June 7982(2!/j
`
`I
`1 r
`
`(". •,
`
`Vo/umr: 3, No . 6
`
`immunology
`today
`
`Receptors, antibodies and disease
`from john Newsom-Davis and Angela Vincent
`d
`· 1
`·
`·
`Biochemi.sts
`, e n ocnno og ists, Immu-
`nologists a nd neurolog ists s ha re a n
`In te res t in cell surface receptors a s
`wa s ev ide nt a t a rece nt symposium
`held by the C iba Founda tion * When
`these rece ptors are targets for a uto(cid:173)
`ant ibod ie·s
`1
`·
`. 1
`.
`, e nc ocn ne or ne u ro og ica
`1
`di sea se ca n res u lt. Att e nt ion foc ussed
`(R ) for
`p a rti c u lar ly o n
`rece ptors
`thyroid stimul a ting horm one (TSH) ,
`Insulin , ace tylcho line (AC h ), pro lac(cid:173)
`tin and ~2-adrenergic ago ni sts .
`.
`The
`1· · 1 ·
`C 111IC<l
`Importance o f a utO-
`.
`fnt ibod ies to receptors, judged pure(cid:173)
`y In numeri ca l terms, varies con-
`Siderab l
`I
`.
`.
`1.
`nsu In-res istant diabete s
`('
`. Y ·
`Insuiin-R a ntibody ), for exa mpl e, is
`ex tremely rare -
`less th a n 50 cases
`
`to
`reported wo rld wide,
`have bee n
`th e other ha nd Graves '
`date. On
`di sease (TSI-1-R ant ibody) a nd myas(cid:173)
`th e ni a grav is (AC h-R a ntibody) are
`modera tely prevalent wh ile as thma
`a nd ot he r ato pic di so rd ers ( ~2 -a dre n­
`e rgic-R ant ibody) a re very common.
`D irect im p li ca tion of the releva nt anti(cid:173)
`bodies in the di sease process see m s
`we ll es tab lished for most of the di sor-
`ders di sc ussed, a lthough som e unce r(cid:173)
`ta inty ex ists a bout the rol e of the
`rece ntly
`id entifi ed
`~2-a drenergi c- R
`ant ibod y.
`In as thm a a red uctio n in lung tiss ue
`~2 adrene rgic receptors, as meas ured
`b y agonist binding or ~2 res ponses,
`ha s been es ta b li shed for so me tim e
`a nd a ntibod ies s pec ifi c for
`the ~2
`receptor can be ide ntifi ed either by
`inhibition o f ago ni s t binding, by
`immunoprecipitation of solubilized
`mntintu:t!onji. !50
`
`.
`.
`·b d '
`• Recepto .. A
`' s "nt1 o 1es a nd Disease (C1ba
`,
`~mposium no. 90) was organized by Dr
`<IVJd Evered a nd chaired by Professor
`N. !\: Mitchi son . Th e procee dings wi ll be
`published by Pitm a n Medi cal Ltd .
`
`1. AChR
`
`2. Insulin -A
`
`4. 8 2-adrenergic-R
`
`5. TSH-R
`
`·
`Fig 1 o·
`.
`b
`d
`su umt
`·
`·
`Iagrammatic representation (roughly to sca le) of propose
`f
`b
`structures
`f h
`·
`·
`·
`d
`· d ·
`·
`d
`•
`J
`hi
`.
`o
`, m 1catmg t 1e s1ze an num er o
`t e prmc1pal receptors d1scusse
`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 a nnu al 'Round Tabl e
`Symposium on Applied Immunology ' wa s
`its 13th year in Axams,
`ca rried
`into
`Au stria , on 25-27 January.
`
`On these occasions small numbers
`of clinicia ns and scientists mee t
`to
`disc us s new developme nts in resea rc h
`that have mutual interest.
`In
`the opening session on cell(cid:173)
`surface antigens H. Bainer (R ij svij k)
`di sc us sed the in-11ivo effec t of mono(cid:173)
`clona l a ntibodies
`(MAbs) directed
`aga inst T-ce ll s ub sets
`in Rhe s u s
`monkeys gra fted with a ll ogeneic skin .
`The MAbs defined huma n lympho(cid:173)
`cy te sub sets that crossreact with the
`equiva le nt rhes us monkey cells. Anti (cid:173)
`bodies aga inst
`the helper/ inducer
`subset (OKT4 eq uiva lent) pro longed
`skin a ll ograft survival whereas those
`directed against the cy totoxic/ sup(cid:173)
`pressor subset (O KT8 equ iva lent) not
`only fa iled to prolong survival but m ay
`ha ve shortened it. T hese res ults are in
`line with rece nt reports that in mi ce
`and rats the T cells in vo lved in initi(cid:173)
`ating gra ft rej ec tion are of the he lper
`(Ly J+ ) phe notype a nd not the cyto(cid:173)
`(LyJ+ 2+) pheno(cid:173)
`toxic/s uppressor
`type . D. Arndt-J ovin (Gottingen) pre(cid:173)
`se nted her use of sophisti ca ted phys i(cid:173)
`ca l method s
`(Auoresce n ce e nergy
`transfer,
`rotat iona l diffusion and
`tra nslational diffu sion) to assess the
`density, proximity and mobility of
`mouse H-2 (Kk) antigens detected
`.with monoclonal antibod ies. The basic
`info rm ation obta in ed
`from
`th ese
`studies should a ll ow direct exa min(cid:173)
`at ion of how H-2 a nti gens 'associate '
`with suc h ext rin sic antigens as viruses
`and thu s the necessa ry co nst ra ints for
`co nsidering the a lte red self versus dual
`receptor hypothes is of T-cell recep(cid:173)
`tors. P. Peterson (Uppsala) prese nte d
`
`cmztinlll'drm fJ . /51
`
`PFIZER EX. 1582
`Page 1
`
`

