immunity, Vol. 13, 37—45. July, zoos, Copyright ©2666 by Cell Press
`
`biversity in the cons Region of trig
`is sufficient for itdest Antibody
`specificities
`
`John l... Xu" and Mark hi. lfiavis’iill
`
`*Department of Microbiology and immunology
`li—loward Hughes Medical institute
`Stanford University School of Medicine
`Stanford, California 94305
`
`Summary
`
`All rearranging antigen receptor genes have one or
`two highly diverse complementarity determining re
`gions (CDRs) among the six that typically form the
`ligand binding surface. We report here that, in the case
`of antibodies, diversity at one of these regions, CERS
`of the VH domain, is sufficient to permit otherwise iden-
`tical igivl molecules to distinguish between a variety
`of hapten and protein antigens. Furthermore, we find
`that somatic mutation can allow such antibodies to
`
`achieve surprisingly high affinities. These results are
`consistent with a model in which the highly diverse
`CDR3 loops are the key determinant of specificity in
`antigen recognition in both T cell receptors (TCR) and
`antibodies, whereas the germllne-encoded CERT and
`CDRZ sequences are much more cross-reactive.
`
`introduction
`
`During lymphocyte development, a large repertoire of
`heterodimeric antigen receptors, both antibodies and
`TCRs, are generated by a variety of mechanisms (Tone—
`gawa, 1983; Davis and Blorkman, i988). ln antibodies,
`the binding site for antigens is formed by six CDRs that
`loop out from the V region backbone termed by two
`sheets of B—pleated strands (reviewed in Davies et al.,
`T996}. Great importance has been attached to the germ-
`line V gene repertoire for the development of effective
`immune responses, as most of the CDRs are encoded
`by the germline sequences (with the exception of CDR3
`of the heavy chain).
`it has been postulated that the V
`region genes are selectively retained in the germline
`during evolution because of their capacity to accommo—
`date different antigens, especially pathogens (eg, Cohn
`et al., 1989; Ralewsky et al., 1987).
`Recent structural analyses show that in up TCRs,
`amino acids at positions equivalent to the CDRs in anti—
`bodies also form the principal contacts between TCRs
`and their peptideiivlilc ligands (Garboczi et al., 19%;
`Garcia et al., 1996; Bing et al., 1998; Relnherz et al.,
`T999). While no complete structure of a ya TCR in com—
`plex with its ligand is currently available, a number of
`studies indicate that 78 TCRs recognize antigens in an
`antibody—like manner (reviewed in Chien et al., 1995; see
`also Li et al., 1998). The binding interfaces between
`antigen receptor molecules and their ligands are gener—
`ally large {over'iEQu A2) and lit—30 side chains from each
`side make close contacts (reviewed in Davies et al.,
`
`iTo whom correspondence should be addressed (e-mall: mdavls@
`cmgmstanfordedu).
`
`1990; Davies and Cohen, 19%; Garcia et al., 1999). How-
`ever, despite the broad interface seen in the crystal
`structures, sequence diversity in antigen receptors is
`not evenly distributed among all six CDRs but is highly
`concentrated in one (in the cases of antibodies and 78
`TCRs) or two (in the case of all TCRs) CDREs {Davis
`and Bjorkman, 1988; Davis and Chlen, 1999). Although
`the skewing of diversity toward CDRBs in up TCRs is
`understandable because these amino acid residues
`
`mainly recognize the antigenic peptide while other CDRs
`primarily interact with Milt: (Jorgensen et al., 1992; Gar-
`boczl et al., l99o; Garcia et al., 1996; Sant'Angelo et al.,
`1996; Ding et al., 1998; Reinherz et al., 1999), there has
`been no explanation for such a phenomenon in antibod—
`ies and ya TCRs. Even more puzzling is the finding that
`in antibodies, both CDR’l and CDRZ of the heavy chains
`and all CDRs of the light chains have only a few "canoni-
`cal" conformations, and only the heavy chain CDRB loop
`has a wide range of variations in both length and shape
`{Chothla and Lesk, 1987; Chothla et al., 1989). Such
`canonical CDR loop shapes have also been seen in up
`TCR crystal structures (CDRZ of a and is particularly,
`but not in CDRSa or CDRBB) (Garcia et al., 1999), al—
`though some of this uniformity may relate to Ml-lC bind-
`ing requirements. The skewing of diversity in all antigen
`receptor molecules has led to the suggestion that the
`highly diverse CDR3 sequences are the principal deter—
`minant of specificity in antigen recognition, at least in
`the primary repertoire (Davis et al., 1997, 1998).
`To further understand the molecular basis of antigen
`receptor specificity, we have constrained mice to use
`a single Vt, gene but full CDRB diversity to generate their
`B cell repertoire. We challenged such mice with a variety
`of protein and hapten antigens and monitored the devel—
`opment of primary and memory immune responses. We
`find that antigen—specific lgiv’l molecules isolated from
`primary response of these mice can differ only in the
`CDR3 of the Vt, domain; furthermore, we find that so-
`matic mutation can allow such antibodies to achieve
`
`surprisingly high affinities upon rechallenge with protein
`antigens. The only antigens that seem generally unable
`to be accommodated by an arbitrarily chosen Vt, are
`bacterial polysaccharides. These results indicate that
`an extensive V (both VH and V.) gene repertoire is not
`necessary for the production of specific antibodies to
`most antigens, and that the V“ CURB plays a very differ-
`ent role in the makeup of the antibody binding site than
`the germllne-encoded CERT and CDRZ sequences.
`These results suggest that the purpose of the highly
`diverse (DRE region of all antigen receptors, both TCRs
`and antibodies, is to determine antigen specificity.
`
`Results
`
`Experimental System
`in order to characterize the antibody responses of an
`organism with a very limited V region repertoire but full
`
`
`
`biversity in the cons Region of trig
`is sufficient for itdest Antibody
`specificities
`
`John l... Xu" and Mark hi. lfiavis’iill
`
`*Department of Microbiology and immunology
`li—loward Hughes Medical institute
`Stanford University School of Medicine
`Stanford, California 94305
`
`Summary
`
`All rearranging antigen receptor genes have one or
`two highly diverse complementarity determining re
`gions (CDRs) among the six that typically form the
`ligand binding surface. We report here that, in the case
`of antibodies, diversity at one of these regions, CERS
`of the VH domain, is sufficient to permit otherwise iden-
`tical igivl molecules to distinguish between a variety
`of hapten and protein antigens. Furthermore, we find
`that somatic mutation can allow such antibodies to
`
`achieve surprisingly high affinities. These results are
`consistent with a model in which the highly diverse
`CDR3 loops are the key determinant of specificity in
`antigen recognition in both T cell receptors (TCR) and
`antibodies, whereas the germllne-encoded CERT and
`CDRZ sequences are much more cross-reactive.
