Antibody Engineering Protocols

440 Pages·1995·26.85 MB·English

by  Sudhir Paul

CHAPTER 1
SEQHUNT
A Program to Screen Aligned Nucleotide
and Amino Acid Sequences
George Johnson, Tai Te Wu,
and Elvin A Kabat
1. Introduction
We have been collecting nucleotide and amino acid sequences of pro-
teins of immunological interest, and aligning them in order to understand
the structure and function relations of these proteins (I). To aid in orga-
nizing and analyzing this collection, a computer program, called
SEQHUNT, was written.
The SEQHUNT program is written in PL/PROPHET (2,3).
SEQHUNT uses a preprocessed form of the database as its search data.
SEQHUNT can pattern match nucleotide and amino acid sequences with
the aligned data, pattern match phrases in the annotation fields of the
sequences, and compare specified regions in similarly aligned sequences.
The SEQHUNT program can be used only on a machine with the
PWPROPHET environment present and with the PL/PROPHET table
representation of the database present. To allow greater accessibility to
the matching capabilities of the program, a partial implementation of
SEQHUNT is available via electronic mail.
2. Materials
The variable and constant regions of immunoglobulins and T-cell
receptors for antigen, and the various domains of MHC class I and class
II molecules have been aligned (I). These aligned sequences and
From Methods m Molecular B/ology, Vol. 51: Ant/body Engmeenng Protocols
Edited by. S Paul Humana Press Inc., Totowa, NJ
1
2 Johnson, Wu, and Kabat
sequences of related proteins (I), together with new sequences published
recently, have been stored in the NIH-supported PROPHET computer
system (2,3) in the form of PL/PROPHET data tables. SEQHUNT uses
this Kabat database (I) for its searching and region analysis.
3. Methods
SEQHUNT is a computer program written in PL/PROPHET for use in
the PL/PROPHET environment. The program performs three main types
of analyses. The first is matching. Given a nucleotide or amino acid
sequence and restrictions on the number of allowable mismatches and
data tables to search through, SEQHUNT will return aligned matches of
all sequences with mismatches equal to or less than the allowable num-
ber. The second function of SEQHUNT allows searching for specified
patterns in the sequence annotations. Name, antibody specificity, T-cell
receptor classification, and reference fields may be searched for the
desired pattern. Moreover, the full implementation of SEQHUNT allows
region analysis of any one or a number of sequence stretches in similarly
aligned sequences, e.g., all immunoglobulin heavy (H) chains. The
program queries for the given region, such as the entire light (L) chain
variable region (positions l-107) or a combination of several com-
plementarity determining regions (CDRs), for example, CDRLl,
CDRL2, and CDRL3 together. All sequences are called as search pat-
terns, and the entire set of sequences is used as the search pool. Redun-
dant matching is eliminated to reduce output. Any number of mismatches
may be specified, although the output for mismatches above 1 or 2 is
usually massive. These three types of searches may be performed on
nucleotide or amino acid data, and matching and annotation searches
also may be performed on unaligned data.
SEQHUNT, as written, must be called from the PL/PROPHET envi-
ronment. To allow greater access to the program, an interface has been
developed that allows specially formatted queries to be sent via elec-
tronic mail for processing. The interface supports all functions of the
original SEQHUNT, except region analysis.
3.1. Sequence Pattern Match
The nucleotide sequence pattern-matching capabilities of SEQHUNT
are shown in Fig. 1. In this example, the nucleotide sequence to match
(TARGET SEQUENCE) is the H-chain variable region of the IgM
SE&HUNT
BALB/c murine monoclonal antibody (MAb) PRl (4), which has speci-
ficity for the PRl antigen on human prostate cancer cells and normal
human prostate cells, This SEQHUNT search was restricted to 12 or
fewer mismatches among the sequences of all H-chain variable regions
of all species currently in the database. In Fig. 1, several sequences with
6,7, or 11 mismatches are shown. They are listed in order of increasing
mismatches. An upper-case base is a mismatch, and all lower-case bases
are matches. Dashes are for alignment (I). To save space, several other
sequences with fewer than 12 mismatches are not listed (see Notes 1 and
2 for other examples).
