CA2183550A1 - In vitro antibody affinity maturation using alanine scanning mutagenesis - Google Patents

Abstract

A method of mutagenizing antibodies to produce modified antibodies, modified antibodies, DNA encoding the modified antibodies as well as diagnostic kits and pharmaceutical compositions comprising the antibodies or DNA the provided.

Description

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W09S123813 P~ 2~
TITLE OF THE INVENTION
IN VITRO ANTIE~ODY AFFINITY MATURATION USING ALANINE SCANNING
MUTAGENESIS
CROSS-RELATED TO OTHFR APPLICATIONS
This is a c-",l;""~ ", of U.S. Serial No. 081206,076 filed March 4, 1994, now pending.
Bl~FF DESCR~PTION OF INVENTION
A method of mllt~g~ni7in~ antibodies to produce modified antibodies, modified ~nhho~irc, DNA encoding the modified antibodies as well as (ii~gnoshr kits and rharm~rel-ti~l c~ o~iLiulls ~UIIIIUli~illg the antibodies or DNA are provided. The method of the invention is a ~y~L~,Illd~iC means to achieve in vitro antibody mo1nr~tinn and uses alanine scanning mutagenesis. The invention is particularly exemplified with a set of single chain Fv (scFv) antibodies obhined by this trrhni~p-~, The resultirlg antibodies are directed against the V3 loop of HIV gpl20, and show altered off-rates agamst the antigen compared to the starLing antibody. Of particular interest are the altered antibodies which show improved (slower) off-rates to the antigen. Observed illl,uluv~lllc~llL~ have been as high as eleven-fold over wild-type.
SUMMARY OF T~E INVENTION
A method of mllt~ni7in~ o~hhQ(R~,c to produce modified ;"";I,o~l;. c, modified antibodies, DNA encoding the modified onhhorii~os as well as di~nr,chir kits and ph~rm~rellti compositions cblll~ illg the ~ntihodiPs or DNA are provided.
BRTFF DESCRIPTION OF T~E DRAWINGS
Figure 1. Alanine-Scanning ~AIIt~n~ci~ Each of the 27 a~mino acids in VH CDR3 of scFv P5Q was converted to alanine by site-directed mllt~ nPcic E. coli clones were induced to express scFv with IPTG. Single chain Fv, which is targeted to the p~ ld~lllic space by the fd phage gene3 signal sequence, was . _ _ .. . _ _ . . . . . . .

W0 95~23813 2 1 8 3 5`$~ C~ ' ' r~ 492 extracted with EDTA~ r~ "i~ extracts were analyzed byBIAcore~M, which measures antibody-antigen affinity by surface plasmon resonance (Fagerstam, 1991), and off-rates 11r~rll.1ill~d against an HIV gpl20 V3 loop peptide. Results of the alanine scan, 5 relative to PSQ, fall into four classes: i) slower off-rate, ii) faster off-rate, iii) no binding, and iv) minor or no change in off-rate.
Standard deviation is + 25%.
Figure 2. Amino Acid RAn~ mi7Ari~n Position 107.
Arginine at position 107 was mutated to all amino acids by site-directed mlltA~nPciC Single chain Fv extracts were analyzed by BlAcore. Percent change in off-rates is shown relative to P5Q.
Pigure 3. Amino Acid ~n~c ,,,;~AIion: Position 111.
Glutamic acid at position 111 was mutated to all amino acids by site-directed ,,, I..g~ ;c Single chain Fv extracts were analyzed by BIAcore. Percent change in off-rates is shown relative to P5Q.
Figure 4. Amino Acid pAIl~llllll;~Ali~n Position 112.
Aspartic acid at position 112 was mutated to all amino acids by site-directed mllrAA~n~cic Single chain Fv extracts were analyzed by BIAcore. Percent change in off-rates is shown relative to PSQ.
Figure 5. Additive Effect of Crmhinin~ Optimized Residues. A double mutànt' C~,..IA;~ Ig the optimized residues, was cu.-~l.u-,~d and analyzed by BIAcore. Percent change in off-rates i~
shown relative to P5Q.
Figure 6. Nucleotide and amino acid s~ql~n~s of scFv 25 psQ with c-myc tail-DETAILED DESCRIPTION OF THE INVE~TION
The gpl20 V3 domain of human immllno~lrri~ yvirus-1 (HIV-1) is a disulfide-linked closed loop of d~ u"u"ately 30 30 amino acids. The loop, in either native or synthetic form, binds tû
and elicits anti-HIV-1 antibodies.
The present invention relates to modified antibodies and methods of making modified. The invention is exemplified with modified HIV-1 immlmo~lobulins and methods of making these .......... _ .. _ . . .......................... ...... .... ....... ... ...... ... .... .

