Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TUMOR-SPECIFIC ANl~ODY FRAGMENTS, FUSION
PROTEINS, AND USES l ~EOF
The present applic~tion is a conlirl ation-in-part of U.S. patent applications
serial llu-llber 08/331,396, 08/331,397, and 08/331,398 all of which were filed on
October 28, 1994 and aU of which are co~ u~l;on~ in part of U.S. patent application
07/767,331, filed on September 30, 1991 which is a continu~ti~n-in-part of U.S. patent
application serial number 07/596,289 filed on October 12, 1990, all of which are hereby
incol~lated herein by reference for all purposes.
BACKGROUND OF THE INVENTION
The subject invention relates to tumor-spPcific reconlbinallt antibody
fr~gmPnt~, to molecules incol~l~ling such fr~gm~.nt~ such as immlmQtoxins and to uses
thereof. FY~ emb~l;.. fnl~ of the invention include i.. ~-otoxins comprising
Pseudomonas exotoxins fused to the Fv regions of monorlon~l antibodies B1, B3, and B5
which have tumor sper-ificity and which may be used in the tr~tm~-nt of m~mm~ n
cancer.
Mc-n~lnn~l antibodies Bl, B3, and B5 are r~cel tly i~l~t~d murine
antibodies d~ecled against a c~ohydl~ antigen in the LewisY (LeY) family (Past~n et
al. Cancer Res., 51: 3781-3787 (1991)). The Le" antigens are found on the surface of
many mucinous carcinomas of the colon, stomach, ovaries, breast, lung as well as some
epidermal carcinomas. R~ e they react with only a limited number of normal tissues,
these antibodies are ideal c~n~ tes for use in the tre~tmPnt and tii~gnn~iS of cancer.
In order to create a ~lot~ ic agent that specifically attacks cancer cells, an
antibody or its fr~gmPnts may be used as the ~geling moiety of an immlmotoxin. In
such immllnotoxins, the ta~geling moiety t-ypically replaces the cell binding domain of a
.;~loto~ in molecule (e.g. domain I of Pseudomonas exotoxin (PE) or the B chain of
Diphtheria toxin) and acts to spe~ifiç~lly direct the cytotoxin to its target cell (as
det~rminPd by the s~-ificity of the large~ing moiety). As a result, only cells which are
recognized by the ~~eling moiety are efficiently killed and cells which are not
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recognized are spared (for a review see Rrinkm-nn and Pastan, Biochem. Biophys. Acta.,
1198: 27-45 (1994)).
Tmmlmntoxins were first made by chPmi.-~lly coupling antibodies to
~;~lot~Aic mnl-c-llPs Thus, for eY-~mpl-;, mnnnrl~m~l antibody B3 has been chPmir--lly
S coupled to at least two different forms of Pseudomonas exotoxin (PE) (IJ.S. Patent
4,545,985). One of these is the full length toxin (PE) and the other a trunr~t~
d~iv~liv-e (PE40) (Kondo et al., J. Biol. Chem., 2~3: 9470-75 (1988) and Pai et al.,
supra). Both of these h.. ~ otu~;n$ have been shown to be seleclivcly C~toAic to
tumor cells that contain the B3 antigen on their surf~re, and these immlmotoxins have
been shown to cause complete tumor lcgr~s~;on in mice bearing human tumor xenografts
(Pai et al., Proc. Natl. Acad. Sci. USA, 88: 3358-62 (1991)).
~lthough rhpmir~lly coupl~ immlmotoxins are useful they have several
nn-lçcir~hle ~,lu~llies. For eY~mple, the ch~.mir~l m~lifir~ir)nc can change the antibody
and affect its binding to the ~ntigPn. FurthPrmore, the purified immlmotoxinc are a
h~t~ugencous ll~lu~c of antibody-toxin moleculPs col-n~c~ to each other via different
positi~ns on the antibody and the toxin. Thus, Pseudomonas eYotoxin~ for eY~mple, can
be coupled either to the light- or heavy-chain of the antibody and to different positionc on
each of these chains.
To overcome the limit~tion.c of ch~Pmic~lly conjugat~d immunr~toxins~
chimPric imml)notoxins have been made as recombinant, single chain, antibody-toxin
fusion proteins. It has been shown that certain single chain antigen binding proteins
made from the Fv portions of the heavy and light chain of antibodies held together by a
polypeptide linker can have the same binding pro~cllies as their full length two chain
coun~~ (Bird et al., Science, 242: 423-26 (1988) and Huston et al., Proc. Natl.
Acad. Sci. USA, 85: 5879-83 (1988)). It has also been shown that, in some cases, fusion
proteins composed of single chain antibodies linked to toxins may retain the binding
ca~cily of the single chain antibody as well as the activity of the toxin (Ch~u-lh~ry et
al., Nature, 339: 394-97 (1989); Batra et al., J. Biol. Chem., 265: 15198-15202 (1990);
Batra et al., Proc. Natl. Acad. Sci. USA 86: 8545-8549 (1989); C~h~ lh~ry et al., Proc.
Natl. Acad. Sci. USA 87: 1066 1070 (1990)).
Receptor proteins have often been used as immlmotoxin targets because
they are cell surface proteins which are often ovc~cA~l~ssed in various cancers
(Rrinkm~nn and Pastan, Biochem. Biophys. Acta., 1198: 27-45 (1994)) and thus provide
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cancer-~perifir, targets. For eYqmrle, single chain immlmot(~xins have been madecon~isting of the Fv domain of an antibody directed at the int~Prle~lkin 2 ~p~r
(('.hA~ lhqry et al., Nature, 339: 394-97 (1989) and Batra et al., J. Biol. Chem. 265:
15198-15202 (1990)) or at the transferrin loeeptor (Batra et al., Proc. Natl. Acad. Sci.
USA 86: 8545~9 (1989)) fused to tr~mr-q-tP~ forms of PE or ~iphth~.iq toYin (Çhqlltlhqry
et al., Proc. Natl. Acad. Sci. USA 87:9491-94 (l990)). ~lthough lcce~tor pr~teins are
o~ p,~ ~sed on many c. ncers, they may still be present on healthy cells and therero~
often do not provide the defined cancer specificity desired for an immlmntoYin.
Since the number of antibodies that react prererenlially with c_rcinomas is
limite~, the irlpntific-qtion and chqrqrtPri7~tin~ of Ad~itionql "cancer spe~ificll _ntibodies
tbat would react with all or most of the cells in a tumor and with relatively few normal
cells and tissues is desirable. In ~d~lition, recomhinAnt i,..",l~notoxins are known to
degrAde over time both in vitro and in vivo. It would be desirtq~ to obtain
i."."~inotoxinS that show a reduced rate of degr~lAti~n and th~iefore require less r,~uenl
15 ~mini~tr~Atj~n. Finally, with repeAted use, murine Antiho~i~os and fusion p,oleins
c4nt~;l-ing murine antibodies, like any other foreign protein, may ultimAt~ly prove
i.""...nogenic and invoke an immlme lesponc~, in the treated or~ni~m. It would be
desirable to pr~luce lar~:li.lg moieties and imm-~notQyinS having reduced Antig~onir
polc~al. As will be eYpl~ined herein, these advantages and others are provided by the
present invention.
SUMMARY OF THE INV13NTION
The present invention provides for recombinant single chain Antiho lies and
fusion proteins such as illllllllnoto~ns employing these antibodies. In particular, this
invention provides for reco.,-billanlly produced antibodies comprising the variable light
and heavy (Fv) chain regions of antibodies that have the binding specificity of
monoclonAl antibodies Bl, B3, or B5. These antibodies provide carcinoma-specific;e ing moi~PtiPs suitable for use in CylOlOAiC fusion proteins.
In one embo limpnt this invention provides for single-chain antibodies
~ 30 comprising an Fv region of both the variable light (VL) and variable heavy (V~ chain
regions of an antibody where the single-chain antibody has the binding sp~ificity of
mono~lonAl antibodies Bl, B3, or B5. Particularly p~ led are single chain antibodies
Bl(Fv), B3(Fv), and B5(Fv).
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In allo~ emho~iment, this invention provides for single-chain antibodies
comrricing an FV region in which position 95 of VH is 1II~ to a serine when it is not
normally a serine. The single-chain antibodies may be c~l,ohydldte-binding antibodies
and more plcf~ bly are LeY-binding ~ntibo~ies having position 95 of VH ~ ~ to a
S serine. In most cases position 95 will be a tyrosine before mllt~tion. Thus a particularly
plcf~llcd antibody is BS(FV): Y95S, ~es~riheA herein.
This invention also provides for single-chain B3 antibodies having various
mutations that increase the stability of the antibody. Particularly plef~ d mut~tionc are
in the VL chain and include a mnt~tion of mrthiQninP to leucine at position 4 (B3(FV):
10 M4L, or a ml~t~tinn of serine to ll~conine at positi~n 7 (B3(FV):S7~ or the comhin~tion
of both mllt~tionc(B3(pV): M4L S7T).