`
`introduction
`
`During lymphocyte development, a large repertoire of
`heterodimeric antigen receptors, both antibodies and
`TCRs, are generated by a variety of mechanisms (Tone—
`gawa, 1983; Davis and Blorkman, i988). ln antibodies,
`the binding site for antigens is formed by six CDRs that
`loop out from the V region backbone termed by two
`sheets of B—pleated strands (reviewed in Davies et al.,
`T996}. Great importance has been attached to the germ-
`line V gene repertoire for the development of effective
`immune responses, as most of the CDRs are encoded
`by the germline sequences (with the exception of CDR3
`of the heavy chain).
`it has been postulated that the V
`region genes are selectively retained in the germline
`during evolution because of their capacity to accommo—
`date different antigens, especially pathogens (eg, Cohn
`et al., 1989; Ralewsky et al., 1987).
`Recent structural analyses show that in up TCRs,
`amino acids at positions equivalent to the CDRs in anti—
`bodies also form the principal contacts between TCRs
`and their peptideiivlilc ligands (Garboczi et al., 19%;
`Garcia et al., 1996; Bing et al., 1998; Relnherz et al.,
`T999). While no complete structure of a ya TCR in com—
`plex with its ligand is currently available, a number of
`studies indicate that 78 TCRs recognize antigens in an
`antibody—like manner (reviewed in Chien et al., 1995; see
`also Li et al., 1998). The binding interfaces between
`antigen receptor molecules and their ligands are gener—
`ally large {over'iEQu A2) and lit—30 side chains from each
`side make close contacts (reviewed in Davies et al.,
`
`iTo whom correspondence should be addressed (e-mall: mdavls@
`cmgmstanfordedu).
`
`1990; Davies and Cohen, 19%; Garcia et al., 1999). How-
`ever, despite the broad interface seen in the crystal
`structures, sequence diversity in antigen receptors is
`not evenly distributed among all six CDRs but is highly
`concentrated in one (in the cases of antibodies and 78
`TCRs) or two (in the case of all TCRs) CDREs {Davis
`and Bjorkman, 1988; Davis and Chlen, 1999). Although
`the skewing of diversity toward CDRBs in up TCRs is
`understandable because these amino acid residues
`
`mainly recognize the antigenic peptide while other CDRs
`primarily interact with Milt: (Jorgensen et al., 1992; Gar-
`boczl et al., l99o; Garcia et al., 1996; Sant'Angelo et al.,
`1996; Ding et al., 1998; Reinherz et al., 1999), there has
`been no explanation for such a phenomenon in antibod—
`ies and ya TCRs. Even more puzzling is the finding that
`in antibodies, both CDR’l and CDRZ of the heavy chains
`and all CDRs of the light chains have only a few "canoni-
`cal" conformations, and only the heavy chain CDRB loop
`has a wide range of variations in both length and shape
`{Chothla and Lesk, 1987; Chothla et al., 1989). Such
`canonical CDR loop shapes have also been seen in up
`TCR crystal structures (CDRZ of a and is particularly,
`but not in CDRSa or CDRBB) (Garcia et al., 1999), al—
`though some of this uniformity may relate to Ml-lC bind-
`ing requirements. The skewing of diversity in all antigen
`receptor molecules has led to the suggestion that the
`highly diverse CDR3 sequences are the principal deter—
`minant of specificity in antigen recognition, at least in
`the primary repertoire (Davis et al., 1997, 1998).
`To further understand the molecular basis of antigen
`receptor specificity, we have constrained mice to use
`a single Vt, gene but full CDRB diversity to generate their
`B cell repertoire. We challenged such mice with a variety
`of protein and hapten antigens and monitored the devel—
`opment of primary and memory immune responses. We
`find that antigen—specific lgiv’l molecules isolated from
`primary response of these mice can differ only in the
`CDR3 of the Vt, domain; furthermore, we find that so-
`matic mutation can allow such antibodies to achieve
`
`surprisingly high affinities upon rechallenge with protein
`antigens. The only antigens that seem generally unable
`to be accommodated by an arbitrarily chosen Vt, are
`bacterial polysaccharides. These results indicate that
`an extensive V (both VH and V.) gene repertoire is not
`necessary for the production of specific antibodies to
`most antigens, and that the V“ CURB plays a very differ-
`ent role in the makeup of the antibody binding site than
`the germllne-encoded CERT and CDRZ sequences.
`These results suggest that the purpose of the highly
`diverse (DRE region of all antigen receptors, both TCRs
`and antibodies, is to determine antigen specificity.
`
`Results
`
`Experimental System
`in order to characterize the antibody responses of an
`organism with a very limited V region repertoire but full
`
`
`
`3212222222252
`3%
`
`Y
`
`3‘1. Heavy 232333222 Mirfiiems‘: HG?
`wVial‘wz‘: KM» 51
`{31“
`Tip: E5: 5:?
`"‘ CY?
`
`
`
`52232.23
`
`2’23.
`
`3‘. Light 2323332323 2.2221223
`
`3 v»: 3:30.23 3:34{:32
`
`‘»
`
`.
`
` Mae. n‘f {he Shin-@6233:
`
`52“: f E15355(ht? tn-‘j
`{23232222229. Mans-3‘. 33 L22;.2 ".
`
`i){Emir98 Hr:5:21.?
`r
`
`
`22‘. 2:25 _
`3253‘. 2‘;2223‘. 583‘.
`.
`
`C.
`252323: {2333mm 1.35222
`
`
`
`{‘22 33323222333: E126
`SCGEEKEBCE 2212223
`CEIEEB {$233325i232 2223:: Lstéfize
`mem- HQ ? whirgh1228:2223“: {mt-3 23:36.2?{12222.22 humer‘.
`‘29
`
`
`{3323222Hanan {‘2‘33-22;S1}. 26::
`f‘mszzimza{H3mgr
`is. 33:3"
`.232;
`
`
`2:; 223g»
`m_,g;2{2{3{2£152. {and {1.32:3 2.5 = 2..) 23.33 {gs-r2322222: 222532222
`
`29932. 2(3'2‘33}.
`'
`'
`2.3222 22.3: 2.333232:- 22‘2
`e2:§._.
`3236'? 22'222‘322232223E2:
`
`
`
`n
`;
` " {g .«:""“‘ {3;32222322332832 {2.‘
`mime 23.255252 brad {mm 322%?‘}
`..~§s{322222032322 {}3£T135‘§“'-'"
`:2 2m22323233033223. 2:322 1332‘:
`2:33:2-
`'
`‘*3325“: 2.22252 9m2°23?
`2’33 {.22 “.322:: F'ég.22“.
`1E;21mm the {2223123233. Eimuxw‘3':me 22:32:233222; {32223-3We
`22:22 the”“2‘" 2223:}:-‘392332332222329232333332232)? {:{ééé‘1.2222
`nasEm-‘Wimhvqum :{2‘3 ‘2‘2‘
`...“cesiinumv
`
`$322233
`533:2 <2xt{{3{2‘e2232
`222232 S ‘.