3 2 1~ ~ ~ g ~ P~ .4~L

modified HIV-1 imml-no~lobulims. The modified immlmoglobulins of the present inventi~n contaim an altered compl~ --~.y . t ",;";"~ region 3 (CDR3) of HIV-I nPlltrali7in~ antibody.
The present imYention also c~ a method of 5 treating of preventing infection through the ~1",;";~1"..;"" of a modified antibody to a suitable host. In one embodiment of the invention, the treatment or ~lcv~ iull of HIV infection through the a~--i--i~L alion of the modified HIV-l immunoglobulim is described.
The present imvention also co --,u.ises ~ gnocti- kits useful for the detection or char~t~ri7ati~n of an antigen. Reagents for the kits may include DNA molecules encoding the modified antibodies or the modified ~ntlho-ii~c or cu...bil.alions thereof.
A method of ml-t~g~ni7in~ ~ntiho~1i.oc to produce modified ?~tiho-lif-s, modified ~-lil,o-lics, DNA encoding the 5 modified ;",lil,û,l;~s as well as ~ gnoStir kits and ~ ""~r~"l;ral compositions Culll~uli~ g the ~l-lil,odics or DNA are provided. The method of the invention is a b~t~ l..dlic means to achieve in vi~ro antibody lllllluu~liiull and uses alanine scanning m~t~gt~nf-ciC The invention is particularly exemplified with a set of single chain Fv 20 (scFv) antihorli~s obtained by this Irl ~,,,i,lu~. The resulting ~ntiho~ s are directed against the V3 loop of HIV gpl20, and show altered off-rates against the antigen compared to the startimg antibody. Of particular imterest are the altered antibodies which sh~w improved (slower) off-rates to the antigen. Observed 25 hll~luvt;lll~llL~ have been as high as eleven-fold over wild-type.
h~ahlr~hnn was achieved through an alanine scan of complcll.~ uy l. r~ .",;";"~ region 3 (CDR3) to identify positions critical to antigen bimding. Critical positions were then r~n~l~mi7l~d to identify amino acids that provided the slowest off-rates. Finally, 30 clones were optimi_ed through the c--mhinin~ of mllt~ti( n~
The underlying principle of the method is the physical and chemical neutMlity of alanine. Alanine is c--hchhltPd throughout a stretch of amino acids, and its effects on binding (such as off-rate and on-rate) are evaluated using conventional methods. The number woss/23813 ~18~550 1~ 492

- 4 -of positions likely to be identified in this manner is relatively small.
Once i/1~ntifif rl these key positions may be r~n~mi7Pd to all amino acids to identify the best amino acid solution at the position. Because all manipulations and evaluations are ct-n~ rtf d in vitro,