Figure 2 shows the results of a search of all H-chain variable regions
for matches with a segment of the human D-minigene D2 (5). Human
D-minigenes sometimes match segments other than the third CDR of
human H chains (6). As shown in Fig. 2, a segment of 14 nucleotides from
human D2 is found in the second CDR of human, mouse, and rabbit H
chains (seeN ote 3). For nucleotide sequencesin the human CDRH3 region,
additional matches are found on both sides of the 14 nucleotides. RF-SJ2
matches human D2 for 24 bases,t tgtugtggtggtagctgctactc, and L42 for 28
bases,g gatattgtagtggtggtugctgctact. The 14 matches in Fig. 2 are under-
lined. Usually, only short segments of human D-minigenes are incor-
porated into CDRH3s (7). When some of these short segments of the
human D-minigenes, e.g., aactgg, a segment of DHQ52 (5), are searched,
identical matches occur frequently over the entire H-chain variable
region (Fig. 3).
3.2. Antibody Specificity Search
An example of antibody specificity searching is shown in Fig. 4 for a
SEQHUNT search called with the specified pattern “HIV,” the abbrevia-
tion for Human Immunodeficiency Virus. Only a few of the matches are
shown. The search was restricted to all H-chain variable region sequences
in the database. SEQHUNT scans the antibody specificities and looks
for exact matches with the “HIV” pattern. This search, Fig. 4, found anti-
bodies directed against ~24, gp120, and gp4 1. Even for the same protein,
the numbers of dashes in the last three lines of the sequences are differ-
ent, indicating that the length of H-chain CDR3 can vary more exten-
sively than those of CDRl and CDR2 (8). Most likely, the antibodies are
directed toward different parts of one of the HIV proteins, Searches of
the name and reference fields are also allowed.
8
3 I I
!IPFLR $EQ"?JNCE FECIES EGIN iFLCIPICITY REPEPENCE
KLS
‘ARGET , yrtytycayctyytygag+c+ggay-
:LQ”ENCL gt~gcctg~t~ra~cctyya~~~~~~
cc.ga~ac.c.cc.ytycn9-c
yyatteyattttsytagotsctggs-
tyaytty~gtcccrgcayyctccagp-
gssay~gctagaatggat+aya aa-
sttaafccayataycayts~ye 8 aa-
actstscyccatctc+rsagg"t~~-
~ttcatcatctccagtyncaac?rc-
aaaaatacgctqtacctgcaastga-
ycaasytyaysfctgaggaca~a~~-
cctttattactytgcer?a
90 GytyAagctTCtCgsytctysa~- OOSE 4 UITI-3-.TJC"S- i LIMURA. H . B”-
ytygcctggtycagcctapay~*t~- !LLncT~SAtIIN-
eaactctcct~tycsycct=a- I "YBRIDOMA
,c;',e tcyattttayfag"taccgya-
toagt------tyyptccpgcaoor-
Itcc ayyyaaaygyctrgost~q~++-
gyagaaattsatcca------qata-
ycaytacystaaactstscoccatc-
tCt."."~lt."~ttC~tC~tCrCC-
131?? OUSE 4 lt;';~yf~$ l- BORDEII F 6 -
?ABAT.k A
1297) FRK Ii-
ATL ACRD SCI-
USA P4,244"-
-2445
OL-126-7 0”SG 4 UITI-AMINOFH- B0NILLA.F A -
:NYL-BLTA-N-- , ZAGHWANI, H-
gtyc"gcctcs- ,cETYLGLocoS- ,P"BIN,M 6-
ggrttcgattttagtagatactgga- MINIIDL. A/F- B0NAA.C (lP-
tgayt------tgyytccygcapgc- L/P/34 IN!-L"- PO ) J . IMMUNO-
tccaggg*aag gctagaatggatt- :NZA VIRUS &,145,616-6-
ygayaaatta* ? cc.------Qa+rl-
gc*ytacgstsaactatacgccatc-