`wmv‘i:
`{323222
`2222;:
`shown} S<22 3.232 2‘22.22%? 2233022}? that $23222?) DEE-gm: and“
`igffi! {221229.22 {E22222 5'22) ii: 382‘} .3{22’2222 flip-18W» 2223232232} arid
`9.3 {'3 552.8 {2.{22‘2222' {E}. 3%22‘} “223 22:22. 223323."eag‘amweiv....« 222852:
`{2322222 3322232 {2332 3222233222}.
`
`3.232233322339323322:: i‘23,iapmmt {2f immune {Eeagzsanses
`
`$8 the H615"11 “$9.”..... £5}:
`.
` z. 1 ‘~ .2'»
`'— waeim‘. Ehigg
`_-.v\ 2:5;{ix/2
`
`2235 i
`
`mm 2:25 pmiz‘m 3224.29
`2‘93““ 22222322 32332222’
`33:22pm 22232252:1."«222m {Ki
`‘2}, {32222 {egggwwwhite {1 2.:312.2222: {BEE}.-
`
`
`
`ifxc
`
`{2 nel'3g2..t«m at
`far”: 3G”. 5.
`‘
`
`3.23.22.2- 2322:2222--
`.,
`Q {3:525 KL; {le 335.343 233.}.- HELL {g}: 13.33;).
`{2H 22::
`3303322222 ‘15.
`{3.2 2223322.:F? 22'; {m
`
`
`
`
`'
`. "m °c 6225‘s.
`m-r‘ “ Sr-
`
`icc‘it‘d‘.‘21223323222232
`{3:26 dilutm
`
`on33.82? {ma2‘.{
`_.n«_..2‘.‘\.‘ 2‘{{f‘:
`31-33. W KL5:5 3:2}. iii-.3. {.22 K125
`Mix}23.2 HEL
`{CEOQNPJ‘335:6:{22' PCQSSA {B}. phi1x2-ESA 02‘
`KL2‘ {E}: 2‘}? f“?v8‘u3. 2332‘ fii‘JPE1555).3“ “mime-2b
`-
`"
`‘
`
`323223.
`22222::3223‘;
`(2&2:qu 222232322342
`
`
`
`
`£551?“
`223:
`&
`E
`E
`E
`
`as {.MLH igM
`1-5 .3 {32422-2 22322
`{.2 {MEL 223223
`‘2‘ WEE?!“
`
`_
`g:
`‘53 "
`
`(3.5
`
`./
`
`
`
`
`
`3:31--
`i E
`i
`r35 as {5.08% mm
`‘1
`3.3% 3. 1302.222 EQG
`‘5
`2:. {MLH 333%?!
`.- (.2 G “W" ‘gfi
`V" ':
`f
`g
`’3'
`855*}
`’f
`f
`,3"
`E
`”.25
`_/‘“'
`
`{3C5 ,
`fimnwxwfiimmfihw»3%....34‘ DJM ,
`i}
`'{ii 15 3% 2:3 Si}
`{1‘
`5
`’
`{‘55-
`fii}
`2':
`‘EG
`3:52
`E
`’2‘
`’3
`f
`i‘
`2
`2’
`KLH
`{EU-i KL}! Nani
`OVA
`U036.
`("WA
`
`
`22‘};Q
`3
`3-9; {3
`2
`35‘ w“
`5 it“? Q“
`i
`‘ "i 85 {IQNFEQ'M
`i
`,2
`‘52 3 32.33 2.33
`22°; 2 222255222922
`2.: 32km 223322
`g
`{2 2213‘»; 222223 f
`2
`.23 22.26.22 2323.
`a G 3223. F5332
`.3‘
`2
`2.
`22
`r
`(‘3‘:
`§ 3'
`2
`m ”i
`'1
`3
`G
`5
`i
`5
`.2
`.5
`:1;
`.
`“'5’:
`"
`2
`
`
`_-
`-
`:W‘5
`{2'
`{'3 Ni
`15
`23"} ES 353 35 at!)
`‘2’
`i‘
`2‘
`2'
`HEEL
`HEEL 5&3.
`HEEL
`
`as
`is"
`
`23""
`If
`3-“
`f”
`;
`
`3/”
`
`/
`13‘
`f
`2"
`2!
`
`P2,:
`
`3°
`
`
`
`.
`.29
`
`252
`
`38‘
`
`33:
`
`{$8
`
`i
`i
`i
`:
`
`
`
`.
`
`‘23:»“$3.33“!
`2: u _
`i5 28 35 35 35 48
`E}
`S‘:
`’i‘
`E
`*
`’E
`E‘NPKLH 8N?-EZLHCERF kLHQNF“{’ELH
`
`‘(3
`
`22.2.2.3“““~------------------------------------------------‘
`
`2.2m“:
`
`”was
`I?”
`(2'
`2
`
`§
`E
`
`.-’~
`.’
`
`‘
`{2: 23323233232.«H
`1 812'
`“ 5 2: 22:23.23 iéfi
`i.2 “2.22m 2
`I 3:- {mm 2922
`2‘
`:3 2.2)i
`i
`3
`
`. ..
`4‘3
`
`i}
`3..
`{18A $1592):‘SA
`p.223»{3
`$3328 :25}:immammtim
`
`?
`
`1.22-
`
`§
`2
`
`2
`
`..
`1 3“ as 27353659.?
`.a ’3 “EEC ‘9'“
`3 {2322222332.22
`2". {{DNWQ‘QC:
`..x”
`
`
`{SE 3.2.x
`
`i}3}2,.m»vm.
`.
`.
`.
`.
`.
`.
`.
`‘
`ii)
`‘55
`EC" 25 36 35 228
`92
`E?
`2‘
`‘E‘
`96233.8
`9(3- E’QLN PSKLH
`{3323222 22223:" immuaimtian
`
`
`
`MrsErsr:s;Eisr Semis. is? E111E11Esrsrsrfirs-Etéuién SEJEEmE‘Ers'E'E};
`3';
`
`“5191251033AQ'KKYS’E" '
`
`‘*\ 11
`
`1' e 111::m53.1113 1:35“ ‘
`.
`Eher mh East-'3. E‘EEe.v .
`€33? 35;)BCG’S WSW: Ehase 1‘:
`GE E'srlmoiagy; SEN-111‘s ark}: :ueitits:
`here are 115 EgngE-E Seetyee.‘Cw
`
`.
`
`_
`
`..
`
`. .
`
`‘
`
`‘ 3. E‘
`
`-
`
`.E‘ti:t.:1ie
`
`EE‘S:\:gEVER“?!
`
`EJVMWWH.
`. {View-:-
`1.x.
`
`mere deseEE hermwu..3131 HE: E""E
`mezewith
`8113gms ene EseEeietE.1*E1rtEerE1e rteHEE KEYS preeeemg
`ehtEgee~E1Sheree“ EQM aetieeiiiec. E11111 seer; 111111111.
`nixed ORE};
`{ENC-1‘3
`t1: mEEEEmEze- CemeiE: 1521;:
`.