5 physiological bias is limited.
Present methods of in vitro antibody maturation are essentially random procedures in which the l~,à~dULl~l generates clones with amino acid substitutions and evaluates them. The problem is that the number of ~ "l;,...~ necessary for a thorough evaluation is extremely large. For example, if one were to evaluate all random substitutions in CDR3, a region typically twenty-five residues in length, one would have to examine 9-1027 poc~ihiliti~.c This is beyond the rsr:lhilitif ~ of present technologies.
Alanine scamling IllaLuld~iull enables the rapid 5 i~lfntifi~ ~ri~n of residues most likely to be illllJolLall~ in binding.
Using the example of a twenty-five residue stretch cited above, only twenty-five a.lll,l;llll;lll~ would be l~CCCa~aly. From this initial screen, amino acid positions likely to be critical to binding may be irlfntififrl The critical residues may then be r~n~nmi7f~d to identify 20 the amino acids that optimize binding. Using this method, scFv ~ntiho~ s with dissociation rates greater than ten-fold slower than the original scFv have been created.
Previous work in in vitro antibody n-q~llr~ti,-n used one of two general d~,ul~L~cllcs. In one approach, PCR l~c~....h;~ n is 25 used to substitute all or part of the VH and VL genes into libraries of scFv clones. In the second approach, random mutations are made throughout a CDR region of ~ scFv clone by the use of lif.~. ..1. ..~.lf' olig~n~ f oti~f ~ In both cases, clones were expressed as a phage fd gene 3 fusion surface protein. Higher affinity clones were identified 30 using a panning assay followed by clonal pllrifir~ti-~n of the phage.
Each approach has dlav~/l)a~,ha. The PCR method is L.,."l-r.,,.. P, limited to the sf-qllfnres of the B cell population, is essentially random in nature, and may introduce unwanted ",l~ "~
through the PCR recombination step. The rqnrl~-mi7~tion app~ach W095/23813 ~183~5~ r~,l/L--_.'17492 produces only a small fMction of the possible CDR changes. Neither approach allows ;" Illlf~ Ir ~ ion of changes rn binding affinity because it is llCC,CS5~y to first generate an enriched population of suitable clones through panning. Both ~I -u~-;læs 5 detect only ch~nges which result in improved binding; they do not identify positions for which the change weakened the binding. The latter class of change may mclude critical binding residues in which the a~rU~ , amino acid solutions leads to iLIlJJluv~ lL.
The methûd disclosed herem is systematic, thorough and unlikely to introduce l-n~rf etf~d or undesired m--t~tilmc All m~nir~ hinnc are done in vitro, which Ill;ll;lll;~.r~ bias due to selection steps. E~valuation of clones is ~u~ liv~. In some cases, a key amino acid position may display poorer binding with alanine, but ran~ ll l may yield an amino acid solution which 5 enables improved binding. Such Illlll;.lillll~ would not be detected by previous methods. Because the method of the present rnvention does not require phage ~)lC~i~iUll for panning, the method can be used on scFVs, Fabs, and full length antibodies. Use is not reshicted to a scFv for phage C;~JI~ iUII. Using the approach of the present 20 invention, an anti-HlV V3 loop antibody was improved uXilllaL~ly eleven-fold.
Alanine scamling Illa~ iOll of ~ntiho~lif c is a general method which may be used to improve bindrng of ~nhhotlif~ to their cognate antigens. The method has been used to identify cribcal 25 residues in the scFv 447 which can be introduced into MAb447.
Such changes may lead to ~i~nifi~nt illl~lUv~ lll of the bindrng affrnity of MAb447 against mulbple species of HIV gpl20 isolates.
This ilIIpIuvt;III~,~Il may increase the nfI-tr~Ii7~ltion capability of the antibody, and ~i~nifir:mtIy lower the effecbve dose.
Although the method and anbbodies of the present invention are exemplified with scFv ~nhho~ , it is readily apparent to those skilled in the art that the method may be used with other types of anbbodies or with antibodies targetted against different wo 9sn3813 2 1 8 3 ;) ~