tctaaaygltsaattcstcatcttc-
agAgacsscgccaaasatacgctut-
acctycaaatgagcasaytgagatc-
tgaggac.c~gccctttattaotgt-
gcragC
5 3A? I1 qaGqtqAaqct?CtCqsqtctqqaq- “ODSE II RNII-GALACTO- SNYDfR,J G ,-
qtqqcctggtqcaqcctqqaqqatc- SYIGLUBOSIDE NENG. A , W-L-
cct aaaGtctcct tqcaqcctca-
99. 2 tcqattttsq e agstactgoa- I$:*; Mh y.z
tqaqt------ tqqqtccggcsqqc- 0)'EUR J ItSi-
tcca999.aa9qqctaqaatggatt- ONOL ,20,267-
.$~aqa.mttaatcc.------qate,- 3-2677
.gtacgCtaaactatGcqccatc-
tctaa.qgataaattcstcatctcc-
ECIaI*at.CACtTzI
? qaqcsaaqtqaaa
tqaqgaoacnqccctttrttsctgt-
qca*ga I
11 gaGqtgAagcCTCtCgagtctgqaq- RNTI-UORPHIN- mss1E.P H.,-
gtggcctqgtqcaqcctqqaqqatc- E-6-HEMISUCC- ANCHIN,J.H ,-
cctgaarctctcotgtqcaqcctca- INATE-BSA suBPAMANxAM, -
gattcg*tttt*gtaqatactgga-
? q.qt------tgqqtccggcsggc- i. 'EE;hk:cI
tcc~CqqaaaCqqctaqaatqqatt- M.D.S. (lPY-
qqaqaaattaatcca------gata- 1) J.mOL-
a~qt*oqat~aCct~tacqo=~t=- 146,4246-4-
? ~taalqqataaattcatcatctcc- 557
l qAgmcarcgccmaaaatacqctqt-
~cotqc~artqsgca~aqt~~~~~~~
Fig. 1. Matching a nucleotide sequence of a H-chain variable region, positions l-94. The sequence, PRl, IS shown m
row 1 labeled as TARGET SEQUENCE. Some of the sequences in the database with 6,7, or 11 mismatches are listed m
the order of increasing rmsmatches as shown in column 2. Names of these sequences are given in column 1. Columns 4-
8 indicate species, beginning position, endmg position, antibody specificity, and reference, respectively.
0 I 2 3 4 5 5 7 8
FO”“Wt? Iw4E DIFFER SCQ”EtlCE SFECIES Er‘SIN END SFE’IPICITf PE~ER?NCE
1 TARGET 0 qtsgtggtqotalc
SEQUENCE
2 014 0 9t---------“o+q- MOOSE S? 5F. “NKNOWIJ LE”Y,N s ,MALlFIERO.O " ,LEBEC-
qtaqtaec P"E.2 G 6 GEARHAPT,F J (l?Rl-
) ST EXF l,El2 ,163,2007-2019
3 PVl1720 0 gt---------aa+3- rABBIT 52 56 “tlnlOWll RC”X,h II ,DRANARAJAN, F , GOTTSC-
9tcl9ta-3= HALh, 7 , MCPOPMACK. A T 5 PENSH-
~lV5R&;03~l~"' J.IMMUt1OL ,14F,-
4 lR/O 0 4t---- --ac7tcl3t3- “WA1 52h 56 ANTI-WA AUT- @EPSIMOtlIAN.H ,SCHWAPTZ,F S ,B-
gtag= OANTIB?DY HY- ARRETT,h J h ST0LLAP.B D. iI+
BPIDOMA 47) J. IMMDNOL ,139,24’=6-25Fl
--I
5 RF-SJZ 0 gtagtggtggtagc HUMAN 98 IlOOB AN-II-IGGl,IG- PASCUAL, V , RANDEN, I , TNOMPSON, -
G2, IGG4 RHCU- K..SIOOD.M.PORRE.O .NATVIG.J -
MATOIU FACTOR i &PRA,k.D (1930) ‘J CLIN- INV-
EST ,86,1320-1328: RANDEN I
R-,D.,TN-SON,K.M.,HDGkLSL~I
0NES.N ,PASCUAL.V ,VICTOR,h ,C-
APRA J D.,PORRL 0 L NATVIG J -
$i992) J.IMtdJNOL ,148,32!+6--
-t
6 L42 0 3tagtggtggtagc Ii- 100 1OOD ADTOANTIBODY K1PPS.T J. L DDFFY,S.F. (lYYl)-
J.CLIN INVEST ,87,2087-2096
Fig. 2. Matching segments of D-minigenes. The format of this figure is identical to that of Fig. 1. The TARGET
SEQUENCE pattern consists of 14 bp from the human D2minigene. It matches identically to nucleotide segments in the
CDRH2 region of 914 (mouse), RVH720 (rabbit) and 1819 (human) as shown in rows 2, 3, and 4 respectively. It also
matches human CDRH3 segments of RF-SJ2 and LA2 shown in rows 5 and 6. For details, see section 3.1.
