 95-11H 5 1\mzcl, L. ivl. and Poljak, R . .J. ( 1979) .-11111. Rn·. Biorhnn. 48,

%1-997 6 Porter, R. R. (1958) ..Vot11rl"( l.rmrlon) 182,670-671 7 Epp, 0., Colman, P. l'vl., Fehlharnmer, 1-1., Bode, W., Schiffer,

M., Huber, R. and Palm, \V. (1974) ,,.llr. ]. lhorhnn. 45. 513-524

H Fehlhammer, II., Schiffer, l'd., Epp, 0., Colman, P. l\1., Lattman, E. E., Schwa~er, P., Steigemann, \.Y. and Schramm, II..J. (1975) JJiojiln·.r. Stmrl . .\fl"lhon/.,111 I, 139-146

9 Epp, 0., Lattman, E. E., Schiffer, M., Huber, R. and Palm, W. (1<)75) /Jiorhnni1lrv 14,4943-4952

I 0 Edmundson, A. B., f~ly, K. R., /\bola, R. R., Schiffer, l'vl. and Paniagiatopoulos, N. ( 1975) JJiorhnnirtrv 14, 3953-3%1

11 Lee, B. and Richards, F. M. (1970)}. ,\/nl.lhol. 55,379-400 12 Colman, P. l'\1., Deisenhofer, J., Iluber, R. and Palm, W.

(1976)}. ,1/oi.JJiol. 100,257-282 13 Marquart, M., Deisenhofer, .J., Huber, R. and Palm, W.

(1980)}. ,\/ol. Bioi. 141,369-392 14 Deisenhofer,J., Colman, P.l'vl., Epp, 0. and lluber, R. (1976)

llnjifit-.~l·riN', -( l'hy.riol. Chnn. 357, 1421-1434 15 Deisenhofer, .J. (19H I) lhorhnnirlr)' 20, 2361-2370 I(> l'vlelchers, F. (1973) lhorhnni,fr)' 12, 1471-147(> 17 Weitzman, S. and Scharft, ~I. D. ( 1976) ]. .llol. lJiol. I 02,

237-252 IS llickman, S., Kulo·.ycki, /\ . .Jr, Lynch, R. G. and Kornfeld, S.

(1977)]. lhnl. Chnn. 252, 4402-440H

PFIZER EX. 1582 Page 8

Fig. 8 Space filling mod el of th e FB (prote in A) - Fe complex . o ra n ~e: FB b lue C H 2 o f c ha in I , C ll 3 a nd ca rbohyd rate of cha in 2 red: C H 2 of c hain 2, C ll 3 a nd ca rbohyd ra te of c ha in I b lac k: pa n s o f C H 2 and car­bohydra te from both c hain s w hic h a re d iso rde red in the crys ta l.

19 Mat sushima , M. , Marquart , ;'vi. , .Jones , T. 1\. , Co lm;1n , P. M ., Barte ls, K. , H ube r, R . and Pa lm , W. ( 1978) }. .\foi. /Jiol . 12 1, 44 1-459

20 Sega l, IJ . M. , Pa d lan, E. 1\ , Cohen , G . II., RudikofT, S , Potter , M . a nd Davies, D. R. ( 1974) !'rot. Nat! r lmrl . ~it. l/.S.il . 7 1, 4298-4302

21 /\bola, E . E. , E ly , K . R. <t n d Edmun dso n , /\. B. ( I %0) /Jiodmn i•l rv 19, 432-439

22 Po lj ak , R. J , 1\mze l, L. M., Chen , B. L. , Phi ac kerlcy , R . P. and Sau l, F. ( 1974 ) l 'ror .. Vol/ clrarl. Sn. { I.S .. ·I. 7 1, 3440- 3444

23 Sau l, f-~., /\mzel , L. M. and Po ljak, R . J ( 1978 ) ]. /J iol. Ci1 1'111. 253, 585-597

24 Wu , T. T. a nd Kabat , E./\ . ( 1970 ) }. F rj1 .. \In /. 132 , 2 11-250 25 /\mzel, L. l'vl. , Po lj ak, R . J , Sau l, F., Va rga, J ,vi. a nd

R icha rd s, F. F. ( 1974) l'ror. Nrtt! rlmrl. .~i · i. { I .S. t l. 7 1, 1427- 1430

26 Ea rl y, P., H uan ~. H ., Davi s, l'vl., Ca lam e, K . a nd H oo d , L.. ( 1980 ) (.i•/1 19, 98 1- 992

27 Schmidt , W ., .J ung, H . D. , Pa lm , W . and l l il sc hm a nn , N. ( 198 1) p ri va te co mmu nica tion

28 Cunn i n ~h am , B. /\. , R uti s ha u sc r , U., Ga ll , W . E ., G o tt li e i>,P . IJ ., Wa xd a l, M . .J. and Ede lm an, G. l'vl. ( 1970) /I ifl(hr·mi• tr)' 9 , 3 16 1-3 170 .