`QameEemehes‘y {3111111.EEEKEEEEE-ees mi?“ mewges.:
`E11
`
`itEEaEEEmeMuthibhzshiimd'
`133E111:{E3306,61?pie
`EEESEEE
`E31: {“twmhmee 113.131.;1
`tees.EEH8 {9CFE}.
`isms? Ehen
`1'onrEEEtE and.
`"
`. E
`E
`'EE1te 13:11:‘EEkE‘i‘DEéSS {Everest
`
`
`tiedzaetse‘ hath were5:“: sari-:3 (1. E1811 ‘EEEéé HE'S? 'EE‘aE1eg1teE1is
`
`ErE tEEisEE heavy» them enti‘ ueat‘eEther \r‘gEJ E tEE“tee-m
`EE1
`their Eight chairs {with 111:: emetic: e: hybrieeme
`HEL» 2. whith egg-ease 19 use1!E412 :11ESE EEe.:1:E“.eEr1:
`:E’ECE date mi 3were} NC: 11>: usage. wee eta»
`
`.
`.
`E;' 21113421181
`
`anti 811$EEEEE. E13533 sequent:EEE; \E‘ifiWN that 311M:
`izxieees
`‘_
`anthem“ EE1 EEe EEEEErEEEErEE: {1.1we isE‘rtE '1EEus;
`Es1.
`tE1isE_i.)R‘1‘: reqitsntt1? 'EEEie heavy E2i1i3irs.i;1
`_
`.
`
`1 Eere :EEE< Ewe Eehtse .‘iGEES?" tii' rsytss‘iithemes 11m
`exei'esi Ediss1EEE'ziEE EEgi'Ei 1:113.Ens e-.
`eEE The fit.“t iset 5E1-
`:EEEtE‘ee E31}:5. EM? .E. 13“?- amen“ i:EMS-i‘12 KLEE.
`,
`51>
`GVAA. QVAJJrse;
`{3‘1E'EIE:1: en:
`'ecenei EncEEgiE‘ee
`,
`SLEEEEAEEE KLHéN‘
`,‘
`3.
`ate-33y: 1:111 eii 0E iheee antieecises .
`:Ei meet the:
`very E13111?:1: am;
`:E1 they were.
`:1 i1g;:aré‘h$t.
`tent 11E -.
`
`
`
`
`r i
`
`eh§e‘~“e1's evaétwmii's {(211951}. :Emiere 1:122:31 3 summit {1‘2 EB}.
`es Ere-1585,-heme:s:’3 haitnétreptsetwi 1E3Ei‘3. :enjugeEeti
`“ KLH} 2ehenyiw‘i e1E101:jmethfiwe’wrmEersegehfiy
`
`{'1
`“1111111193site«3 EC}111:K131“. ”are.1311211111111 .cee). amt phesv
`U
`E13! sEahQEEEne (EEC. E:E311§EE~E§EEE 2dit: KLH}. “NEE ‘3. 3311311121
`Eeeeitsr
`111‘
`1211;111:1111EEEEEteiE Emma:
`'.E.1EE:EE2E1E 113183;:
`M».”a
`,
`
`E ‘SAE 11? “fling"'E'E Ehiwe E'twgzsim
`AstygumzumsEmisi
`Ehsmtmer" #0143115: Egh’i ersrstmvir
`" {QE-
`
`
`.t1w<v~31131':t
`31 ASE EECE mere 11er
`eniigehe EeSEmimere Ehe {1131:1191:iw
`
`emieirs to. MESEE hm}; theee WEE)? rEEE1e-r
`.
`.
`EHEetecEeEEEe res-wage Estate 110Egee-11m}. 5119
`
`J
`
`
`were E'iss)E1:Emi Ehai En mime wEE E1 a fleet}
`
`V?)3-1
`' ‘E'EEE-E'se E‘iE-isgaiazxeshesaand emhisii:
`
`9,131“:
`a SEgrEEit:eE“s; EetreH‘“ EEEE1::
`
`i531:
`E'eeeE“EeEEis {Cieeceth:.« eE'
`€311.51?
`is ..
`e1 3%.. 3995‘) EE we: there re
`page
`eerre:ngemehieeehiifer::mat1:“:
`£33;
`Esteiethe anthee}; EEBSEROE‘ESE
`52235-38 E51 Ems-.1;
`
`‘ fie: " ~‘:e roceeimé far1 ES.
`‘
`33$ weii 21:;
`is.)
`
`iriIi2'EE1ie Eexprmiiini; onishmrm Er.
`
`
`
`
`
`
`3m:31:311:;
`.13:
`
`
`
` Figure 4. 33333216:ch “m
`
`
`
`
` 33me (33*. '31 13.33531 735:4 .1352:15:33:; '33
`6:31 0 sw'rwss (sswqqnwux («Nb
`
`
`{33123-33 333::I shown}. T139335 INSNIIS' 53323337 I135? NINNI‘NII}; 131
`the 321:2}?3 33331013 (3f ‘3’. 313(33'1915 SNIIININNI 1:3 GNININ Nghéy
`spec.INN INN antiINN-dies In these magmas
`Amqnq the NNIhsmcéIIN hybrkiamag is
`FRIES} 1W0
`seque..LNS {BNI‘8 23m 0NP«’1‘11 were 38 NININN:
`1N
`(3321(1311'33'3. 0NI3—é‘z and? LIN? II ease. CRIN:NNI at 13:31};
`(331:3
`NNSEIENN {i-smieucine- 3333:3333: 331233333333}
`I33 ‘3‘.. CBRS‘ , Fur
`
`11383::.‘.(338 ‘ha 1'.
`I133nz3i {fixer-3:383; 11‘. I1N23h£2=3xiy #323133 I3
`
`
`3333mm“ For 33Ka3mpfi3 8;} {333:
`(33333 hyhnfimm I136
`Idemticai {38113 13333;;313, (3:163:3(1 .1-2 gem: (3'33gmené. 321:3
`thcx‘ésasésti Qiuiamma at {3053213333 9’3 {333355 I33?tami'm 33:1(1
`I33 133:3 ju33cticnai {@1933}: 133 IIINI{gm 333333233 23!! I333: NIIN
`Nazi Ieucine aI 33(333113333 ‘36 {INN 31:5.5*: 333311343 acid In Use
`33333513331331 :egiqn}, wen though «gamma;:9. IN encadafi.
`(31‘ that msé'IIN-N in the germIéne J 1 :'2331313 33331331Max:333:
`33393231.“: IN: {3333333111 afifigmt. mm? :VNIN 333313133535 IN
`g3Ne3I:: (is WNII: 3‘33; exampIN sequences: (IagauzaeIIIi'd 01!
`
`
`RH 1,3314%?