- 6 -epitopes or antigens. Other types of antibodies include but are not limited to fr~mPntc of antibodies and full-length antibodies.
The molecular biology and immunological techniques of the present invention can be performed by standard tP, l",i.~ well-known in the art. See, for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Mo~ecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).
Because the genetic code is ~ tld~, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA olignn--cl~oti~lP,c The cloned DNA molecules obtained may be expressed by clonmg the gene encoding the altered antibody into an expression vector cnnt~inin~ a suitable promoter and other alu,ulululi~lt~
l.~ls.;.i,ulion regulatory elements, and ,-d -~rt--~d imto prokaryotic or eukaryotic host cells to produce re~",.,~ modified antibodies.
Terhniq~-Ps for such m~lnir~ tions are well-known in the art.
In order to simplify the following FY~mrl~-s and the Detailed D~sc.iluLio--, certain terms will be defined.
EA~ iUII vectors are defined herein as DNA sPql~Pn~ es that are required for the ~IdllS~ liUll of cloned copies of genes and the translation of their mRNAs in an a,u,ulu,ul;~ host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells.
25 Expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
Spe~.ifi~:llly designed vectors allow the shuttling of DNA between hosts, such as bacteria-yeast or bacteria-animal cells.
DNA encoding ~ntiho~1iPc may also be cloned into an 30 expression vector for expression in a host cell. Host cells may be -uhll-yolic or eukaryotic, imcluding but not limited to bacteria, yeast, m~Tnm~ n and insect cells and cell lines.
The expression vector may be introduced into host cells via any one of a number of tP~hniqll~c including but not limited to W095/23~13 2 1 8 3~5 ~

- 7 -- transformation, tPnCf~e~inn, protoplast fusion, and el~ u~ lion.
Expression of cloned DNA may also be performed using in vitro produeed synthetie mRNA. Synthetie mRNA ean be effieiently tr~ncl~tPd im various eell-free systems, ineludimg but not 5 limited to wheat germ e~traets and reticuloeyte extracts, as well as efficiently translated in cell based systems, mcluding but not limited to ll~i~luillje~lion mto frog oocytes, with micro-injeetion into frog oocytes being preferred.
It is also well-known that there is a ~ amoumt redundancy in the various codons which eode for speeifie ammo aeids. Therefore, this invention is ~Iso direeted to those DNA
se-l~7rnrec which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this crerifir~tinn, a sequence bearing one or more replaced codons will 15 be defmed as a 11~ r variant.
The following examples are provided to further define the invention without, however, limiting the imvention to the particulars of these eY~n~
EXAMPLE I
Construetion of IlI-.l;lti~ .c Plasmid pP5Q was the starting vector for all mllt~nic studies. Plasmid pP5Q is a derivative of p5H7 (('~mhri(l~-25 Antibodies). Plasmid pP5Q contains the VH and VL regionsoriginally derived from MAb 447 (Gorney et al.) cloned as a simgle chain fragment variable (seFv).
Table I lists some of the olig- m-rl~ oti-l~ primers used for site-direeted mllt~nrcig of eomplementary ~rlrllllillill~ region 30 3 (CDR3) of MAb447. Primers were ~ylllll~ ,d on either a model 381A DNA Syll~ el (Applied Biosystems, Foster City, CA) or a CycloneTM Plus DNA Syll~ r (MilliGen/Bioseareh, Marlborough, MA). Mutagenesis was performed with the Tl~l~ lTM ~llt~Pnrcic Kit (CLONTECH, Palo Alto, CA) .. _ . . . _ _ . . . .. .

2 1 8 ~
WO 95/23813 - 5 ~ r ~ 492

-8 -according to the mqmlfqrlllrer's instructions. All mutations were verified by DNA s~q~l~nrin~ using the Sequenasei~ V2.0 DNA
Seqllt nrin~ Kit (United States Binrh~mirql, Cleveland, OH).
Table I
Primers:
Pqn~nmi7qtinn of position 107:
CTC GGA GAC TCC C/GNN AAT CAT AAT AAA
~qntlnmi7qtinn of position 111:
GTA GTA GTA GTC C/GNN GGA GAC TCC CCG
E~qn-lnmi7qfinn of position 112:
GTC GTT GTA GTA GTA GTA GTA C/GNN CTC GGA GAC