`

`lmlllllllology Tod11y, 11ol. 3, N o. 6, l!l82
`
`[J (immunology today J
`
`Vol u m e 3, no. 6
`June 1982
`
`Guide for Authors
`Althnu .~h most of lrmmmologv Todrty '.r co n(cid:173)
`te nts a re commi ssioned, unsolicited a rticl es
`are welcome, preferably a ft er consultation
`Wit h the editori a l office. Authors should be
`awan~ of the diversit y of their readers'
`CXJ)crience of immunology a nd should
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`possible.
`Review articles offer a sy nth es is of
`cu.rrent knowl edgt~ in a fie ld where rapid
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`not exceed 3000 words, 40 references and 6
`fi gures/tables.
`Compass articles a re c rit ica l com(cid:173)
`mentaries on one or several rt>cent research
`papers that contain res ults of substa ntial
`;mportance. The text should not .exceed
`_0
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`Rostrur~ articles offer hypotheses, state(cid:173)
`~hents of persona l opinion, or spec ul ation.
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`rnmrmum necessary number of references
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`Letters to the Editor offer comment on
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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
`Eli zabeth S im pson
`
`letters
`l nterleukim: a rose ts a rose .
`Steve n G illis
`
`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 Hre nt
`C:om.jJlenwnt and .\olubili.;;_alirm o/ irmmme comjJiexes·
`Nevin 1-1 ug hes-J ones
`
`reviews
`i\11 mwclonal antibodies - tools to dis.1erl lhe nervmo· system
`Colinj. Barnsta ble
`The Llzree-dimemional .1lmcture o/ antibodie.1
`l'vlarkus Marq uart and Johann Deisenhofer
`The lll(llll711ary gland man immunological organ
`I ,ars A. I-ianso n
`
`books
`Coj~zng with the !Jimnedical l ,iterature
`(ed. Kenneth S . Warren)
`J ack Fra nklin
`The lrmmme Systenz . A Crmr.l'e on the AI/ olewlar and
`C'tlfular /Jasii of l mnumology ( by I. M cCrmnefl , A. Jl4mzro
`and II. Waldmann )
`N . !\.Sta ines
`
`Edito rial O ffi ce:
`l ·~l scv i c r lliomcdil';tl Press,
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`T el. (0221) 1 15% 1
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`technical focus
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`classified
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`149
`
`149
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`152
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`153
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`155
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`156
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`157
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`160
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`168
`
`172
`
`173
`
`I.Ji. llt
`IV
`
`v
`
`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 enor(cid:173)
`mous variety of foreign substances (antigens) and sub(cid:173)
`sequently 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 frag(cid:173)
`ments1·'.7·111·12-1' led to an advanced understanding of
`the way in which antibodies meet these opposing