In another embo lim~nt, this invention provides for c~im~ri~ single-chain
antibodies compricing a variable heavy chain of a first antibody and a variable light chain
of a second antibody where the first and second antibody are dirr~ antibodies and the
15 heavy and light chain are recombinanlly fused to form a single-chain antibody which
spe~ifi~ally binds a LewisY carbohydrate ~ntig~on, In a particularly pr~f~lcd
emho~liment, the single-chain antibody has the binding s~ificity of mo~orlon~l antibody
B1,B3, or B5. In one e~.hc~;...çnt, the first antibody is B1,B3, or B5. In another
embo~lim~-nt, the second antibody is B1,B3, or B5. Particularly pl~,f~lt;d single chain
20 ~ntiho lies include BSVH~B3VL and B3VH_B5VL.
This invention also provides for recombinantly produced humqni7~ single-
chain antibodies comrricing h~ ni7~d variable light and heavy (FV) regions of
~ntibolies that have the binding speçificity of monorlc~n~l antibody B1,B3 or B5. These
antibodies provide c~c~ o",a-~ific ~E,~t;ng m~ es suitable for use in CylOl(~uC
25 fusion proteins. Particularly plef~lled are hnm~ni7~d single-chain FV regions of Bl, B3
or B5.
In one embo~lim~nt~ the single-chain antibody is a hum~ni7~d B3(FV).
Particularly preîelled is an antibody compricing a hum~ni7~A variable heavy chain, more
specifically a hu m ~ni7~d variable heavy chain having the amino acid sequence desi~n~t~d
30 ~umB3VH in Figure 11. Another plc;fe~l~d variant is an antibody comprising a
h~ ni~d variable light chain, more spe~-ifin~lly a hl-m~ni7~d variable light chain having
the amino acid sequence decign~tçd HumB3VL in Figure 11. Yet another pl~fe.led
hllm~ni7~d antibody is one comprising both a hnm~ni7~A variable light chain and a
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s
hl....~ni7P~ variable heavy chain. Particularly l~efel~d is an antibody comI~ri~ing a
hl)mqni7~ variable heavy chain having the amino acid sequence de~ n~t~~ mR3VH inFigure 11 and a hu---~-; ~ variable light chain having the amino acid S~ Uc~
d~ nqt~l ~umR3VL in Figure 11. Still yet another plcf~lcd hllm~ni7~ ~ntibody is
S one comprising a h~ d variable heavy chain having the amino acid sequence
de~ n~t~ mR3VH in Figure 11 and a hum~ni7~d variable light chain having the
amino acid sequence desi~n~ted ~llmR3VL in Figure 11 in which the serine at the
poCitirm ~e~ nate~ as 82b in Figure 11, is replaced with arginine.
In any of the single chain antibodies described above, the variable heavy
10 chain region and the variable light chain region may be joined by a linker. One
particularly plcr~lcd linker is (Gly4Ser)3.
This invention also provides for single-chain fusion proteins incûl~l~ g
any of the above~l~ribe~ single-chain ~ntibo~ os~ The fusion proteins comprise the
single chain antibodies recombinar~tly fused to an erreclor mol~ul.o. The err~r
15 mo!~ may be a Cy~ l such as Pseudomonas exotoxin and more preferably is
either PE38, PE40, PE38KDEL, or PE38REDL.
In a particularly ~ f~l~d e-..bo~;l..Pnt, the Fv region is a Bl(Fv), a
B3(Fv), or a B5(Fv) region or any of the mor~ifi~d Bl(Fv), B3(Fv) or B5(Fv) regions
described above. Thus, plcr~lcd fusion pluleins include B3(Fv)-PE38, B3(Fv)-PE40,
B3(Fv)-PE38KDEL, B3(Fv)-PE38REDL, Bl(Fv)-PE38, Bl(Fv)-PE40, Bl(Fv)-
PE38KDEL, Bl(Fv)-PE38REDL, B5(Fv)-PE38, B5(Fv)-PE40, B5(Fv)-PE38KDEL,
B5(Fv)-PE38REOL, B3(Fv): S7T-PE38, B3(Fv): S7T-PE40, B3(Fv): S7T-PE38KDEL,
B3(Fv): S7T-PE38REDL, B3(Fv): M4L-PE38, B3(Fv): M4L-PE40, B3(Fv): M4L-
PE38KDEL, B3(Fv): M4L-PE38REDL, B3(Fv): M4L S7T-PE38, B3(Fv): M4L S7T-
PE40, B3(Fv): M4L S7T-PE38KDEL, B3(Fv): M4L S7T-PE38REDL, B5VH-B3VL-
PE38, B5VH-B3VL-PE40, B5VN-B3VL-PE38KDEL, B5VH-B3VL-PE38REDL, B3VH
B5VL-PE38, B3VH-B5VL-PE40, B3VH-B5VL-PE38KDEL, and B3VH-B5VL-PE38REDL,
HUMB3(Fv)-PE38, HUMB3(Pv)-PE40, HUMB3(Fv)-PE38KDEL, HUMB3(Fv)-
PE38REDL, B5(Fv): Y95S-PE38, B5(Fv): Y9SS-PE40, B5(Fv): Y9SS-PE38KDEL and
B5(Fv): Y9SS-PE38REDL.
The fusion proteins may also include a linker b~lw~n the variable heavy
(V~ and the variable light (VL) chain regions of the Fv fr~gmPrlt One p~ef~lled linker
is the peptide linker (Gly4Ser)3. The fusion proteins may also include a cQ~nP~
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~lwe~n the Fv region and the ~rreclor molecu1P A particul. r pler~ nn~ll~r is
SGGPEGGS.
All of the embo~imPnt~ described above are recombin~llly t;Apr~ss~d as
single chain fusion proteins. Thus, this invention also provides for recomhinqnt DNA
5 m~l~culPs that encode any of the above-described single-chain antibody Fv regions and
fusion pf~t~ins.
In another emho;~ t this invention provides for a ~h~r",~xul;rql
co."pGsiLi,on compri~ing any of the single-chain fusion proteins described above in a
COI~ .*l;Qn suffiri~pnt to inhibit tumor cell growth together wi,th a phqrmqreutirqlly
10 acceptable carrier.
This invention ~imilqrly provides for a method of killing or inhibiting the
growth of cells bearing a LewisY antigen in a patient. The method incl~d~Ps the steps of
~~mini~t~ring to the patient a pharm-q-r~ut,irql composition compri~ing any of the fusion
proteins desc,ibed above in an amount sl~ffiri~nt to kill or inhibit the growth of the cells.
This invention also provides for a method of de~!;~g the pl~nc~ or
absence of a cell bearing a LewisY carbohydlate antigen in a patient, the methodcompri~ing the steps of removing a tissue or fluid sample from the patient, adding any of
the single-chain antibodies ~leseribe~l above to the c~mplP, and dete~ting for the presence
or ab~nce of a binding co rleY b~l~n the antibody and the ~ntigPn
In yet another e-.. bo l;~e~t~ this invention provides for a method of
illlp~ving the binding affinity of antibodies that lack a serine at position 95 of the VH
region. The method includes the step of replacing the amino acid at position 95 of VH
with a serine. The antibody is preferably a carbohydrate-binding antibody and even
more preferably an anti-LeY antibody. The amino acid to be mutated at position 95 of
25 VH will, in most cases, be a tyrosine.
BRIEF DESCRIPIION OF THE DRAWINGS
Figure la illustr~tes the strategy for the cloning of the heavy and light
chain Fv genes of monor4 n~l antibody B3 and the construction of ~ ession vectors
30 (e.g., pl~mi~s) for the c;~-pl~ssion of B3(Fv) immunotoxins. The cloning ~llategy is a
variation of that previously described (Ch~udh~t~ et al., Proc. Natl. Acad. Sci. USA, 87:
1066 70 (1990)). The pl~mi~ pVC38H, which is used as a vector for construction of
otoxins from heavy and light chain Fv regions, cont~in~ an NdeI and a ~in~TTT
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ecognilion sequence pr~ g the PE40 gene (Ch~ lh~ry e~ al., supra (1990)). (*)
in-lic~tPs a PCR-gencl~ted mutation and was l~p~l~ by site dil~c~d mut~en~cic; (L)
in~iç~s the region enco-ling the (Gly4Ser)3 linker (Sequence ID No. 32) which serves to
join heavy and light chains of the immllns)tosin.
S Figure lb shows the construction LMB7, the i.l.. ~ otc,~ B3(Fv)-PE38
with a "C3 com eclol" belween the Fv region and the PE38 ~;~ ~toAill.
Figure 2 shows the nucl~Qti~e sequences enr~ing the heavy and light
chain Fv region of monocl~n~l antibody B3 (Sequence ID No. 33). (a) The heavy chain
Fv coding region eYt~n~c from position 30 to 383, the light chain Fv gene from position
433 to 767 and the linker from 384 to 432. The d~uc~l amino acid sequence is shown
in plain letters (Sequence ID No. 34); below in italic letters is the protein sequence
del~ ~ by Edman sequenring of the antibody. The first amino acid enr~ded by the
cloned heavy chain Fv gene is Asp instead of Glu due to the oligonl~c1~Qtide primer used
at position 456-465. This is the region where the PCR cloning artifact was repdil~d.