Pl~alaliull ûf extracts and BlAcore analysis of scFv Extracts:
~Ailltq.~eni7~d plasmids were introduced by C~ lul~ulalioll into bacterial strain Escherichia coli TG1 for iUII. Simgle colonies were inoculated into 10 ml of 2X-YT
(which contains per liter of water 16 g tryptone, 10 g yeast extract ar~d 5 g sodium chloride) supplemented with 2% glucose. Cells ~/ere 25 grown overnight at 30C with vigorous shaking, collected by centrifugation in a Beckman GPR ç~ntrifil~ at 2500 rpm, and re~llcr~n-1f.d in 10 ml of fresh 2X-YT supplemented with 1 mM
isopropyl-beta-D-thiogala-,~ul~ylalloside (IPTG) to induce expression.
Cells were incubated at 30C for an q.(l~litinnql 5-6 hours with 30 vigorous shaking, collected by c~ u~ ion, r(~ rçnrl~d in 1 ml of ~hn~rhqt~- buffered saline: ethylen~ A",~ t~ âcetic acid (PBS:EDTA; 10 mM sodium phosphate pH7.0, 150 mM sodium chloride I mM EDTA), and incubated on ice for 30 minutes to 21835~
W09~123813 - P~ '5~

release periplasmic proteins. Extracts were clarified by centrifugation and stored at 4C until use.
., , EXAMPT F. 3 Off-rate 11~ t~ .."i"~lifms of the scFv antibodies were 1~ t~ ,llil-rd using the BlAcore system (Pharmacia Biosenser). HIV
gpl20 V3 loop peptides, Al-l variant (Ala-l peptide) were covalently immobili_ed on a carboxylated dextran/gold matrix via the primary amino group. The carboxyl-dextran matrix was first activated with N-ethyl-N'-(3-diethyllllil,~plu~yl)carbonfiiimi~iP
(EDC) and reacted with N-hydroxylcllrrinimi~i~ (NHS). HIV gpl20 V3 loop peptides such as Ala-l peptide were covalently immnhili7Pd via the free thiol of a cysteine placed at the N-terminus. These 5 peptides were reacted with the EDC-NHS activated matrix which had been reacted with 2-(2-pyridinyldithio)~ . Rrm~inin~
unreacted NHS-ester groups were displaced by addition of ethanolamine. EDTA extracts were added in a flow passing over the immobilized antigen. The refractive index changes, in the form of 20 the surface plasmon resonance caused by the binding and subsequent ~ijc~oci tirln of the scFv, were monitored cnntiml~l~cly. Off-rates were r~lr~ trd from the d~ll.,.,.,.l;..Ally collected data using the Plldlllla~;s Kinetics Evaluation software.

Alanine scanning of CDR3 identifies residues which modulate scFv-antigen bin iir~
Alanine scanning ml-t~Pnplcic was used to identify 30 residues within the VH CDR3 region of scFv clone P5Q critical for binding. It was lly~ u~le~,d that effects on binding by alanine j,..l .~l ;l ,.~ ;. lll would lead to four broad classes of effect: class i) slower off-rate; class ii) faster off-rate; class iii) loss of binding; and class iv) minor or no change in off-rate. Class i) and ii) were wo gsn38l3 2 1 8 3 ~ ~ n ~ PCrllJS951024g2 Qpf r~tinn~lly defined as critical. Class iii) was defined as obligato Class iv) was defined as nrmf ritir~l ry The 27 positions that comprise VH CDR3 of scFv clone `' P5Q were individually changed to alanine by site-directed 5 mllt~f nf~cic rf ~ DIIlic extracts were prepared from the alanine replacement clones and assayed for off-rate A~lr~ onC against the AL-1 gpl20 V3 loop peptide (Fig. 1). Alanine substitutions at positions 107 and 111 resulted in 1.7 and 2.7 fold illl~JlOVt;lll~llLD in off-rate, I,_DI,e.;~ively. These positions (class i) were judged critical and sllhsçq~lf~ntly r:tn~lnmi7fd to identify optimal residues. Alanine s~hstitlltir)nc at positions 102, 112, 113, 114, and 118 led to faster off-rates (class ii); two of these positions were selected for further evaluation. Alanine substitution atpositions 98,101,115,116,117, and 121 resulted in no binding (class iii). Alanine s--hctitlltion at the 15 remaining fourteen positions had only a min~r effect on the off-rate (class iv). The class iii and iv positions were not evaluated further.