29 Sa ka no , H ., M a ki, R., Kurosawa , Y. , R oeder, W . a nd T o negawa , S. ( 1980). Volttrr ( l .onrlon) 286 , 6 76-683

30 M ic hae lson , T. E. , h·a n ~ i o n c, B. and Frank lin , E. C. ( I !J77)

]. !hoi. U wm 252, 883-889

Fi15. 7 Conforma t io n o f th e hinge rc'Sion as see n in IgG I Ko l. IJI; I( k : li ~ ht c k1ill C:-tnm in i ,,.rj · him:•·lwp ti ck IJitw : lw<Jv y- h,· ;JI'Y and he ;JI ' y- li ~ht

r h:t in cli ~t dphick ll!'id ~e~

) I El y, K . R ., (:ol lll <ll l, 1'. \ 1., /\ 1)()1<1 , 1 ·~. 1 ·~ .. l ie ~-;.;\ ( ., Pcahoch. 1) .. S., P ;JJT , 1) : \ 1., C:riJHH'I I, C . 1·:., l. ; ttt ~rhin~n . C: . 1\ . ;u;d l ·:dm und ~ • >n , 1\ . II . ( I 'J7H) 111111 h,·"'"tn· 17, H20-H2 )

.12 .S il vC' rto ll , I·> W ., . ;w i<J , ,\ I . t\ . iiiH I l> a viC' ,, 1> . R. ( I 977) l 'un. .Va t/ clmrl. Su . ( I. S .. I. 74 , S l ,10 1 1·1·1

33 \ l uc llc r- l·:l>n h;lf'(l, I I. J. ( I '}7 )J . IIIII. u.'/'. fJt,nl/1'111 . ·1-! , 6')7-72-! 1·1 Poncr, R. 1\ . ii!HI l{ci d , K . H. \ 1. ( ICJ7 'J ) . ld1 ·. f',ut. (.'!,<Ill. 1:\.

1-7 1 Y) Con ne ll, (; . 1·: . a nd Po r te r, !{ . R . 11')7 1) 1/ ,u!f, ,·lll.]. 12·1, 'i3 P 36 Ya -; rnc•·n , D ., l·: lkTm n , .J. 1\ ., l )orrin ~ton , K . j . a nd Painttr . R

II. ( I 976) J. l ll ll/111111!/. I I r, , 'i I H-126 37 S jocd ah i, .J. ( 1977) l-.'ur.J. !lulllu•m. 78, ·17 1-.J<)()

31) Uei sc· n iH> fn , ) ., J onc,, '1'. 1\ ., ll ul)(' r , R ., Sjocdahi , .J. and .'ij oc­q ui s t , .J. ( I 97H) .::: l 'hv"ol. (,l,, .,t, . 359, ')71-9H'i

39 .vl ctz~c r , I I. ( 197 K) ( ,i!li/ . Tufi. .l!ol . /nnnu11ol . 7, 11 9- 148 40 .J aton , J.-C. , ll usC'J' , II. , Braun , 1) . (; ., (;i vo l, U ., l'c c ht . J. and

Sc hk,s i n ~n . J. C: . ( I<J7S) /ittll !/1'1/ll >trv 14 , S3 12-'i3 1.5 4 1 Brau n , D . c;, an d ll u -;c r , II. ( l lJ 77J inl'ro t!,l'•'" 111 1111 11 /lllto!o!',l Ill

(;'vi and ·1, '1'. F., C: h ecr~. C:. I !. , ll osk in ~. C:. S., \ ld<.cmic , I. 1:. C. a nd Nossa l, (; .J. V, cd s) pp. 25)- 26 -1, EJ ,cl' icr :\orth -1 !ol la nd , /\rn"C' rd ;llll , Ne w York , O xfo t·cl

42 Koe hl e r, C . and \ l ils trin , C: . ( I<J7'i) y ,,t/11• r l .ondo11J 2)(>,

49 'i-4CJ7 43 M e lch c rs, 1:., !'otte r , B. \ 1. and Be thesda, N . \\ '. (c d ~) I ICJ7H)

Curr . T of'· ,\•l u roll! of . l lllltllrllol . HI 44 Co lm a n , P. ;\ I ., ( ; ou~ h , K . II ., Li lley , (; . (; .. Bl a~rov(', R . J.

W c l>s tc r , R . (; , an d l. ii VCI' , W . (. . ( I 'J8 1J J. .\ l ui. ! Ito!. 152. (>()<)_ (, 14

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