`requirements.
`
`is a schematic drawing of an antibody
`I
`Fig.
`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 con(cid:173)
`served. 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 frag(cid:173)
`ment Fab, and the Fe fragment (Fe was the first anti(cid:173)
`body fragment obtained in crystalline form) 1
`'.
`
`A
`
`8
`
`F
`
`E
`
`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.
`
`H
`
`G
`
`c
`
`D
`
`X
`
`IMI1UNOGLOBULII< DOI·IAII<S
`IN
`ARRMGEI1EIH OF STflANDS
`X N-TERMINUS UP •• C-TEfU11NU3 UP
`
`Fig. 2 Schematic drawing of the strand topology in a V(cid:173)
`domain viewed parallel to the strands.
`(x) and (e) indicate N-and C-tenninal ends of the strands point(cid:173)
`in~ 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
`
`161
`
`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 eli(cid:173)
`sulphide 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 con(cid:173)
`siderably.
`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(cid:173)
`globulins: lateral (or trans) interactions and longi(cid:173)
`tudinal (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 immuno(cid:173)
`globulin 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 hyper(cid:173)
`variable segments arc involved. A comparison of V(cid:173)
`domain 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
`
`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 con(cid:173)
`tact and presumably helps to stabilize the Cl-12-
`domain. The branched carbohydrate forms a few
`hydrogen bonds with the Cl-12-domain, but the dom(cid:173)
`inant 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(cid:173)
`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 con(cid:173)
`formational changes in antibodies affect those inter(cid:173)
`actions.
`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
`and may bE
`at the
`~ubje;:t USCoP"yright Laws
`
`PFIZER EX. 1582
`Page 4
`
`

`

`Fig. 3 Ste r·eo dra w ing of a s pa ce
`fi llin ~ m o d e l o f human Fc-fra g -
`!11C lll.
`t\\ O
`from
`Th·· molt· < uk cs bu ilt
`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; ,·
`llotlt
`lt ;ik c; ; 11 ·c rr·l a tc<i
`ll\
`L\ ~"""Jl ~.
`.q>pro xi lll iltt· rl i;, rl s.
`,,.cl , l, l;l{'k : (: JJ 2- domain s of<hain I
`<IIll i < lt;,in 2, n· ~ p<T t
`i v c l ~.
`hill <', o ,·;lnt;e : C l ll-d ont;Jill'i and< .tr(cid:173)
`I ;md c h;tin 2
`hnlt\ d rat•· o f c lt;1in
`!'I 'Sjl< T l i\'t· Jy.
`
`F il{. -~ II{G I molec ul e K o l.
`T h<·
`l·;ti> pa ri s ;11H I th\' hint;c Sl'L(IllC'Ilt <1r•:
`well ordcTn l 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.-rlo m ;,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
`
`Fig. 5 Amino acid comparison of residu es 98- 119 (Eu num(cid:173)
`bering) of M603, New, Kol and Eu h eavy chain s. T he
`und erlined res idu es we re left out in Fi g. 6c .
`
`End of VH
`
`98
`Cys Ala Arg
`Cys Ala Arg
`Cys Ala Arg
`Cys Ala Gly
`
`M603 :
`New
`Kol
`Eu
`
`0 segm ent
`
`Asn Tyr
`Tyr
`Asn
`Leu
`lie
`Asp Gly Gly
`Gly
`Tyr Gly
`
`Gly Ser Thr
`Ala Gly Cys
`His Gly
`Ph e
`lie
`Tyr
`Se r
`
`li e
`Cys Se r Ser Al a
`
`Se r Cys
`
`I )C
`
`PFIZER EX. 1582
`Page 5
`
`