15 This sequence encodes the same amino acids as the ori~in~l B3 light chain gene but uses
other codonQ Homology coll~p~;Q-ons to the known nucleotide sequence of PACT Ig
kappa chain ~aub et al., J. Biol. Chem., 264: 59-65 (1989)) which is most homologous
to the B3 light chain in-lir~tes that the origin~l sequence was most probably
~-l~-l~CCTG (Sequence ID No. 37) instead of ITGAGTTTA (Sequence ID No 38).
20 Thus the natural B3 light chain gene has a sequence repetition
5-(CCAGTCT[CC)ACTCTCC]-3' (Sequence ID No. 39) belween po~itiQnC 445 and 465
which is responsible for the incorrect primer ~nn~ling in PCR. (b) Sequence at the
3'-end of the light chain for eA~ ion of the single chain B3(Fv) alone (Sequence ID
No. 35 and amino acid sequence Sequence ID No. 36). (SD) - Shine Dalgarno conQ~n25 sequence; (*) - tr~nQl~tion stop signal. (Term) transcription ~ lor.
Figure 3(a) r~nts the toxicit,v of B3'(Fv)-PE38KDEL on different cell
lines. C,vtotoxicity assays were pelro,-l,ed as described in FY~mI)le 7. (b): Inhibition of
the C~lotoAicity of B3(Pv)-PE38KDEL by monoclonal antibody B3. Co...~elilion by
monoclonal antibody B3 was performed on A431 cells as described in F ~mI~le 7.
Figure 4 shows the ADP-ribosylation and cytotoxic activities of Bl(Fv)-
PE38, B3(Fv)-PE38, B5(Fv)-PE38 recombinant imm~motoxins. (A) ADP-ribosylation
activity was determined by the inco,~,~ion on l4C-NAD into acid-yre~ Ahle m~teri~l
using elong~tion factor 2 enri~l wheat-germ extract (Collier and K~n~el, 1971). (B)
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CytotoYicity ~v~uds A431 cells was measured by the inhihition of incol~.dtion of3H-leucine into cell protein, following 2 hours (open symbols) or 20 hours (solid
symbols) of inrub~ti~n of the cells with serial ~iluti5)nc of immunotnyinc in PBS + 0.2%
BSA.
Figure 5 shows antigen binding of Bl(Fv)-PE38, B3(Fv)-PE38 and
BSlFv)-PE38. Antigen binding was e,~ .A~d by con,pelil;r~n of tl25Il-Bl IgG (A) or
tl25Il-B3 IgG (B) binding to A431 cells at 4~C.
Figure 6 shows stability data for Bl(Fv)-PE38, B3(Fv)-PE38, and
B5(Fv)-PE38. The immlmot~yinc were diluted in PBS to 0.1 mg/ml and in.;ub~l~d at37~C for 4 hours. (A) The m~l~c~ r forms of the immllnotoxinc were than analyzed by
size eYclllQ;on chr~llatoEla~ly at 4~C. The monomer peak elutes at 18-20 ml, while the
aggrcgat~s elute at 11-13 ml. Chl~,l,a~l~l~s of the proteins prior to in~ub~tinn at 37~C
are shown by broken lines. The proteins after the in~u~ n at 37~C are shown by solid
lines. (B) Cytotoxic activity of i~ o~ins before (open symbols) or after (solid
symbols) incub~tion at 37~C. Other details are as in Figure 5(B).
Figure 7 shows blood levels of B3(Fv)-PE38KDEL in mice. Balb/c mice
were injected intravenously with 10 ~g of B3(Fv)-PE38KDEL and immnnotoxin levelswere measured at different time perio~s~ Bars ind~ t~ the standard deviation.
Figure 8 illustrates the effect of B3(Fv)-PE38KDEL on the growth of
A431 tumors in nude mice. Mice were injected with 3 x 106 A431 cells on day 0 and
treated begi,-ning on day 4 with inllavenous injectionc every 12 hrs x 6. A: (O)unt~aled; (--) 10 ~g B3(Fv)-PE38KDEL;B: (O) 2.5 ~g B3; (--) 5 ~g
B3(Fv)-PE38KDEL; C: (~) 2.5 /lg anti-Tac (Fv)PE38KDEL; ( ~ ) 2.5 ~ug
B3(Fv)-PE38KDEL; (--O--) 0.5 ~g B3(Pv)-PE38KDEL; D~ -nt began on day 7
with intravenous injectionC every 12 hrs x 8. (O) unlleated, (--) S ~g
B3(Fv)-PE38KDEL. Bars = 1 standard deviation.
Figure 9 illustrates the construction of pl~cmitlc for ~;A~lession of B3-B5
c~im~-ric Fv single chain immunotoxins. L in~ tes the (Gly4Ser)3 linker which connects
the VH to the VL in the single-chain Fv configu-~ti~n.
Figure 10 shows the ~ oluAic activity of immunotoAins B3(Fv)-PE38,
B3VH-B5VL-PE38 and B3(Fv)-PE38: VL M4L S7T following in- ub~tion in PBS at 37~C.A431 epidermoid carcinoma cells were incub~ted with aliquots of the immlmotoxinc
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which were diluted in PBS +0.2% BSA following incubqtinn at 37~C. 3H-T PucinP was
added 20 hours after ~litinn of imml)notoxins.
Figure 11 illlJstr~t,Ps the hllmqni7~qtiol~ of B3(Fv). .AlignmPnt of the amino
acid sequences of (A) B3 VH, 56P1'CL VH and HUMB3VH (Sequence ID Nos. 45, 46
and 47) (B) B3 VL, GM607 VL, and HUMB3VL (Sequence ID Nos. 48, 49 and 50). B3
amino acids that differ from the residues of the colr~sl,onding position of the human
antibody are in(lir~ted by vertical lines above the sequence. Inter-domain residues that
were not hl~mqni7Pd are in~ qt~ by qctPriclr~ below the sequence. Heavy chain residue
82b is un~PrlinP~l. Nu~bel~ above the sequence in~i~qte the positions of residues that
were hllmqni7~d.
Figure 12 illustr~tes the plq~mi~l~ utili ed for eA~ ression of hllm~ni
B3(Fv)-PE38 imml)notoxins. Single-stranded uracil-co~ ining pULI7 DNA (A)
en~ ;ng wild type B3(Fv)-PE38 was the t~mplqtP for the mutagenesis according to the
m~.tho~ of KlmkP.l, Proc. Natl. Acad. SCi. USA 82,488-492 (1985). Single str~q-n~ed
uracil-col-t~ ing pB3VH-HUMVL-PE38 DNA (C) was the temrl-qtç for the genPrq-tion of
pHUMB3(Fv)-PE38.
Figure 13 shows the ADP-ribosylation activity, ~;ylotoAicity, and antigen
binding of B3(Fv)PE38 and of the hllmqni7~d v~iar~ts. (A) ADP-ribosylalion activity was
det~ ed by the incol~".~;on on l4C-NAD into acid p~e~ hle mqtPriql using
elong,qtifn factor 2 enr~-~ wheat-germ extract. (B) Cytotoxicity towards A431 cells
was Ille~ur~ by the inhibition of in~.~ol~tion of ~ -leucine into cell protein. (C)
Antigen binding was estim-qtp~ by co...~ilion of [~ -B3 IgG binding to A431 cells at
4~C with each imml~lnotoxin.
Figure 14 shows the reactivity of pooled monkey anti- B3(Fv)-PE38 sera
to B3(Fv)PE38 and h~ ni7P~ variants. B3(Fv)-PE38, B3HUMVH-HUMVL-PE38 and
HUMB3(Fv)-PE38 were immobilized on a 9~well microtiter plate. Sera that were
preincubqt~d with PE38 as a co.ll~elitor at a mol. r ratio of 1000 to 1 over theimmobilized proteins were added in an equal volume at a dilution of 1:50. Percent
reactivity was calcul~ted by setting the mean reactivity with B3(Fv)-PE38 obtained from
four ind~PpPnd~Pnt experimpnt~ to 100% and adjusting the relative reactivities with the
hum~ni7~ variant accordingly.
Figure 15 provides the nucleotide and deduced amino acid sequences of Bl
heavy (A) and light (B) chains. Unde~linPd nucleotide sequences cG~rc~ond (at the 5'
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WO 96/13594 PCT/US95/13811
end) or are comp~ nl~. ~ (at the 3' end) to the PCR primers which were used to PCR
amplify the fr.qgmPnt The amino acid sequence is in single-letter code; below is the
amino acid sequence del~l,lmed by Edman seqU~Pn~ing shown in italics. CDRs are
lmrlk.l;ned, and co~cl~nl region amino acids are struck through.
Figure 16 provides the nucleotide and deducPd amino acid sequence of B5
heavy chain (A) and the variable region and the beg;~-ning of the con~tqnt region of the
light (B) chain. Other details are as in Figure 15. The struck-through C~lbUAY1 te. ",;n~1
amino acids in the heavy chain col~c~ond to the beginl-ing of the (Gly4Ser)3 linker used
to c~nnp~t the VH and the VLin the single chain configur~qtinn.
Figure 17 prûvides the amino acid (peptide) sequence of the B3 single
chain Fv. The figure provides the sequences for the VH region, the linker and the VL
region ~spectively. CDRs are in parenthçses.