20 ~ nA~mi7 ition at critical positions to identify optimal amino acid solutions The two critical class i) positions (107 and 111) were individually l~.,A-.",i,. d to all amino acids, and off-rates against the AL-1 peptide ~lf t .",i"rA In addition, two class ii) positions (112 25 and 118) were also selected for r~nAf~mi7~tion studies.
The results for position 107 are shown in Fig. 2. The slowest off-rate was observed with the negatively-charged glutamic acid, which decreased dissociation 2.5-fold. ~llh~tihlti-f)n of other polar and charged amino acids had no ~i~nifir~nt effect on 30 .l;~c~ " With the exception of alanine, ~llhstitlltion with hydrophobic amino acid resulted in complete loss of binding. These results are consistent with the preprlnAfAr~n~e of surface ligand-contact residues being hydrophilic.

` ~83~0`

~ ~n~lnmi7~*nn of position 111 (Fig. 3) showed that the aromatic residues tyrosine and ~lyyl~pLIl prnduced the slowest off-rates (~ oci~tinn rates decreased 4.2 and 4.7-fold, ~e~Liv~ly).
However, sllhstitllti~n with any l~ydlolJhOl)ic amino acids increased 5 affinity relative to wild-type clone PSQ.
Class ii) positions 112 and 118 (faster off-rate upon alanine ~ Stitlltinn) were also selected for amino acid ,,..,~i..",;, .l;n~ Forboth position 112 (Fig. 4) and 118, the residues present in the original scFv PSQ, aspartic acid and ~r~r~ inf were the best solutions-Im~rovements at positions 107 and 111 are additive A double mutant that combined the optimized residues at positions 107 (E) and 111 (W) was c~ Llu~ d to rlf,t -Ill;ll` whether or not the individual illl~ v~ ,llL~ are additive. Figure 5 shows that the double mutant has an off-rate 9-fold slower than wild-type clone PSQ. The off-rate value ~~ ,Ailll~t~,s the product of the fold illll,l.,v~.ll"ll~ observed with the individual optimized residues (2.5 for 107E and 4.7 for 11 lW). One i lL~ Lion of this result is that for these two positions, the C~ to scFv-antigen affinity are in~lf p~n~f nt and additive.
~XAMPLE 7 Mf thod of makin~ modified antibodies An antibody is mutagenized by alanine scanning mllt,~ nf ciC to produce a modified antibody. The binding of the 30 modified antibody to its antigen is .1~ I~..lll;llP~I Binding f.t~ .",;~".l;nn~ may be made by conventional methods and include off-rate IllC~ lc;lllc;llL~. Modified antibodies havmg desired t~ l;( S are selected and Ill~ ;llrrl WO95/23813 2183550 P~ g!

Method of using modified antibodies The modified Antiho~1je5 or r~ ",~ ;rAI compositions 5 thereof are used for th~ ic or ~ a~ iC treatment of diseases caused by their antigen. Methods of treatment include, but are not limited to, intravenous or i~ dpc~ ,al injection of the modified antibody.
o EXAMPLE 9 Di~nn~ti~ kit employing modified antibodies The modified antibodies of ExaTnple 7 are used as reagents in iiA~nnstir kits. The modified antibody reagents may be 5 further modified through tPrhni~ which are well-known in the art, such as radiolabeling or enLyme-labeling. The liq~nc-stir kit mây be used to detect or ~ ;t~liL~ the antigens.
F~xAMpLE 10 DNA encoding tnodified ~.~il,odi~s The DNA encoding the modified antibody of Example 7 is used as a reagent for the production of modified antibodies. The DNA may be incorporated into an expression vector. The 25 expression vector may be used to transform a host cell. Cultivation of the host cell under cnn-1itinn~ suitable for the expression results in the production of modffled antibody.

w09s~?,8l3 21 8 3S5 0 P~ ' '192 DNA encodin~ modified antibodies The ~NA encoding the modified antibody of Example 7 5 is used to detect DNA encodmg the antigen in test samples. Methods of detection include, but are not limited to, llyl~ inn under selective con~ ion~ Test samples include, but are not limited to, samples of blood, cells, and tissues.