`

`Fig. 6 Antigen binding region of
`lgGl Kol.
`third hype r(cid:173)
`(a) Th e exte nded
`va ri a ble loop or the heavy c ha in
`(res idu es d raw n in b lue ;1re pre(cid:173)
`suma b ly cod ed
`for by
`the D
`seg m e nt 21· ·~'1 ) folds into the puta (cid:173)
`t ive a nti gen bindin g pocket fo rme d
`b y VL (bl ac k ) and VH (reel ).
`(b) C a backbone a nd sidec ha ins
`o f Kol a nti ge n binding pocke t.
`Co lours h ave 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
`in
`th e
`third h y p e r(cid:173)
`r es idu es
`·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 c urved cl eft.
`
`Gly Pro
`
`Try
`
`Tyr
`
`1 10
`Try Gly Ala Gly Thr
`Phe Asp Val
`Try Gly Gin Gly Ser
`Asp Val
`Try Gly Gin Gly Thr
`Asp Tyr
`Pro Glu Glu Tyr Asn Gly Gly
`
`Thr
`Leu
`Pro
`Leu
`
`Val
`Val
`Val
`Val
`
`Thr Val
`Thr Val
`Thr Val
`Thr Val
`
`119
`Ser Ser
`Ser Ser
`Ser Ser
`Ser Ser
`
`J seg m ent
`
`PFIZER EX. 1582
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`
`

`

`164
`
`!mTmmolot;v Torl(lv, 1'111. i, Xo. li, /')82
`
`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 con(cid:173)
`veniently 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
`• In Fab
`'1 ~ 1
`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 binding 21 . Indeed, crystal structure analyses
`of I g fragment-hapten complexes show that hap tens
`bind in a cleft or depression formed by the hyper(cid:173)
`variable 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 1
`2".
`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: L(cid:173)
`chain hypcrvariablc regions one and three, and all
`three H-chain hypervariable regions. The second
`hypcrvariablc region of L-chain is screened from the
`
`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 hyper(cid:173)
`variable 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, electro(cid:173)
`static 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 hyper(cid:173)
`variable 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 com(cid:173)
`pared in Fig . .S. The scquciHT alignment and clas-;ifi(cid:173)
`in VII, J) ;tnd .J segment 2'• 2''
`c;ttion
`is somewhat
`arbitrar·y, especially for the beginning ofthe.J segment
`as a nucleotide scqtrciH'l' has been determined only f(>r
`1\.·1603 21'. The ;rdclitional residues in Kol with the
`nearly palindromic amino acid sequence -Gly-Phe(cid:173)
`Cys-Ser-Scr-/\la-Scr-Cys-Phc-Giy fold into the puta(cid:173)
`tive 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(cid:173)
`~en binding ;m~;t lg(; Kol thus looks <ts if it c<HTied its
`own hapten in forrn of an extended third hyper(cid:173)
`variable 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 disulphiclc(cid:173)
`linked 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
`
`165
`
`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 struc(cid:173)
`ture, 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 inter(cid:173)
`actions, 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 Fc(cid:173)
`part of antibody mol~cules of certain classes and sub(cid:173)
`classes, 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<lrbo(cid:173)
`hydrate. 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 require(cid:173)
`ments which <lrisc on binding of antibodies to multi(cid:173)
`valent antigens.
`The unclcrstancling of the effector functions of anti(cid:173)
`body molecules is much less complete. One of the
`central problems is
`the explanation of the strong
`enhancement of Clq binding to antigen-antibody
`
`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 immuno(cid:173)
`globulins 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 strepto(cid:173)
`coccal 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 speci(cid:173)
`ficity12·11. Structural studies of 'natural' antigen-anti(cid:173)
`body 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 t