Figure 18 provides the amino acid (peptide) sequence of the humqni7~d B3
single-chain Fv. CDRs are in parentheses
DETAILED DESCRIPIION OF THE INVENTION
Definitions
Abbreviations used here for the twventy naturally occurring amino acids,
the five nqtl~r,qlly OC~U~ ~ ;ng nucleic acids and the eleven nucleic acid de~ene; ~ s
(wobbles) follow conve~.l;Qnql usage. In the polypeptide notation used herein, the left-
hand directiûn is the amino te ",inal direction and the right-hand direction is the C~bUAY_
t~-....n~l di-~ction. In the nucleic acid notation used herein, the left-hand directiûn is the
5' direction and the right-hand direction is the 3' direction.
The term "nucleic acid" refers to a deoxyribonucleotide or ribo~ cl~otide
25 polymer in either single- or double-strqn~led form, and unless otherwise limited, would
cllcolllpass known analogs of natural nucleotides that can function in a manner similar to
naturally OC~;U--;ng nucleotides.
The phrase "nucleic acid enC~ing~ or "nucleic acid sequence enr~ing"
refers to a nucleic acid which directs the eApression of a specific protein or peptide. The
30 nucleic acid sequences include both the DNA strand sequence that is tr~qn~rihed into
RNA and the RNA sequence that is trqn~lqt~d into protein. The nucleic acid sequences
include both full length nucleic acid sequences as well as shorter sequences derived from
the full length sequences. It is understood that a particular nucleic acid sequence
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11
includes the degen~-ALe codons of the native sequence or sequences which may be
introduced to provide codon p~fe;~cnce in a spe~ific host cell. The nucleic acid in~ des
both the sense and ~nti~n~ strands as either individual single strands or in the duplex
form.
The terms "i~ol~ted" or "s~ 1y purifiedn, when rerf .;,~g to
recombinantly produced proteins, means a ch~mic~l co-,-position which is esse-.L;~lly free
of other cellular co...ponf n~ Such a composition is preferably in a homogeneous state
~lthough it can be in either a dry or aqueous solution. Purity and homogeneily are
typically d~te ~..in~d using analytical ch~mistry techniques such as polyacrylamide gel
elecl,~phoresis or high ~,r~.. ~nc~ liquid chlonl~togl;1phy. A protein which is the
pled~l,lil ant species present in a prep~ ;on is subst~nt~ y purified. Genp~lly~ a
SubSI; nt;~lly purified or i~ ted protein will comprise more than 80% of all
macrom~l~ul~r species present in the p,~ l;on. Preferably, the protein is purifiPd to
r~pr~~l t greater than 90% of all macrom~ ul~r species present. More p~ft;lably the
15 protein is purifi~ to greater than 95%, and most preferably the protein is purified to
~r,t;~1 ho,l,ogen~ily, wh~,~;n other macromrlcc~ r species are not ~et~t~d by
conventiQrl~l techniques.
The term "labeled antibody" as used herein refers to an ~ntibody bound to
a label such that det~tion of the pl~sence of the label (e.g. as bound to a biological
20 sample) in~lir~tes the pl~scnce of the antibody.
Cyl~tc~ refers to a mnlf~clll~ that when cont~t~t~d with a cell brings about
the death of that cell.
The phrase Nbinding sp~-ificityn, "specifically binds to an ~ntibody~ or
nC~cifi~lly immlmQreactive with," when lefelli. g to a protein or carbohydrate, refers
25 to a binding reaction which is del~,l-.inative of the presence of the protein or
carbohydrate in the presence of a heterogeneous population of proteins and otherbioloeics. Thus, under d~Pcien~tPA imm~mQ~cc~y con~itions~ the srPrifiP~d ~ntiho liPs bind
to a particular protein or carbohydrate and do not bind in a cignific~nt amount to other
proteins or carbohyd,d~s present in the ~mplP Specific binding to an antibody under
30 such con~itionc may require an antibody that is SPlPct~pd for its cpe~ificity for a particular
protein or carbohydrate. For e~mple, antibodies raised to the LeY antigens may be
SPl~tPd to provide antibodies that are spe~ifi~lly immunoreactive with LeY proteins and
not with other proteins. A variety of immlmo~sc~y formats may be used to select
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antibodies ~er-ific~11y immlmoreactive with a particular protein or c~l,ohydlatt;. For
example, solid-phase ELISA immunn~c~ys are fuulii ely used to select antibodies
s~ifir~lly ~ nQreactive with a protein or carbohydrate. See Harlow and Lane
(1988) Ar~ibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York,
S for a desc~ip~ion of immllno~cs~y fol..-als and contlition~ that can be used to det~ ...i..P
sper-ific immlmnreactivity.
The terrns "recombinant DNA,n "leco,-,binallt nucleic acid" or
"~...bin~r,tly produced DNA" refer to DNA which has been j~ol~tPA from its native or
endogenous source and mo lifiPd either rhpmic~lly or cnzy~ ;r~lly by adding, dPlPting
10 or ~ltPring n~tllr~lly~cu. . ;ng fl~nkin~ or intPfn~l nucleotides. Fl~nking r~llrlPotides are
those nucleotides which are either upstream or downstream from the desrrib-pA sequence
or sub-sequence of mlclp~tides~ while intPrn~l m~cl~tides are those nucleotides which
occur within the described sequence or subsequence.
The terms "recombinant protein" or "recoml-in~nlly produced protein"
15 refer to a peptide or protein produced using non-native cells that do not have an
endog~.~vus copy of DNA able to express the protein. The cells produce the protein
be~use they have been geneti( ~lly altered by the introduction of the applo~liate nucleic
acid se~lu~nce. The l~co.l.binant protein will not be found in ~csQc:~l;nn with ~n,teins
and other subcP~ r co---rn~-nL~ normally ~ccoci~teA~ with the cells pro~ucin~ the
20 protein.
Mu~tion~ in proteins are dPci~n~eA by nomPnr1~t-~re consi~tin~ of the
peptide sequence in which the mutation occurs, a l~pr~se~.t~lion of the non-mut~t~P~
amino acid, followed by its positiQn, followed by the l~ple~nL~ n of the mut~te~ amino
acid. Thus, for example, a mutation design~tPA B3(Fv)VL S7T is a mutation from serine
25 (S) to lhr~onine (T) at position 7 of the VL chain of B3(Fv).
Sin~lP Chain Antibodies
This invention relates to recombinantly produced single chain antibodies.
In particular, this invention provides for recombinant single chain antibodies that may be
30 joined to one or more erreclor molecul~ps and, because of their ability to ~.ifir~lly bind
to a particular presPl~P~t~ target molecule, these antibodies are useful as ~gelhlg
moieties which serve to direct the joined effP~ctnr mo1~P~ulPs or compositions to a cell or
tissue bearing the prese1~PctPd target molecule.
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13
As used herein, the term "antibody" refers to a protein con~i~ting of one
or more polypeptides ~lbs~ 1ly encoded by immunoglobulin genes or fra~m~nt~ of
i... -oglobulin genes. The r~o~ nized i.. ,.. -oglobulin genes include the kappa,
l~mb-~, alpha, g~mm~ delta, epsilon and mu c~nct~nt region genes, as well as the5 myriad immllnoglobulin variable region genes. Light chains are c~ ifi~l as either
kappa or l~mb~l~ Heavy chains are c~ ified as ~amm~ mu, alpha, delta, or epsilon,
which in turn define the immlln~globulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
The basic immunoglobulin (antibody) structural unit is known to comprise
10 a t~.1,i~"~er. Each tetr~mPr is co~llposed of two idet~ti~l pairs of polypeptide chains, each
pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-te~ s of each chain defines a variable region of about 100 to 110 or more a-m-ino
acids primarily res~n~ihle for antigen l~co~n;~;on. The terms variable light chain (VL)
and variable heavy chain (V~ refer to these light and heavy chains r~ ely.
Antibodies may exist as intact immlJnoglobulins, or as mo lific~tions in a
variety of forms inclu-lin~, for example, an Fv fr~mPnt con~;~h~ only the light and
heavy chain variable regions, a Fab or (Fab)'2 fr~m~nt con~in;ng the variable regions
and parts of the c~nst~nt regions, a single-chain antibody (Bird et al., Science 242:
424-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988) both
20 inco,~Glaled by reference herein), and the like. The antibody may be of animal
(espe~i~lly mouse or rat) or human origin or may be chim~nc (Morri~on et al., Proc
Natl. Acad. Sci. USA 81, 6851-6855 (1984) both incol~ted by reference herein) orhl...l~ni~ aones et al., Na~ure 321, 522-525 (1986), and published UK patent
application #8707252, both inccl~ldted by reference herein). As used herein the term
"antibody" includes these various forms. Using the guidelines provided herein and those
methods well known to those skilled in the art which are described in the references cited
above and in such publi~tions as Harlow & Lane, An~ibodies: A Laboratory Manu~l,Cold Spring Harbor Laboratory, (1988) the antibodies of the present invention can be
readily made.
The term "Fv" region as used herein refers to a single chain antibody Fv
region co~ ,in;ng a variable heavy (V}~ and a variable light (VI) chain. The heavy and
light chain may be derived from the same antibody or dirr~rent antibodies thereby
producing a c~imPnc Fv region.