`QVNJ WW‘VFEMSGM ,
`'
`, £13m,
`QVA I and (311A?3333 (Iii'fz3rz3r'13'snéy 33,: NMIIINN933-133 {I'm
`13223333333:ham. WEN: ESIQIIIIIIK? 1N EVAN (333:3 '_\;s33333 In {EVA2:
`{3(311333'3313313393:333 13:5»333 msks‘ues 3:333: QNSIEI3333Iy 33331333335.
`
`High-M1331“: 133331 Antibecfissss I33: I‘rqteir: Rmigens
`fmm INN HCI“~"" IgH'~'"' ng"-" 11:31:23
`Having 33331333“(:1 the“ Ihe NC?“INN” 1333"“ 3331233
`(3383:33331310 of (mum.33:1. h1ghi'ysNNc'II‘IC Eifii'112353338 1Nw
`
`3123013623113 mam, 33" 3301 ii1-1.G§ {he amEg-a '
`
`
`WIIINII '~'\(33:I3 5363333:er fiftyiwk‘high-311131113;Kifiibfld"
`
`We {s-u'rsgeti 21113 questém 3133333 1'3); qammting 333333333»
`321133323? 1336 athNdIes through: hypssrimmunizaIEqN and
`by 3333:11‘33133g 3391;335:6333: 1-9323 antibadies 32:10:33 antisera. We
`Nina?(391:1 1WGI§§31 manocmnai 511’13'180513333.QN3 SNNNIIIN
`I0: NB. {H1513} and 311:3.
`(33133: IN:
`(.1939. I‘Q‘JN‘II. 3:313
`manuaisnafl qnitzbmiezs used 1113? Vgg'swfifiwfitfi
`EN 3333
`
`
`
`heavy chain 3m} V31u5.1,-\‘1« ,,._’1
`133 I
`..
`iIghi 3'11 1'33. BNIII:
`
`Has-‘3 532131 {NIKKI ewviibsxmag 2315(1 '
`‘IIIKIII‘ 3333313393303 (31,"
`Namath:1:333:333'33313323303: in {mm I:I3'\ 33": and A chairs-:3 with
`
`33361510? INN INNIEIIIGNE; being; 1(33:
`Im‘ 133 IN: £11193 2:3(3133335
`{FE-333353 II}. 233 Is 33:91:23}.
`W32 3113333 ezaNIeyed Surface 3:1135.333(31'3 rNanance {3133?}
`36331333133139 @31qu1»:I. W393} 313333measure IN'N aI'IINIIV NI
`INNSN ,sImINNNINS. We Immebmzed the MEL3 afiiihfitfiy
`0:3 INN. 1.3303 <0S3‘3'1‘ chip“and 3353523an qu‘ININm :;ancnntfa~
`IINNS (II MEI. (332e,: ho Immamem (333131313613; {FameSA}
`
`Fitimq 3,3? the I;
`{3333.
`,,
`'-
`3N asguctatian .332:
`
`
`'
`:aIian mks (31" 2.5
`'
`
`(3'1..."I5.“. \ 11‘1'"-
`.,
`,
`
`M N:253:; 331V}. (squ 33233331 10 INN hIghNSI affinity anti»
`
`
`IIEL 2333IEE3N33188 13.3133333'33 {flaw-“1&5 and 83313533? INN} Far
`NNKNNWN INEESGIIS, IENINNNEIIINIINN NI {EVA ‘3 3:333 :3 biqssmn
`3:3: chip 3331133" G1§31h3”i11*8*i0*13t1&§3¥3 3333313333.;311113,
`we 1mmnt31Izefi {313% an Ihe 1210:533331333.
`'Nip and (33:33:83?
`IJVAI to 131333 10 III-N smmsmizgfi Iggand {Figure ‘58}.
`Vhfie at.32.33.311.36 fitting (33‘ the Nivefimiz 15,313 NINN'INI} '33 (3:33:
`
`
` "
`
`
`
`Newman{32:39.22{95.1%
`
`, "
`
`,
`
`,
`
`13:33.0 {3'
`
`,
`
`
`
`33331.3;
`
`(3333.3
`32.33:)
`
`SKIN
`12033.3:
`32:13::
`
`{3:53
`
`‘
`'
`
`-
`13
`
`mm _
`
`1135
`
`'
`‘
`-
`330% £395 6C1}
`£536 3 "‘
`Tirmis)
`
`'
`7‘01}
`
`' «w.v..::3::§mfifii L312: «.-
`C‘
`11.98
`95% 330 «GS 5331‘ 5339 7(3)
`7833933:'",1
`
`' 4 Amima$1333 133':~
`
`E~:(3:133‘3’ H1311?AIIIniIr3:31
`
`{31:39:}. {Nam {ha 531'.‘
`533‘: NT
`
`mms'JCm-nai H
`'
`51. 3320:: (31' ..-,E IN"
`
`
`3333333133}. 83‘62513311 «N33153:;
`.51 RT:Wrat:I!
`NI 3333‘} 3353mm.
`
`r311 wcrs {3:3533*0: (315‘: Emma-
`{F31 QVN'{"333“
`EIEIINN‘ {WA mm Ru.
`15339.31 circies) (3.2
`
`
`
`3153333933(3 '*: Iii”
`E3333. Cz‘a‘é sssrss(33-:33133 33 B CJL‘IICGN
`Sig-{mi
`
`
`
`
`
`
`
`
`
`5.3353 H‘smm,‘
`551115;,“ 3,
`(.5
`«“5559 51511115315)"; 51155)“ 5:11.
`
`4)
`
`5).
`E
`
` 3.0WWm'W‘“
`
`:5 1.11:2;
`i
`‘P
`
`5.55.:55‘55555535 85515. 5:55 1:11111151‘555g;§5;:~i.‘éu§=551“.5.15,=5,‘~35“Er:'155;
`3‘5
`
`\
`$11153115111). 5111) <111:5 5111515511515 (”5' 11‘--
`~12 3991):;5‘15 151;;51115551211‘5 511515 5.“. 5. 5} \ 5
`in
`.
`.{ASE
`5311.5.15551 51155151951115); 1135553 15‘:35511253151151;ates 555.1131; .3;:
`3.3515151551359355 “5“- (3513‘ 31*1111555 51131: 5:911 .551;5).)».53551115 5‘51:
`55.51:;51181155 {5157525353555 am‘ Wiffiw
`HiéE.)
`5'
`5391131‘511815155‘13853551)5555151“15521511115135”):
`55135155; 333.)
`.)< 15.1“" L‘ ($11511?)3535:1153; as:5.113515:
`(11' 6‘5“ )1-5535) 5115“ ‘3” 5.15 ‘35:15?51-;11<1.5.111 5555::
`3552153011. 59532,) 5115:, K; 1.15 Q‘!!.-«? 5555151111
`
`3*2/w ".11
`g. 5:3
`5555.55 5.1151115) 1:111).;>e11.51551 111155151)-<11)<11‘:551rea5
`2‘3
`5155551155155 555‘15255915 5.511511:)‘5111112$55111115).