Preparation of mntlifil~d li~ht chain ;mmlmnFlobl]lin~
The light chain of an i",...,.l,~,L,Inbulin is mllfsl~ni7~.d by alanine scamling mllt~ n~ ciC to produce a modified immlmnglnblllin 5 having modified binding ~ c~.i~ s. The modified immuno-globulin is used as a reagent for ~ gnn~tic kits or as a 11- ,.l,c"li~
agent.

WOgS/23813 ~ ~ 8 3 ~ ~ P~ 1492 .

SEQUENCE LISTING *
~.
( 1 ) GENERAL INFORNATION ~
i) APPLICANT: LEWIS, CRAIG M.
LUDMERER, STEVEN W.
HOLLIS, GREGORY F
(ii) TITLE OF INVENTION: IN VITRO A-NTIBODY NATURATION
(iii) NU~BER OF SEQTJENCES: 2 (iv) U~;~ONU~N~k; ADDRESS:
(A) ADDRESSEE: CHRISTINE E CARTY
(B) STREET: P.O. BOX 2000, 126 E LINCOLN AVENUE
(C) CITY: RAHWAY
(D) STATE: NJ
(E) COUNTRY: USA
(F) ZIP: 0706S
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC comp~tible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: P~tentIn Release #1 0, Version #1 25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION N~qBER: US 08/206, 079 (B) FILING DATE: 04-MAR-l99 (C) CLASSIFICATION:
(viii) ATTORNEY~AGENT IrlFORMATION:
(A) NANE: CARTY, CBRISTINE E
(B) REGISTRATION NUMBER: 36,090 (C) REFERENCE~DOCKET NUNBER: 19190P
(ix) ~T~ r ~ rIoN INFORNATION:
(A) TELEPHONE: (908) 594-6734 (B) TELEFAX: (908) 59~-~720 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE ~ Rr~"l~R~CTICS:
(A) LENGTH: 816 ~lse p~irs (B) TYPE: nucleic acid (C) s~rRr~ m~ single (D) TOPOLOGY: line~r (ii) MOLECULE TYPE: DNA (genomic) 218~5S~
WO 9~/23813 - r~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GCCATGGCCG AGGTGCAGCT GGTw-AGTCT rrrrr~-~r~r TGGTAAAGCC L~i~iWWll ~ 60 CTCAGACTCA CCTGTGTAGC ~ l~L~ lL~ ACGTTCAGTG ATGTCTGGCT GL4ACTGGGTC 120 rrrrPr~r,r~r rPrr.r7~7lr.rr. GCTGGAGTGG ~ l~ TTAaaAGCGC CACTGATGGT 180 rnrar~Prpr~ ACTACGCTGC ATCCGTGCAA GGCAGATTCA CCATCTCAAG AGATGACTCA 240 Dpap7~rprnr TATA'L~ iL.D~ AATGAATAGC CTGAAAACCG ar~a~prpnr (7111rLLl-~, 300 TrrParPrPr- ATGGTTTTAT TATGATTCGG GGAGTcTccr~ AGGACTACTA r~arrrprTpr 360 AACGACGTTT r~rr~rP~ r~ GACCACGGTC ACCGTCTCCT CAGGTGCAGG CGGTTCAGGC 420 GGAwTGGCT ~ L~i~Wl~i CwATCGCAG l~L~ lL~ L rnrPrrrr~rr CTCAGTGTCT 480 Grr~rrrPr GACAGAArGT CACCATCTCC TGCTCTGGAA GCAGCTCCAA CATTGGGAAT 540 AATTATGTAT TGTGGTACCA GCAGTTCCCA rrl~arPnrrr CCAAACTCCT CATTTATGGC 600 paTPa~ rr GACCCTCAGw GA~rTCCTGAC CGATTCTCTG GCTCCAaGTC TWCACGTCA 660 GCCACCCTGG GCATCACCGG ACTCCAGACT r~r~r~a~nar~r CCG~LTT~TTT CTGCGCAACA 720 TGwGATAGCG GCCTGAGTGC TGATTGGGTG TTCGGCGGAG rrarrP~IrrT GACCGTCCTA 780 w~i r~r7~ArP~71a ACTC~LTCTCA GAAGAG 816 (2) INFORNATION FOR SEQ ID NO:2:
(i) SEQUENCE ruavprTRRTcTIcs (A~ LENGTH: 272 amino acids (B) TYPE: ~mino ~cid (C) SlrR~ cs: single (D) TOPO10GY: line~r (ii) NOLECULE TYPE: protein (xi) SEQUENCE l~;a~l~ : SEQ ID NO:2:
l~ ~et Al~ Glu Val Glx Leu V;L1 Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Ar~ Leu Thr Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Val Trp Leu Asn Trp V~l Arg Gln Al~ Pro Gly Ly~ Gly Leu Glu Trp V;L1 Gly Arg Ile Lys Ser Al~ Thr A~p Gly Gly Thr Thr Asp 21835~G
WO 95/23813 r~ 492 Tyr Ala Al~ Ser V~l Gln Gly Arg Phe Thr Ile Ser Ar '~Asp Asp Ser 65 70 75 , 80 ys Asn Thr Leu Tyr Leu Glx Net Asn Ser Leu Lys Thr Glu Asp Thr 85 90 g5 l~ Val Tyr Ser Cy~ Asn Thr Asp Gly Phe Ile Met Ile Arg Gly V~l Ser Glu Asp Tyr Tyr Tyr Tyr Tyr Asn Asp Val Trp Gly Lys Gly Thr Thr V~l Thr Al~ Ser Ser Gly Al~ Gly Gly Ser Gly Gly Gly Gly Ser 13 0 135 14v Gly Gly Gly Ser GIn Ser V~l Leu Thr Gln Pro Pro Ser Val Ser Ala 145 150 . 155 . 160 la Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser A~n le Gly Asn Asn Tyr V~l Leu Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Asn s Ar Pro Ser Gly Ile Pro 195 200 Ly g 205 Asp Arg Phe Ser Gly Ser Lys Leu Leu Ile Tyr Gly Al~ Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly Asp Gln Ala As r Phe C s Ala Thr 225 230 p Ty Y 240 rp Asp Ser Gly Leu Ser Ala Asp Trp V~l Phe Gly Gly Gly Thr Lys eu A~r V~l Leu Gly Al2 Al~ Al~ Glu Gln s Leu Ile Ser Glu Glu 260 265 Ly 270