0 1 2 3 4 1 8
Pownme NAME DIFFER SEQOBNCE SPECIES ZEGIN LD SPECIFICITY RECBRENCl
BEES
1 TARGET 0 aactgg
SEQUENCE
CLL4 0 asctqg HUMAN 3 5 VNIWOWN CAI,J.,HDMeHRIES,C-
RICHARDSON,A. C -
+~cKER,F.W. aaozb-
J.EXP.MED.,176,10-
73-1081
3 257-D 0 aactgg Hvl4AN 32 33 ANTI-HIV TY-PE 1 SF- ANDRIS J.S JOHNSO-
ECIFIC FOR TIE PRI- N,S., 26IJA-bAENER, -
NCIFAL NBUTRALIZIN- 9 4 CAPRA,J D. (l-
G DCHAIN OP gpl20 - 991) PROC NA?L.ACA-
OF lS4 AND MAP TO R- 97;$I.VSA,88,7783--
ESIDVES XRIHI
4 VHVI 0 aactgg HvMAN 358 36 VNRNoim BOLUW3LA,L. L RABB-
IT'IS,T.H. (1988) E-
VR J.IMMUNOL.,18,1-
843-1845
'1 CLL-27 0 aactgg HvNAN 44 46 O?WNOWN BP,RMF.N,J.E. RDMPHR-
IBS,C.G.,EdTH,J -
ALT,P." ‘ TUCKER:-
6 Ab21 0 asctgg HUMAN 50 52 POLYRBACTIVE AUTOA- SANf,I.,CASALI,P ,-
NTIBODY TIiOtlAS,J.W.,NOTKIN-
S,A.L. ‘ CAPRA,J D-
I 4m6 0 ractgg MOUSE 93 95 VNKNOMi GV&;$p"I=-.i-i-Eg-l
TV K. c ~OR!~TER I -
(i991, J.EXP MtD -
,173,1357-1371
8 L22 0 .llC2tgg HUMAN 1OOE loo? ADTOANTIEODY KIPPS,T.J. L DDFPY-
,S.F. (1991) J.CLI-
luvEsT.,e7,2087--
Q 4611 0 ractgg MOUSB 102 103 ANTI-IDIOTYPIC ANT- TAOB,R.,HSJ,J.-C.,-
IBODY AGAINST THB - CARSKY V.M. HILL,B-
TEYROTROPIS (TSR) - .L.,ldANGCk,B.F. -
RECEPTOR b KOHN,L.D. (1992)-
J.BIOL.CBEM ,267,-
5977-5984
Fig. 3. Matching of a segment of the human D-minigene, DHQ52 (TARGET SEQUENCE) with different regions of
mouse and human H chains.
1 2 3 4 5
0wn.m. NAME SEQUENCE SPECIES SPECIPICITY REIERENCE
BAT123 --- gaa gtg c.g ctt tag gag tcg gga cct ggc ctg gt- MOUSE ANTI-GLYCOPR- LIOW,R -S ,ROSEN,E.H-
g a.. cot tct c*g tct ctg tee ctc *cc tgc act gtc - OTEIN qpl20 - .,mNG,H s c.,suN,w -
act ggc t-0 tu at0 *cc 'gt g.t tat gco tgg .*c --- OF HTLV-IIIb- N.C. SUN C.,GORDON W-
- tgg .tc egg c.g ttt cc. gg. .*c .** ctg gag tgg - STRAIN OF H- .,CHhG,k.T. A CHAfW
atg ggc tat .t. age --- --- --- tat sgt ggt ogc .o- IVTXFE 1 ,T (1. (1989) J.IMMU?i-
t *cc t.0 sac cc. tot ctc *.a agt cg. .tc tct ate - OL ,143,3967-3975
act cga ac no. tee sag 1.0 otg ttc ttc ct.$ C.?.Z”I
g agt to 8 gtg act tct g*g g.c acn gee ac. -at
tgt gc. agg ggg agt ttc gg. gac --- --- - -- -- - - --
- --- --- --- --- --- --- --- --- --- tgg ggc a* -
ggg act ctg gtc act gtc tot get
CB-mab-,x24/ --- tag gtc ..I ctg tag gag tct ggg gg. ggc tta gt- MOUSE ANTI-P24 COR- KUTTNER,G ,GIEBMANN,-
13-S g eag ctt gg. ggg tee ctg ..a ctc tee tgt go. gee - E PROTEIN OF- E ,NIEMNN,E ,WINKLE-