`
`
`
`511531 1§G5 251551150151855 51555).1 .511: H113”? 1‘ 535‘s"""" .5g31:"~'"'
`5)
`1
`115515) 5511515151 5.):
`.‘.5::§‘.’e“5.u 52L) 55515515553115.5391)
`
`5'553:):es 853511151 5535551:) data 51515515111115);
`£553.35.) 5’ Hm»)
`515555151;:
`555152;);5315111311155 59:315515553151'1111511
`55'5“? £5553). Hiff““"" 5gH“"“ .5955“““ 5515525.).
`53551111 5515155 135101;);.3556:
`51515551121).5)5553553511511 <‘5551, 5351=1;1;).551>51.JESS-511115555511555111?
`5:15
`“5' 50‘" s‘(F5q155‘ :i‘ ‘15.? 355551“5:15. "551
`<2. 5:51
`'2‘
`
`51:35.15 55152.:5: 111151535(1155::
`1515121515<5 .1550 1152
`<.-; 8 5._5;§)1<;3E 1555135585:
`
`1mm”;
`
`‘1'.1:'551155)g;55511e1‘ 5115‘) 21111111§955 {)5.‘555111;; (5.1;: 550111 511.551
`5:)
`.553.:5 551115 (NR ‘1’.31155115355)1'31; 5115111). pmyammé 55115
`$5355“: 355115555: 551-.55 15115151515: 51511551115111 1:35). 31155.. 25111551535115);
`
`559;) 551% H“‘5 ‘5‘ FgH ‘ 1511.)" 5115913111 aw‘sw very; high
`15‘5‘11‘15‘.‘18L) 1153355155 211;
`11:5 psfiieén $551398“551‘{3111.1155135.155:
`51:5,.155121“ 51515.51 25555151.? 125.3155515') 5593155501111.
`
`\:
`
`
`
`B
`
`3'
`
`,5
`5"
`,5
`5“.
`
`5
`
`u =11)5;5<11_55.5,53~5
`5) 511DEXWSJ$E$
`.5 515551311?» 1115-)“
`15 51115315 5515.1, 5:); 5
`
`51"
`.1
`
`:
`g
`E
`
`3
`
`
`1.5 \ :xn£\ WM HC'E'“-‘l\§35'5”“ E‘Wf‘l'
`5,5 5)£3E\E.)u. WNW 11551“
`,.
`
`
`3
`515' 178$), 5,3215 55"1""“<3"I"‘ 1g:
`‘
`35155 5 11£5§\ 5.513. 55515-151311" 1551-)"
`‘55
`
`
`
`
`
`
`{5C5
`53.)}
`
`
`3
`xxxx~~xx~~~w
`{3‘
`1:5 1111:0235)!
`
`
`.-
`I,
`y.
`.4 K'
`('
`
`
`,3 151.1515.an 55Ci"5v"1‘5g15‘3‘=51x)‘1
`5:5 955555.11 55113, 55535530 igHV‘ 135W?
`
`5.51121) 1) 5593;35151
`$385552) :15 5515‘: 55“?"1‘ 53H""" 19H
`c: 55515551551
`55511353121255’5-515111211);
`.,
`..
`i:15) 1:5 3.51551 11) 1151:131115111132155) 5151):1 1115155 5)?
`
`5.5155131 215. 551155;) 31) 11151513 55155ere~555‘1.-51,111,555);
`..3'15)51511512 gensmw ,.Ni'5.552155552115535 5513113515 5151.15
`
`
`).(1135,1{15155'5V‘5' 53351.55 5.55;;15351335: 5.0 51:5.2:5955191:5535»-
`11551115515...
`r151 55115111558115
`515515521:511151 51)
`155.5)
`155155‘55511’555 51.111551511555 21115555:115-355.“’11???“
`Sun‘s
`
`
`
`f}:
`
`_
`e HG?"‘~"“5‘Q.."‘"5’5)
`_
`51351151155121») 511511305113515).53:11::13519151615851) .
`51351155155 antigens; 55155535115
`5‘15:
`'
`~'
`~
`535:2: anti 511511 055535.551 {B5
`
`25E§EXE 555‘),.1 5.111.585.551559:35.15: 5515552
`25‘: 1;5511::11;1:
`{3515!3515111111553.55 551% 15;
`
`11:115.).1111 521555 55511511955111 1‘215555151156 5:3,5 El.
`15‘.
`5555555559):51155515115111355m1w15511.251511‘ 5515‘s 511‘5111111;51111111).
`
`£18};'=11)5551‘51=551‘. 5165,35,). 15533111551311.5113 951555515855
`‘5“.595.55
`155551163? ‘EgH‘ 1;,1')” 11):135-5159114155 151555;
`65:51 5)? 1.1551g511'5) $111 The H517?""“5gH"553x)“
`r'
`)5) .5ibdtawsgmdiww- 5131111155153 155155.)”
` .m...
`5.1"
`"égF(551512:5.5311? 1.15511 5153‘
`5;}:“““ {3351515551511 586} ...51‘_:
`
`555(15)§INEC51"‘5 5111.51}; 1).. 51552 255515.151).
`(TM?)- F.i<:5)55 (531135155);
`53552
`5151125 65‘}.
`551,55
`2,4,51~‘55‘11115:51:1g)55
`
`”511515151)».E.1<k111:5-5p151wv111’19w51551151599155111259151.15-
`a
`“5'1111‘11511;?1‘15511‘5511a51" 5‘‘ ’5‘;H“~"‘ My map5:51551m;
`)51‘515.. .5) 531:? (1'1" C5155.$1.53‘11:. 531115.11 355 25511551515 mam)-
`’55:
`1151.1.)51151mdse.s~‘5
`1155:)55155.5)5115=5~5~«11151.51.35155116):L111
`5‘7;.1» 11:
`5.115815: Wang 555‘ 35. 5959.}'E $11315 5151; 51.;L51);3515151551
`D.)
`1511515111511m3511 :51) 5151315111.) =1s1tiig:5.5).-55c::1)515211255 111
`W”1:
`.11311511505155“)51155515551535, 1155551), 11; {3515531135551 515551
`
`)
`M m-5.:5‘)
`5,5}5H‘“ 51555
`$515151
`,,
`1.181115
`2?:
`
`{5..,,1‘);<<5555‘,){1551515511515355515mg11{315151555
`
`
`
`‘1‘55'
`7:1;wM '371.).)
`
`5313);; 5555511 5151155555551555115155
`
`315;;55151)‘ HUT "
`.‘
`511111555: 5921-“: 52555.5«1111-55v1‘15 551-53--
`
`Ficaii 51515 H515 1'11 535.155.
`
`..
`,5
`'
`115551) 155
`15g. 511;» {A 11555.5 8} ~35 5553.H5311:1135 {1i
`115; 515111
`
`51'; ,555351315.35‘ 55‘,211111 C5}53.11 1111);“ ‘
`'
`
`
`51115535155253} 5551
`_
`1.55 31);} 15351:?