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14
The term nerr~lo~ molecul," or "erreclor comroeition" as used herein
refer to agents having a particular biological activity which is to be directed to a
particular target mn'~cu1~ or a cell bearing a target m~A.1AcukA. One of skill in the art will
apprcchte that err~tor mAl~A-clllPs may include various drugs such as vinblA-etin~,
5 d~u-~l-lycin and the like, ~y~,uns such as native or moAifiPd Pseudomonus exotoxin or
Di~hll.~ toxin, enr~ llAting agents (e.g., Lposo...es) which th,me,lves contain
phA~rm~ eir~l co."~ inne, r~io~rtive agent such as l25I, l3'Cs, 32p, 14C, 3H, and
35S, target moieties and lig~nds.
As used herein "ligands" are mol~ul.os capable of reacting with or
otherwise recognizing and ere~ifir~lly binding a "target" molecule. T.ig~nde and their
~c~l ectivc target molcc~ll,s ~p~sent paired sr~~i s. Typical paired species incl~lde, but
are not limited to, enzyme/s~sllate, ~ceptor/agonist, antibody/antigen, and
lectin/carbohydrate. The binding between a ligand and its target may be m,Ai~t-A by
covalent or non-covalent int~~tinnS or a comhin~tinn of covalent and non-covalent
int,~ti~1ne When the in~ ;nn of the two species produces a non-covalently bound
complex, the binding which occurs is typically elecLI~s~lic~ hyd~ugen-bonding, or the
result of h~dluJ~hilic/lipophilic int,~çtin~e Accordingly, ''spe~~ific binding" occurs
belween a ligand and its target mol~cllle where there is interaction between the two
which produces a bound complex having the çh~r~rtPrictirs of an ~ntihody/antigen or
cn~ s~sLlatc interaction. Spe~~ific~lly, ~ rles of ligands inçl~de, but are not
limited to antibodies, lymrhokinrs, cyt~kin~s"ecep~or proteins such as CD4 and CD8,
solubilized ,~tor proleins such as soluble CD4, hormones, growth factors, and the like
which speçifir~lly bind desired target cells.
The chûice of the particular errcclor mol~cllle or comrosition depends on
the pa~ular target mol~clll, or cell and the biok)gir~l effect it is desired to evoke.
Thus, for eY~~mple, the erf~;lor m~ e~ule may be a .;ylolc,Ain where it is desired to bring
about death of a particular target cell. Conversely, where it is merely desired to invoke
a non-lethal biological response, the erreclor molecule may be a conjugated non-lethal
pharmacological agent or a liposG--,e con1~ining a non-lethal pharmacological agent.
In a particularly prerelled embo~imPnt, the antibodies may be joined to an
err~;lor moleculp that is a drug or to a ~;yloloAin to form an immunotoxin capable of
selectively illing particular target cells. Numerous cytotoxic compounds are known to
those of skill in the art and include, but are not limited to, ricin, abrin, Pseudomonas
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exotoxin (PE), Dirhthe~i~ toxin (DT), and the like. P~rel.ed toxins are PE or DT.
Native PE and DT are highly toxic coll-pounds that typically bring about death ~ ough
liver toxicity. PE and DT, however, can be mo~lified into a form for use as an
olo,un by removing the native ~,e~ing component of the toxin (e.g. domain Ia of
PE and the B chain of DT) and repl~r-ing it with a different antibody ~eLing moiety.
The term "Pseudomon ls e~otoYin" (PE) as used herein refers to a full-
length native (n~tllr~lly oc~. . ;ng) PE or a PE that has been m~1ifi~ . Such
mo lific~tion~ may incl-lde, but are not limited to, eli"~in~liQn of domain Ia, various
amino acid deleti-~n~ in domains II and m, single amino acid substitution~ (e.g.,
replacing Lys with Gln at positiQn~ 590 and 606), and the ~ lition of one or more
sequences at the ~ubo~yl tel",illus such as KDEL (Seq. ID No: 51) and REDL (Seq. ID
No: 52). See Siegall et al., J. Biol. Chem. 264: 14256-14261 (1989). Thus, for
e~mple, PE38 refers to a tl..nr~t~ Pseudomonas exotoxin co,nl)osed of amino acids
253-364 and 381-613 (see CGI-----Only ~igned U.S. Patent ~pplir~ti/m Serial Number
07/901,709 fi1ed June 18,1992 incol~ld~d herein by reference. The native C ~ JS
of PE, REDLK (residues 609-613, Seq ID No: 53), may be rerpl~e~ with the sequence
KDEL, REDL, and Lys-590 and Lys-606 may be each mut~t~d to Gln (see comlllonly
~ign~ U.S. Patent App1i~ tion Serial Number 07/522,563 filed May 14, 1990,
incol~ dled herein by reference).
The term "l~irhth~ri~ toxin" (DT) as used herein refers to full length
native DT or to a DT that has been mo~lified~ Modifi~tion~ typically include removal of
the ta~ i, g domain in the B chain and, more specific~1ly~ involve truncations of the
carboxyl region of the B chain.
The recombinant single chain antibodies of the present invention may be
fused to, or otherwise bound to the erre~or mol~cule or co"lposition by any method
known and available to those in the art. The two co",ponents may be ch~mic~lly bonded
together by any of a variety of well-known ch~--mi~l p~lur~s. For ex~mpl~ the
linkage may be by way of heterobifi-nctis)n~l cross-linkers, e.g. SPDP, carbofliimide,
g1ut~r~ldehyde, or the like. Production of various immun-,toxins is well-known within
the art and can be found, for example in "Monoclonal Antibody-Toxin Conjugates:
Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine,
Ac~demic Press, pp. 168-190 (19~2) and W~l(lm~nn, Science, 252: 1657 (1991), both of
which are incoll,u,dled by reference. To use the recombinant PE molto~ul~s with an
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WO 96/13594 PCT/US95/13811
16
antibody, a form of the PE mol~cul~P with cysteine at amino acid position 287 is
pl~f~-cd to couple the toYin to the antibody or other ligand through the thiol moiety of
~;y~
In a ~lere.~ed embo~iment~ the antibodies of this invention may also be
5 fused to a protein erreelor mol~culp by recomhin~nt means such as through the use of
recombinant DNA techniques to produce a nucleic acid which encodes both the antibody
and the err~P~lor molecule and GA~r~ssing the DNA sequence in a host cell such as E.
coli. The DNA e -co~ g the chimeric protein may be cloned in cDNA or in genomic
form by any cloning P1~1U1G known to those skilled in the art. See for eY~mp
10 Sa"l~luok et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
labol~ly, (1989), which is herein incorporated by reference.
As il~ r~tpd above, in ~itinn to CylO~O~ S, the single chain antibodies of
the present invention may be fused or chPmi~ ~lly conjugated to a wide variety of eLrGc~r
mol~ lps Thus, for ey~mpl~p~ the antibody may be conjugated or fused to b~ tP~ l or
15 plant toYAins, or to other err~ ot~r agents to treat or rli~nt~sP~ human cancer. For eY~mpl~,
r; diol-u( lides conjugated to ~ntibo~ip~s that bind to tumors can produce cell killing based
on the high local con~ ;on of r~ tinm Chemo~ -Ape~ c drugs, for eY~mpe,
vinblastine or daunomycin, can be coupled to the antibodies and delivered at high
con~nl.ation to cells that react with the antibodies. Simil~rly, the antibodies of this
20 invention may be utilized to specifically target a vehicle that enc~ps~ tes a the~ul;c-
agent. For e~amplc, the antibodies may be conjugated to a liposo",e which itself carries
a drug (e.g. doYAorubicin) and Ill~eby spP~ifiç~lly targets the liposollle to a specific tissue
or cell. ~lt~...;.l;.~ely, the antibodies may be recombinantly fused to a melllb,d, e-
inserting protein and thereby inco,~oldted into the liposo~lle for delivery of th~ld~ulic
25 agents.
Fusion or conjugation of the antibodies of this invention to various labels
produces a highly sperific detect~hle marker that may be used to detect the presence or
absence of cells or tissues bearing the particular molecule to which the antibody is
detect~d. ~ ely, the antibodies may be chemic~lly conjugated or fused to an
30 er~ec~r mol~ule that is another s~çific binding moiety, e.g. a ligand such as those
dese~ibed above. In this form the composition will act as a highly specific bifun~tion~l
linker. This linker may act to bind and enh~nce the inter~Ction between cells or cellular
components to which the fusion protein binds. Thus, for example, where the fusion
CA 02203236 1997-04-21
W O96/13594 17 PCTrUS95/13811
protein is a growth factor joined to an antibody or antibody fr~gmPnt(e.g. an Fvfragment of an antibody), the antibody may sp-p~ifir~lly bind antigen posilive cancer cells
while the growth factor binds lc~e~tol~ (e.g., IL2 or IL4rccepl~l~) on the surface of
i.. ~J~ cells. The fusion protein may thus act to enh~nre and direct an i.. l"~
S response toward target cancer cells.
One of skill in the art will appreciate that the antibodies of the present
invention may also be utilized as multiple ~geling moietiPs Thus this invention also
provides for co~ ilions in which two or more antibodies are bound to a single er~r
mrl~clllP Where the erre~:lor mol~cule is a cyloto~in, the presence of two or more
antibodies may increase sper,ificity or avidity of binding of the immllnotoxin.