tct gg. ttc act ttc agt ago t.t tn.2 atg tct. --- --- HIV-1 R K ,GRUNOW,R , HINKU-
- tgg gtt cgs c.g act cc. g.g ..g agg ctg gag ttg - I,& J , ROSEN, J , WAHRE-
gtc gc* gee .tt ..t agt --- --- aat ggt ggt ago gc- N,S 5 VW 6AEHR.P. -
c tat tat cc. a0 l ct gtg a.g ggc cg. ttc *cc at= - j;;fX\-r;'Mk IllMUNOL ,-
tee ag. g.0 a. ? gee ..g a.0 .CC ctg t-0 ctc c.. .t-
g age agt ctg ..g tct g.g g.c .c. gee ttg tat t.c -
tgt gc. ag. ct. ccc ctt --- --- --- --- --- --- ---
- --- --- --- --- --- --- --- g*c i-*c tgg ggc a* -
ggg ICC asg gto *cc gtc tee tea
0 S-BETA --- tag gtt csg ctg 0-g ug tct ggg get a.0 ctg gt- MOIlSE ANTI-FRINCIF- MATSVSRITA,S.,P!AEDA,-
f at Cf. .t. gag --- ---
- tgg ntg a.. c.g ..t c.t ggg arg ago ct. g.g tgg - PND) OF HIV-- .,TOKIYOSHI,S. C TAK-
.tt gga *at ttt c.t cot --- --- t-0 agt gst gat .c- 1 gpl20 ATSUKI,K (1992) AID-
t ..c tat ..t g.. . . . ttc ..g ggc a. S RES.HUMAN RETROVIR-
.ct gta g.. I.. tco tot age .c. gttc ? .cg ee ttg *** gagt t? t- - USES,8,1107-1115.
c age cga tt. .c. tot gat g.c tot get gtt tat tat -
tgt gc. at. c.c t.0 ggt agt gee t.0 got atg --- --
- --- --- --- --- --- --- --- gac t*c tgg ggt aa -
gg. *cc tc. gtc *cc gtc tee tea
257-D ANTI-HIV TXQ- ARDRIS J.S. JOiiNSON,-
E 1 SFSCIFIC- S.,ZOLh,-FAkNER,3 b-
tct gg. t.0 *cc ttt ..C g.c ..C tgg .tc ggc --- --- FOR TflG FRI- CAFRA,J D (1991) P-
- tgg gtg cgc erg .tg ccc ggg . . . ggc ctg gag tgg - ~~C~GNSUSU- ROC NATL.ACAD SC1 US-
atg ggg .tc .tc tat Cd --- --- grt g.c tct g.c .g- - A,80,7703-77R7
c aca gtc .gt ccg tc. ttc c.. ggc tag gtc .EC .tc -
tu gee g.c arg tee .cc .gc .CE gee t-0 ctg c.g tg-
0 *go .gc ctg ..g go0 tog 9.0 .CC gee at= tat trc - F TO RESIDW-
3 KRIHI
--- t.c ttt g.c t.
gg. *cc ctg gtc .cc gtc tee tea
71-31 --- 0.g g =*g "t? =w -g tct p7g=p~.~*w&3~ HtJnAN ANTI-HIV TYF- ANDRIS J.S. JOMSON,-
g arg cc F ggg gee 0. gtg a.. at G 1 SFSCIFIC- S.,ZO&-FAhNER.5. L-
tct ggc ttc tee ttc .tc ICC t.c t.t ttt-cat 2- --- FOR ~24 CAFRA,J D (1991) P-
- tgg gtg cg. c.g gee ccc gga tag ,gg ctt 0-g tgg - ROC NATL.ACAD SC1 US-
.tg gga gt. *to ..C --- --- -- cc .gt ssf cw= *c- A,B0,7703-7787
. *cc tn.2 to. tag ..g tto tag ggc ag. gtc gee stg -
*cc .gg gac .cg tee .cq ago go. gtg tat rtg g.g tt-
ago .gc ctg ag. tct gta g.0 .cg gee at. tat t-c -
9 St get 99. gtt -g 999 stg g=c egg a=t tt. ggg w-
,= - -- -- - - -- --- --- --- --- --- -- tgg ggc ag -
gg. tee ctg gttc ICC gtc tee tu
--