`
`55135525115:‘.5-).r 55
`““351-151‘. 519131551 51111535511155):
`
`
`.
`BSA {E (1115‘! 1'}. 5111515125:
`.~.11.11115.5‘5 5119111151 531’ man-3a-
`1L1113565<5 g5).115‘
`and 53111155591) 55;“5159):).."5-1'55).s:55.1155:111;)5).<1$;p5525 5:55: 5:5
`5W5):-
`.L5¥}‘1€E$‘E
`'3: 135353?8512':-551511.315 SG-CGHCSEEW 3111=b<1555.5)..5.
`
`..
`
`{.1 .sshiiw 1‘53: T5311???
`V15 65115511,E's-‘1': 5515. 5:52”. 5511‘s
`
`3.9m
`Le:.25,w :2?5':j
`,W.
`5511105501135?5621555111511;
`Ea
`
`511555151; 5115113155551 5..
`.j:1~s""" 1£55.“-'} 515-:
`
`15) 1121551135115 51:1 {BF-X
`.... 2‘52515:1:‘1‘5. 5515161355115.)
`
`51522511151§3515~"“‘155'
`51555511 .1 .111155.51.- -)5=5)<
`3
`5511; 251151;;91'1. as); 1555.))! 551111.15r€55; 111151515155 11" ..
`
`51153 513“
`
`511:) 51513555531591) 151:5:{55'9H'"""‘555353):.“ .Hi: 5")=‘ 19,,“““
`§g1<"‘~".a).555 H5351‘51‘5H)‘55H5),).5::-« ‘55 53555)) .1.) 51.5;01115
`
`'
`=35 51...,‘-')~£51 551.11 any 115‘ ‘11!) 11‘
`
`155051.111! ‘515;
`555155;}.
`'1
`
`r-1.".5 .w «4:
`1);
`$512155)151151355115555595‘;'
`
`
`5555 {.1555155515 5‘5). 3152155,: 55‘111:
`
`
`..
`_- “35511‘55;
`{551111155515 55511
`
`51555.5.
`
`138?}: I: .11).“..501'155‘5
`
`1“ 55511:) 5,5135 111 5353555 51551155
`
`1::15515110g11155‘1“ .1151); 5555511p1‘5351015551115555113115 ‘1’ 1153.515).
`"
`1.5555115.)”555': 35113.5 5:511; 555111511551.5.; 2551,55 \1‘ games. are
`
`
`
`immunity
`42
`
`used in immune responses of inbred mice to many hap—
`ten, polysaccharide, and even protein antigens, includ-
`ing p—azophenylarsonate {Pawlak et al., 1913; Makela
`et al., 197a; Manser, 199d), phenyioxazolone (makela et
`al._, 1978; Kaartinen et al., 1983; Berek and Mlistein,
`1987),
`(4-hydroxy-3—nitrophenyl)acetyl
`(NP)
`(lmanishi
`and ivtakela, 1914, 1975; Biler and Bothweii,1988), DNP
`(Dzierzak et al., 1980, 1985), phosphorylcholine (Potter
`and Lieberman, 197d; Sher and Cohn, 1972; Barstad et
`al._, 1974; Crews et al., 1981), phosphatldylcholine {Seidi
`et al., 1997), dextran (Biomberg et al., 1972; Riblet et
`ai., 1975; Wang et al., 1990), gatactan (Mushinski and
`Potter, 1973), Staphylococcal nuclease (Fathman et al.,
`1977), and influenza hemagglutinin (McKean etal.,1984;
`Clarke et al., 1985). T cell recognition of peptide/Mlle
`complexes is often (but not always) fairly restricted with
`respect to V gene usage (e.g., tiedrick et al., 1988).
`However, it is not clear whether the preferred V gene
`segmentts) are absolutely required for such epitopes or
`are merely the result of extensive clonal expansion
`where a slight advantage in binding would allow the
`most optimal VHVL combination to predominate. in the
`current study, we find that mice having only one func-
`tional VH and (effectively) two 11L gene segments are able
`to mount highly specific antibody responses to most
`antigens. it thus seems likely that the restricted V gene
`usage observed in most immune responses represents
`the consequence of antigen—driven clonal expansion of
`kinetically favorable antigen receptor clonets).
`in the H6?” lg "" lga+ mice, we find a remarkable
`restriction in VH CDR3 diversity with respect to both
`length and amino acid composition in the primary im-
`mune response to both hapten and protein antigens. in
`the tgl‘vi antibodies, there are many identical or very
`similar sequences. A restrictive pattern of VH CDRS
`would normally be interpreted to mean antigen-driven
`clonal expansion; however, considering the tow eff“-
`ciency of cell fusion, the isolation of identical repeats
`suggests that certain antibody specificities could be
`specifically expanded even prior to immunization {Ra-
`iewsky et at, 1987).
`it is not clear at present whether
`the expansion of a particular specificity occurs as a
`result of preferred ViD)J rearrangement event or is the
`result of some type of selection.
`Gur results also indicate that the antigen binding site
`of nonsomatlcally mutated antibodies is not uniformly
`specific but instead consists of one highly diverse CDR
`loop {VH CDRB) and at least four “generic” antigen bind—
`ing regions (Stilt-“2s 1+2). Even the somewhat diverse VL
`CDR3 region seems to play a very minor role. Using a
`phage display library, Winter and colleagues previously
`showed that a range of hapten and protein binding activ-
`ities could be isolated from a repertoire of antibodies
`comprising 5:) human 12H gene segments in combination
`with a fixed light chain (Hoogenboom and Winter, 1992;
`Nisslm etal.,1994).'t‘hatVH CDR3 might play a dominant
`role in antigen binding was also suggested by earlier
`studies showing that a heavy chain alone or even single
`VH domains can bind antigens with a comparable affinity
`as the intact antibody (Ward et at, 1989; Noel et at,
`19%). in addition, it has been found that randomly muta—
`genlzing the VP; CDRB of an anti—tetanus toxin antibody
`enables the isolation of a new specificity lie, for fluores-
`cein) {Barbas et al., 1992). but results are also consistent
`
`with the findings that murlne B cells that have a re—
`arranged heavy chain immunogiobulln gene in the germ—
`iine always change the CDR3 sequence and often the
`VH as well when they deviate from the original antigen
`reactivity (Cascaiho et al., 19933; Lopez—Macias et al.,
`1999). (Eur data may explain the long—standing puzzle
`of “canonical" CDR structures in antibodies (Chothia
`and Lesk, 1987; Chothia et al., 1989). We would suggest
`that uniform CDR shapes are important for the stability
`of a binding site, with the exception of Va CDR3, which
`has to "fit" the antigen surface much more precisely.
`These results are also reminiscent of the case of human
`
`growth hormone and its receptor, where structural anal-
`ysis shows that about so side chains from each protein
`are in close contact but only a fraction of those residues
`have any contribution to the binding affinity and only
`two account for more than three—quarters of the free
`energy (Wells, 19%).