Conversely, ml~lt~ e err~:~r molccllkps may be fused or otherwise joined to a single
antibody. CGIII~S;~;r)nC of this nature may provide two or more kinds of biological
activity with a single l~,cling moiety.
In a particularly pref~llcd embo~iment, the antibodies of this invention are
antibodies that sper-ifir~lly bind LewisY (LeY) carbohydrates (LeY carbohydrate ~ntigPns).
As used herein, the LeY carbohydrate ~ntigen~ include natural or synthetic LeY
c~bohydrd~s or fr~mPnt~ thereof that contain e~i~opes recognizable by LeY binding
antibodies. Also inrlude~ are c~bo}lydlates, glycoploteins and other glycoconjugates
which contain or mimic the LeY carbohydrate or ~ilopes cor.~ ed within the LeY
~bohydldle(see, Pastan et al., CancerRes., 51:3781-3787(1991) and Hoess et al.,
Gene, 128:43-49(1993)). Such mimics are known by their ability to speçifir~lly bind to
known arlti-LeY antibodies such as Bl,B3,B5,BR64 and BR96 (Hellstrom et al.,
Cancer Res., 50:2183-90(1990)), and the ~ke.
Of the LeY binding antibodies, particularly prerelled are antibodies having
the tissue binding spe~ificity of Bl,B3 or B5. The term "tissue binding spe~ifirityll as
used herein refers to the particular distribution of tissues to which an antibody binds and
does not bind as determined by immunohi~torhPmir~l analysis. l~etho~s of de~lllining
tissue binding speçificity as well as the binding sperificities for Bl,B3 and B5 are
described in U.S. Patent 5,242,813 (see, e~ lly Tables I, II and III) which is
incolyolated herein by reference.
The antibodies may be derived from the monoclonal antibodies de~iEn~
Bl,B3, and B5 (see U.S. Patent 5,242,813). These antibodies have been shown to
specifiç~lly bind to LewisY and LewisY-related carbohydrate antigens that are typically
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18
found on various carcinomas inc~ ng carcinomas of the breast, colon, cervix, and
p~st~le.
The antibodies of this invention may be Fv regions comrrising a variable
light (VL) and a variable heavy (VN) chain. The light and heavy chains may be joined
5 dir~lly or through a linker. As used herein a linker refers to a mol-cllle that is
covalently linked to the light and heavy chain and provides enough sp~ring and flexibility
be~ween the two chains such that they are able to achieve a conformation in which they
are capable of spe~ific~lly binding the epitope to which they are directed. Protein linkers
are particularly p~cr~lod as they may be t;~ ssed as an intrin~ic c4l"ponent of the
10 fusion protein.
Preparation of Antibody Fv Fr~ ts
Single chain Bl, B3 and B5 Fv regions may be cloned from the hybridoma
cell lines Bl, B3 and B5 which were depocited on October 10, 1990 with the ~merir~n
Type Culture CollP~tion (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, wherethe depo~ils were granted the n~cescion numbers ATCC HB 10572, HB 10573, and HB
10569, lc;s~ ely. The depocitc were made ~ul~lt to the provisions of the Rud~rP~st
Treaty.
The Fv regions may all be cloned using the same general SLI~tCg~.
Typically, for CA . rle, poly(A)+ RNA extracted from the hybridoma cells is reverse
tranc~rihed using random h& . ~.. ~ as primers. The VN and VL domains are ~mplifiPd
s~ t~ly by two polymerase chain reactions (PCR~). Heavy chain sequences may be
~mplifi~ using 5' end primers which are d~PcignP~ according to the amino-tell-linal
protein sequences of the Bl, B3 and B5 heavy chains respectively (Sequence Listing ID
Nos. 19, 17, and 21 l~ s~clively) and 3' end primers according to concPncus
immlmoglobulin collct~nt region sequences (Kabat et al., Sequences of Proteins of
Irnmunological Interest. 5th edition. U.S. Department of Health and Human Services,
Public Health Service, National Tnctitutes of Health, RethPsd~, MD (1991) incorporated
by reference). Light chain Fv regions are amplified using 5' end primers decignPd
according to the amino-tPrmin~l protein sequences of Bl, B3 and B5 light chains
l~eclively (Sequence ID Nos. 20, 18 and 22 ~cs~eclively) and in combination with the
primer C-kappa (rable 1 and Sequence ID No. 14). Suitable primers are specifically
illllctr~t~d in F~mp~eS 1 and 2 although one of skill in the art would rccognize that other
suitable primers may be derived from the sequence listings provided herein.
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WO 96/13594 PCT/US95/13811
19
The crude PCR products are subcloned into a suitable cloning vector.
Clones con~;~ining the correct size insert by DNA restri~tion are identifiP~l. The
nucleotide sequence of the heavy or light chain coding regions may then be d~ ,lined
from double st~nded pl~cmid DNA using seqUPn~-inp primers ~ r~nt to the cloning
site. Commercially available kits (e.g. the Sequenasen' kit, United States P;ochemi~l
Corp., Cleveland, Ohio, USA) may be used to fa~ilitatP sequPn~ing the DNA.
Of course the sequencing steps are llnnpc~ss~ given the sequence
information ~i~losed in the present invention. One of skill will a~l~iate that utili7ing
the sequence information provided for the Fv regions of Bl, B3, and B5 (Sequence ID
Nos. 17-22), nucleic acids encoding these sequences may be obtained using a number of
mPtho~s well known to those of skill in the art. Thus, DNA encoding the Fv regions
may be prq?~red by any suitable method, in~ ing, for eY~mplP, amrlific?~ti- n
techniques such as ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4: 560
(1989), T ~ n, et al., Science, 241: 1077 (1988) and R~rringer, et al., Gene, 89:
117 (1990)), ~n~crirtion amplific~tion (see Kwoh, et al., Proc. Natl. Acad. Sci. USA,
86: 1173 (1989)), and self-svsl~;n~d sequence repli~tion (see Guatelli, et al., Proc.
Natl. Acad. Sci. USA, 87: 1874 (1990)), cloning and restriction of a~ul~liate sequences
or direct chPmi~l synthesis by metho~s such as the phosphotriester method of Narang et
al. Meth. Enyrnol. 68: 90-99 (1979); the phospho~iPstP-r method of Brown et al., Meth.
Enymol. 68: 109-151 (1979); the diel}~ylphosrhor~mi~i~ method of Rp~lc~e et al.,Tetra. Lett., 22: 1859-1862 (1981); and the solid support method of U.S. Patent No.
4,458,066, all such references in this paragraph inco,~oldled by reference herein.
ChPmi~l synthesis produces a single stranded oligom~cl~Potide. This may
be converted into double st~n~ed DNA by hybri(li7~tion with a com~;~~m~Pnt~l9
sequence, or by polymPri7ation with a DNA polymerase using the single strand as a
template. While it is possible to chPmic~lly synthe~i7~ an entire single chain Fv region,
it is preferable to synthe~i7~ a number of shorter sequences (about 100 to 150 bases) that
are later ligated together.
.Alle...~ ely, subsequences may be cloned and the appr~p,iate
- 30 ;,~s~u~nces cleaved using a~p,u~.;ate restriction enzymes. The fr~gmPntc may then be
ligated to produce the desired DNA sequence.
Once the Fv variable light and heavy chain DNA is obtained, the
sequences may be ligated together, either dir~;lly or through a DNA sequence encoding
CA 02203236 1997-04-21
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a peptide linker, using techniques well known to those of skill in the art. In a ~l~f~.led
emho-l;...PI.t, heavy and light chain regions are c~nnPct~P~ by a flPYi~lP peptide linlcer
(e.g. (Gly4Ser)3) which starts at the C~hbOAY1 end of the heavy chain Fv domain and ends
at the amino ~"-""us of the light chain Fv domain. The entire sequence enrocles the Fv
S domain in the form of a single-chain antigen binding protein.
,ation of Antibody Fusion Protei~s
Once a DNA sequence has been i~lPntifiP~ that encodçs an Fv region
which, when eA~ a~d shows spP~Aific binding activity, fusion pr~eins comrricin~ that
10 Fv region may be p,c~d by mPtho~$ known to one of sldll in the art. The Fv region
may be fused direclly to the erÇ~clor molA~AulP (e.g. .;~ ,un) or may be joined dileclly
to the ~Ain through a peptide con.-~lQr. The peptide connector may be present
simply to provide space be~ the ~ling moiety and the effP~tor molAculP or to
f Ailit~te mobility b~l~ncen these regions to enable them to each attain their OptilllWIl
15 confo....--lion. The DNA seque,lce comrricing the connP~Ior may also provide sequences
(such as primer sites or restriction sites) to f~ cilit~Ate cloning or may preserve the reading
frame ~el~n the sequence çnco~ling the targeting moiety and the sequence enr~ing the
err~-lo~ mcl-clllP The design of such cQnne~tQr peptides will be well known to those of
skill in the art. However, one particularly p,efe"ed conne~tor is the peptide
20 SGGPEGGS (Sequence ID No. 44), ~içcign~b~d herein as the C3 col~np~clor.
Mçthods of produc-in~ fusion proteins are well known to those of sldll in
the art. Thus, for eY~mpl~, Ch~l~dh~ry et al., Nature, 339: 394-97 (1989); Batra et al.,
J. Biol. Chem. 265: 15198-15202 (1990); Batra et al., Proc. Natl. Acad. Sci. USA,
86: 8545-8549 (1989); ~h~ltlh~ry et al., Proc. Natl. Acad. Sci. USA, 87: 1066-1070
25 (1990), all inco,~,dted by reference, desçribe the prep~alion of various single chain
antibody-toxin fusion pr~leins.