Claims (12)

WHAT IS CLAIMED IS.

1. A DNA molecule encoding a modified antibody, the modified antibody being derived from a native antibody by alanine scanning mutagenesis and the modified antibody having binding characteristics different than binding characteristics of the native antibody.

2. The DNA molecule of Claim 1 wherein the native antibody is MAb447.

3. The DNA molecule of Claim 2, the DNA
molecule being selected from the group consisting of P5Q, DNA
encoding modified antibodies of Figures 1, 2, 3, 4, 5, combinations thereof, derivatives thereof and degenerate variants thereof.

4. A method of modifying an antibody to make an modified antibody comprising replacing at least one amino acid of the antibody with alanine to produce a modified antibody.

5. The method of Claim 4 wherein the modified antibody has improved binding characteristics.

6. Modified antibodies produced by the method of Claim 4 or homologues thereof.

7. The method of Claim 4 wherein the antibody is MAb447.

8. The method of Claim 7 wherein the amino acid replaced with alanine is located in complementary determining region 1, complementary determining region 2 or complementary determining region 3 of MAb447.

9. The modified antibodies of Claim 6 selected from the group consisting of P5Q, the antibodies of Figures 1, 2, 3, 4, 5, combinations thereof, derivatives thereof, and homologues thereof.

10. Diagnostic kits comprising the modified antibodies produced by the method of Claim 6.

11. Diagnostic kits comprising the DNA molecules of Claim 1.

12. A pharmaceutical composition comprising at least one modified antibody of Claim 6 or DNA encoding at least one modified antibody of Claim 6 or combinations thereof.