`Another interesting issue raised by the data presented
`here is the role of somatic mutation in producing higher—
`affinity antibodies upon rechallenge with antigen. Qur
`data shows that, at least for protein antigens, having an
`arbitrarily chosen human tit, and one of two mouse it
`chains as a germltne repertoire is no barrier to generat—
`ing very high—affinity lgfs antibodies. This finding is con—
`sistent with work on the chicken immunogiobulin genes,
`where only a single functional VH and VL are utilized, but
`goes beyond this in two important respects. Cine is that
`chickens have somatically mutated antibodies prior to
`immunization (Weill and Reynaud, 1987; McCormack et
`al., 1991), thus allowing the argument that this sequence
`diversification takes the place of a large V region reper—
`toire. $econd, the germline VH and Vi: genes utilized
`have been selected over many millions of years for their
`compatibility with many different antigens and thus they
`may represent the most polyfunctionai VHV-L pair. in our
`system, we have chosen our VHVL pair at random, sug—
`gesting that all or most such pairs are capable of binding
`a large number of different antigens, given a single,
`diverse V“ CDR3. This indicates that an antibody binding
`site excluding VH CDRB is exceptionally cross-reactive,
`at least until acted on by somatic mutation (Patten et
`ai., 1995; Wedemayer et at, 199?). This interpretation
`is consistent with the observations that the residues
`
`characteristic of antibody binding sites have an unusu—
`ally high proportion of asparagines, iysines, and aro—
`matic amino acids, more typical of the interior of a globu—
`lar protein than to its surface and capable of making a
`wide variety of different contacts (Kabat et ai., 1977:
`Janin and Chothla,1990; Padlan,1990; Mlan et at, 1991).
`it also has some similarities to the proposal of Pauling
`and others that a single antibody molecule could fold
`around different antigens in different ways to achieve
`specificity (Pauling, 1946).
`The data presented here, as well as previous work,
`strongly suggest that the purpose of highly diverse
`CURB regions in all antigen receptors is to provide anti-
`gen specificity.
`it supports our earlier speculations
`(Davis et at, 1997, 1998) but is not consistent with the
`Protecton model of Cohn and colleagues, which argues
`that the primary antibody repertoire is solely defined
`by germline VH and V-L gene sequences (i.e., CDR‘l and
`CURE) and that CDR3 is not important in the initial deter-
`mination of antibody specificity (Langman and Cohn,
`
`
`
`Molecular Basis of immunoglobulin Specificity
`43
`
`1987: Cohn and Langman, 1990). The Protecton model
`also argues that in the primary antibody repertoire, each
`specificity is expressed by a large number of B cells.
`Our data support the notion that certain antibody speci-
`ficities may be relatively abundant prior to antigen stimu-
`iation in these mice. Thus, it would be interesting to test
`whether some CDR3 sequences are expressed at higher
`frequencies than others in the preimmune repertoire in
`the mice analyzed here and in wild-type animals.
`The only deficit we have detected in the immune reper-
`toire of mice with limited if gene-(s) so far is in their
`inability to respond to bacterial poiysaccharide anti-
`gens. interestingly, chickens are unable to mount robust
`antibody responses to carbohydrates as well (Jeurissen
`et ai., 1998),. although this point is controversial (Gran-
`fors et at, i982; Jaikanen et at, 1983). if the failure to
`respond to bacterial polysaccharides is due to the lack
`of appropriate VHNL pair(s) to accommodate these moie—
`cuies, the 5461”“ lgfr" igrd‘ mice may have difficulties
`in clearing bacterial infections. The system described
`here will provide an experimental test for the idea that
`unique VHNL pairs could be selected by pathogens dur—
`ing evolution and then fixed in the germline (Langman
`and Cohn, 198?; Cohn and Langman, 1999).
`
`Experimental Procedures
`
`Mice
`as mice were originally obtained from the Jackson Laboratory (Bar
`Harbor. ME) and bred at our Animal Research Facility at Stanford
`University. Mice transgenic for the human heavy chain minilocus
`HCT were previously described (Taylor et ai., 1992, 19%). Mice
`containing targeted deletions of the JH region or the JK‘CK region
`were also described and are referred to in this paper as lg
`’“ or
`:96" (Chen et al., 1993a, 199%). The H61 transgenic mice were
`bred onto the lgH"‘ 196’“ background. Details of mouse breeding
`and screening will be furnished upon request.
`
`Reagents
`BSA, CTB, CSA, DEX (MW 599,909), HEL, KLH, levan, EVA. FCC.
`and phOx were purchased from Sigma (St. Louis, MG). DNP-KLH
`and DNP—BSA were from Caibiochem (La Jolla, CA). TNP-Ficoil and
`TNP—BSA were from Biosearch Technologies,
`inc. (Novato, CA).
`phOx—CSA and thk-BSA were prepared as described (Makela et
`al., 1978). PC—KLH and PC—BSA were kindly provided by Dr. Leonore
`A. Herzenberg (Stanford University).
`
`immunization, ELlSA. and Hybridomas
`Mice at 6—8 weeks of age were used in all experiments. To elicit
`immune responses, 1% pg CTB, HEL, KLH, FCC, GVA, DNP-KLl—i,
`or phOx-CSA in either complete Freund’s adjuvant (for first injection)
`or incomplete Freund's adjuvant (for subsequent injections) was
`administered by intraperitoneal injection; 59 pg levan was adminis—
`tered in phosphate buttered saline (PBS) intraperitoneally without
`adjuvant; and 16 pg DEX, 1i) pg TNP-Ficoil, or 103 pg PC—KLH (in
`PBS) was administered by intravenous injection. To detect H61—
`encoded antibodies in the H61+mlgH"‘ lgk""‘ mice. diluted serum
`was added to mlcrotiter wells coated with monoclonal mouse anti—
`human iglVl (Clone CH6. The Binding Site, Birmingham, UK) or mouse
`anti-human lgGi antibody (Clone unease. Fc specific. Calbiochem),
`and p
Accessing this document will incur an additional charge of $.
After purchase, you can access this document again without charge.
Accept $ Charge
Still Working On It
This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.
Give it another minute or two to complete, and then try the refresh button.
A few More Minutes ... Still Working
It can take up to 5 minutes for us to download a document if the court servers are running slowly.
Thank you for your continued patience.
This document could not be displayed.
We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.
Your account does not support viewing this document.
You need a Paid Account to view this document. Click here to change your account type.
Your account does not support viewing this document.
Set your membership
status to view this document.
With a Docket Alarm membership, you'll
get a whole lot more, including:
- Up-to-date information for this case.
- Email alerts whenever there is an update.
- Full text search for other cases.
- Get email alerts whenever a new case matches your search.
One Moment Please
The filing “” is large (MB) and is being downloaded.
Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.
Your document is on its way!
If you do not receive the document in five minutes, contact support at support@docketalarm.com.
Sealed Document
We are unable to display this document, it may be under a court ordered seal.
If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.
Access Government Site