Generally producing imml)notoxin fusion proteins involves se~a~dlely
prel~ing the Fv light and heavy chains and DNA encorling any other protein to which
they will be fused and l~co-"bining the DNA sequences in a pl~mid or other vector to
30 form a construct encoding the particular desired fusion protein. However, a simpler
approach involves inserting the DNA encoding the particular Fv region into a construct
already encoding the desired second protein.
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21
Thus, for Pl~mple~ DNA enr~ing Bl(Fv), B3(Fv), B5(Fv) or chimPric
Fv fusion proteins is most easily pl~ed by inserting the DNA çncoding the B1, B3,
B5 or çhimPric Fv regions into constructs already cont~ining DNA enr~ling the desired
cytotoxin. The e~ssion pl~cmid pVC38H cont~inc the gene from the immllnotoxin
S TGFa-PE40 under control of the T7 plo.llo~r, the T~ transcription ~~ nator at the 3'
end of the PE40 coding region and the single strand repli~tion region F+, to generate
single str~ndP~ phage DNA by contransfection with (M13) helper ph~g~Ps, if desired to
create d~ivati~es of the pl~cmid by site dil~d mutagenesis (ch~l)dh~ry et al. Proc.
Natl. Acad. Sci. USA, 87: 1066-1070 (1990). Simil~rly~ the pl~cmid pRK79K encodes
10 the Pseudomonas exotoxin PE38KDEL (C'h~ h~ry, et al. Proc. Natl. Acad. Sci. USA,
87: 308-312 (1990).
The DNA sequence enr~ling the Fv region is inserted into the construct
using techniques well known to those of skill in the art. Thus, for ~"~...ple, to create a
pl~cmid for e~ s~ion of the immlmotosin B3(Fv)-P_40 (pULEE3), the TGFa gene is
removed and rep~ d by the B3(Fv) gene in a 3 fr~mtont lig~ti()n~ using an NdeI/BamHI
fragment of the heavy chain coding region and the R~m~TlFTintlm fragmPnt encoding the
light chain Fv (Figure la) as ~lesçnbe~ in Example 1.
Simil~rly, a pl~cmi-l enr~1ing B3(Fv)-PE38 may be produced by removing
the PE40 coding region from pULI1 from the T~in-lTTT site to an EcoRI site positioned
just beyond the P_40 gene and repl~cing it with a ~TinflTTT/EcoRI fra~m~nt from pRK79K
described by Ch~ ry et al. supra. This approach is described in greater detail in
FY~mp~
A particularly pr~fellt;d approach involves the use of pl~cmi~ pULI7
which encodes the B3(Fv)-PE38 immlmotoxin (Benhar et al. Bioconjug. Chem., 5: 321-
326 (1994)). For each Fv, the VH and VL sequences are PCR ~mplifi~d using the heavy
chain and light chain in their l~ e pl~cmi~ls as t~mpl~t~o-c. The ~mplifi~ti~n
primers are decigned to have at their ends sequences that are complempnt~ry to the
tr~n~l~tion initi~tiQn, peptide linker and Fv-toYin junction (col n~tor) which are common
to the single-chain Fv-immlmotoxin e~yltssion vectors. The PCR products are purified
and ~nn.o~led to a uracil-col-l~;ning single st~nded DNA col~s~nding to the pULI7
DNA ~ ~cd by rescue of pULI7 with a helper phage. The ~nnP~led PCR products are
extended using the single stranded DNA as a templ~te (see, for eY~mple, MI~TAGENE0
mutagenesis ~lo~ocol, Biorad, Hercules, California, USA). The intact DNA may be used
CA 02203236 1997-04-21
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to transform cells and express the new fusion protein. In a p.~r~l-ed embo~imPnt,
bccause qnn~-lin~ çffiriPncy to the templ~tP is low, the r~ inil-~ intact "I~nm~ifiPd"
DNA may be digested using a rçstrictiQn PndQ~ rlPqcP~ which has a unique site in the
B3(Pv) template but that is absent from Bl and B5. This destroys any residual B3(Fv)
5 sequences leaving only the modified sequences. This approach is dP~rihed in greater
detail in Fyqmrle 2.
The ~pa~lion of DNA E~co l; ~ Variable Domain Shumed Fusion Proteins.
It was observed that the stability of monoclonal antibody B3 could be
10 improved. In the form of a single chain Fv jmml)nntoxin, B3 is con~ pr~qhly more
stable, however it still undergoes inactivation, mainly by aggreg,qtion~ esreriqlly upon
incu~qticn in 0.15 M NaCl, 0.01 M NaPO4 pH 7.4 at 37~C. In contr~qct to B3(Fv)-PE38
in.. nn~Ain, B5(Fv)-PE38 is more resistant to inactivation under these con~itions (see
Figure 6). Based on these observations, the stability of chimPric Fv immlmotoxins was
15 ~ ;nfd.
It is an un~-l~c~d discovery of the present invention that chimpri~ Fv
regions con~;n.ng variable heavy and light chain domains from different, albeit related,
antibodies may show ci~nifi~qntly greater stability in vitro and in vivo than Fv regions
where both the heavy and light domain are derived from the same ~ntihody. Thus, for
20 eY~mple, a fusion protein comrricin~ a B3 variable heavy region and a B5 variable light
region fused together and to PE38 shows higher activity and longer term stability than a
B3(Fv)-PE38 fusion protein.
Nucleic acids encoding ~himP1ic Fv regions are easily pl~palt;d using the
techniques desçrihed above. The VH and VL sequences are PCR amplified using the
25 heavy chain and light chain in their ~s~ e pl~cmitlc as tem~ tPs as dçsrrihed.
However, instead of using the VH and VLDNA from the same antibody, the VH and VLDNAS are SPlP~t~Pcl from different antibodies. Thus, for example, one may combine a
B3VH with a B5VL or a B5VH with a B3VL and so forth. The DNAS are ~nnP~lPd to a
uracil~~ ining single str~nl1~P~ DNACO11e~ ;n~ to the pULI7 DNA and the
30 synthesis of the chimeric Fv-c~lotoAhl fusion protein DNA is completed as described
above and in Fx~mrl~s 2 and 12.
One of skill will appreciate that it is possible to Plimin~tP the C~lotuAi
moiety and express the chimPric or single antibody Fv regions alone. These may be
CA 02203236 1997-04-21
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23
used in various rhPmi~ conjugates for e~mple, either dir~clly with toxins or other
thf~r~u~;c agents, with c~l;~r~ for thc ~r~ul;c agents such as liposo,l,es, or with various
labels and 1~ 7 such as fluol~scenl labels.
S Stabili7in~ Mutations of B3
When a more stable related form of an antibody is idpntifip~l~ site directed
mut~genP~i~ may be used to identify the dirre.Gnces bcl~n the more and less stable
forms. Thus, for example the B3VH-BSVL-PE38 im.. ,-otoAill shows greater stability
than the B3-PE38 (B3VH-B3VL-PE38) immunoto~in. To identify the amino acid residues
10 contributing to the increased stability one ~rOll"s a sequence analysis to identify those
regions of the particular light or heavy region (in this case the VL region) that differ from
the coll~sponding light or heavy chain in the non-~-himPri~. antibody. Once the
dirr~.lces have been id~PntifiP~, mut~til n~ rPfl~P~ting those dirrer~nces may be
s~ lly in~luced into the coll~cron~ing region of the non-chi..~f-;c antibody.
15 Com~ricon of the activity and stability of the mut~t~ antibody with the chimPri~
antibody fusion protein will inrlir~tP which mut~tinn iS rc~nsible for the increased
stability. For eY~mple, it was discovered that replacing the B3 VL mPthioninP,4 with
leucine st-l ili7~1 the immlmQtoxin as much as the B3VH-BSVL-PE38 combination
whereas repl~ing VL scrine 7 with ll.,~onine had no stabili_ing effect. Thus, in a
20 particularly ~ler~lcd ernbo~ t the fusion protein comI-ri~Ps either B3VH-BSVL-PE38
or B3(Fv)-PE38: VL M4T.
Mutations that Increase Antibody Binllin~ Affinity
An ~-nr~ ~ result of the present invention is the discovery that
25 mUt~tion.~ at position 95 of the VH region can alter the binding affinity of the single chain
antibody. More speçific~lly~ it was discovered that mUt~tion~ that altered the serine at
position 95 in B3(Fv) to tyrosine or to phenyl~l~nine, which are the most common amino
acids at this position in other antibodies, reduced the binding affinity of B3(Fv) by
ap~)r~ Ply l~fold (see Example 18). Conversely, when the tyrosine at VH 95 in
30 B5(fv) was ...ul~ted to serine showed a for-fold increase binding activity as analyzed by
~i~/lotoAicity assays. B5(Fv) differed from B3(Fv) in having a completely dirr~ t
binding site. Thus the effect of the mut~tion is independent of the particular binding site.
Without being bound to a particular theory, it is believed that a serine
CA 02203236 1997-04-21
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24
located in the VH/VL;ntPrf~r~ slightly destabili_es the intp~ce cQnt~rtC enabling
muv~ nt of VH relative to VL. This movement f~rilit~tPS a so called "in-luced fitH
binding mode. This destabili7~tion mPch~nicm would be eYI~ect~Pd to function in any
antibody in which the VH 95 position is not normally a serine. Thus, this invention
provides a new mP~h~nicm for increasing the binding affinity of ~ntiho~ips that do not
normally have a serine at VH position 9S.
e-l B3(Fv)
RPc~se mon~lon~l antibodies B1, B3 and BS are mouse antibodies,
np~~ ~lministr~tinn of either labeled antibodies or the immlmotoxins inrln~in~ these
~nti~iPs as ~E5c~ g moic~i~os will result in the formation of anti-mouse antibodies
(Parren et al., Hurn. Ar~ibod. Hybridornas., 3: 137-145 (1992)), in addition to the
productinn of ~ntih~o~ip~s to the toxin moiety. This immlmç req~once may preclude long
term tre~tm~pnt in some cases. Therefore it is desirable to produce less immllnogPnir,
mnlc~u1~s
As a first step in making less imm~mr~gPnic moleculPs the Fv portion of
the mou~ antibody is hlnn~--i?Pd so that it may then be used to replace the Fv portion of
the murine antibody in the fusion p~leins of the present invention. Hllm~ni7~d
antibodies are non-human antibodies in which some or all of the amino acid residues are
re~laced wit~h the co~ Jonding amino acid residue found in a similar human antibody.
~um~ni7~tiQn thereby reduces the ~ntigpnic potential of the antibody.
Antibody variable domains have been ~llm~ni7Pd by various methods, such
as CDR grafting (l~i~hm~nn et al., Nature, 332: 323-327 (1988)),reP1~rPrn~~nt ofexposed residues (Padlan, Mol. Irr~nunol. 28: 489~98 (1991)) and variable domainres--rf~çing (Roguska et al., Proc. Natl. Acad. Sci. USA, 91: 969-973 (1994), all
incol~ldled by reference. The minim~lictic approach of res--rf~ring is particularly
suitable for antibody variable domains which require preservation of some mouse
framework residues to ~ int;1in m~xim~l antigen binding affinity. However, the
straightrulw~-d CDR grafting approach has also been succ~ccfully used for the
hllm~ni7~tion of several antibodies either without preserving any of the mouse framework
residues (Jones et al. Nature, 321: 522-525 (1986) and Verhoeyen et al., Science, 239:
1534-1536 (1988)) or with the preservation of just one or two mouse residues
CA 02203236 1997-04-21
WO 96/13594 PcTruS95113811
(p~i~.hmqnn et al., Nature, 332: 323-327 (1988); Queen et al., Proc. Natl. Acad. Sci.
USA, 86: 10029-10033 (1989), all incc~ ted by reference.
To improve the Bl, B3, or B5 antibodies or the ehimtoric antibodies of this
invention, for th~p~ulic applications, the Fv portion is hl....~ni~d by a method rerelled
5 to as "framework e-Ych~nge". In this approach, fi~ullewclh residues are identifi~d that
differ from human framework residues in highly homologous human VH or VL donors.These differing rla..lt;wcl~ residues are then simultaneously l~-u~ed to human reC;~Ies.
The mut~tions are introduced onto a single-str~nde~ DNA t~mrl~te pl~ ed from a
single-chain imml)notoxin c~sette which may be t;,.pf~ ssed in E. coli and allows the
10 rapid pllrifi~tin~ and analysis of the r~sultin~ hllm~ni7~d va~iant~.
This approach combines, yet deviates from the principles of CDR grafting
or from the re~ ment of eYpose~ recidues, as some residues that are not normallye~sed are hlum~ni7~d, while some other residues that are normally exposed are not
h~lm~ni7~d Decici~n~ to preserve certain mouse residues are based on knowledge
15 ~galdil~g the effect of ...u~ g these particular residues on the binding affinity of the Fv
fra~ment, or on the possible interactions of these residues with other Fv residues
observed in a structural model.
More sp~ifir~lly~ hum~ni7~tion is accomrlish~d by ~li&nin~ the variable
domains of the heavy and light chains with the best human homolog identifi~l in
20 sequence ~i~t~ ~s such as GENBANK or SWISS-PROT using the standard sequence
cc...~ on sorlw~ as ~lesc-ribe~ above. Sequence analysis and cc...~ ;son to a
structural model based on the crystal structure of the variable domains of monoclonal
antibody McPC603 (Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989)
and Satow et al., J. Mol. Biol. 190: 593-604 (1986)); Protein Data bank Entry IMCP)
25 allows i-~ntifi~ation of the framework residues that differ between the mouse antibody
and its human count~
In a p,er~,~d embodim~ont, the mouse residues at B3 VH POS;t;On~ 1, 3, 19
24, 89 and 91 (see coordina~s in Kabat et al. supra.) and B3 VL positions 2, 3 and 41
(Figure 11) are preserved. In a particularly pfef~,ed embo~1imçnt~ residue 82b in B3 VH
30 is l~u~ted to a~inine.
VH and VL gene se~m~nt~ (e.g. in pl~mi(l pULI7) en~ling wild type
B3(Fv)-P_38 may be in~lependçntly hu...~ni~d by site spe~-ific mutagenesis (see FY~mpl~
14). One of skill in the art will appreciate that once the Fv region has been cloned and
CA 02203236 1997-04-21
W O 96/13594 PCTrUS95/13811
26
sequenr,ed, alteration of various residues by site specific mutagenesis is routine using
s~nd~d terhniques well known to those of skill in the art (Klml~l, Proc. N~l. Acad.
Sci. USA, 82: 488-492 (1985)).
S Expression of Recombinant Proteins
The recombinant Fv regions and fusion yr~teih~s incoll~u,dling these
~ntihody regions may be c;A~ sed in a variety of host cells, inrlu-ling E. coli, other
b~~Pri~l hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and
HeLa cells lines and myeloma cell lines. A particularly pl~;fell~d host is E coli. The
10 recombinant protein gene will be operably linked to appl~lidle ~A~lesaion control
~ uences for each host~ For E. coli this incl~ldes a promoter such as the 17, trp, or
lambda promoters, a riboso,l,e binding site and preferably a ~nsrnption ~. ",in~ion
signal. For eukaryotic cells, the control sequences will include a promoter and
preferably an enh~ncer derived from immlmQglobulin genes, SV40, cytomegalovirus,15 etc., and a polyaden~laLion sequence, and may include splice donor and ~r~ptor
sequences.
The pl~mirl~ of the invention can be tr~n~fçrred into the chosen host cell
by well-known m.-th~s such as c~lci-lm chlorille tranaÇol"lation for E. coli and c~lcinm
phosph~te tr~tm~nt or elec~upcl~ion for m~mm~ n cells. Cells transformed by the
20 pl~smi~ls can be s--l~ted by re~i~t~nce to antibiotics conr~led by genes c~nt~inFd on the
p1~mi~S, such as the amp, gpt, neo and hyg genes.
Once c:~.plessed, the recombinant fusion proteins can be purified according
to standard pr~lures of the art, i~cll)ding Ammn~ m sulfatepl~i~ ;nn, affinity
columns, column cl r~",alog,aphy, gel electrophoresis and the like (see, generally, R.
25 Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deul~r-h~r, Methods in
Er~mology Vol. 182: Guide lO Protein Purification., A~1emic Press, Inc. N.Y.
(1990)). Subst~nti~lly pure compositions of at least about 90 to 95% homogeneity are
ylcrelr~d~ and 98 to 99% or more homogeneity are most prefe~led for pharmAr~utir~l
uses. Once purified, partially or to homogeneity as desired, the polypeptides may then
30 be used thclApeulir~lly.
One of skill in the art would recognize that after cllemi~Al synthesis,
biological t;A~r~ssion, or punfi~tion, the single chain Fv region or a fusion protein
comrricing a single chain Fv region may possess a conformation substAnti~lly different
CA 02203236 1997-04-21
W O 96/13594 PCTAUS95/13811
27
than the native protein. In this case, it may be ne~esc~ to denature and reduce the
protein and then to cause the protein to re-fold into the ~l~r~l~d conform~tion. ~etho~s
of red~1cing and ~en~tllring the protein and in~uring re-folding are well known to those
of skill in the art. (See, Debinski et al. J. Biol. Chem., 268: 14065-14070 (1993);
Kçt;il~ and Pastan, Bioconjug. Chem., 4: 581-585 (1993); and RUChn~Pr, et al., Anal.
Biochem., 205: 263-270 (1992) which are incol~l~ted herein by reference.) Debinski
et al., for example, describe the denat~l,dtion and reduction of inclusion body proteins in
gll~ni~inp~-DTE. The protein is then refolded in a redox buffer corl~in;ng oxirli7~d
~lut~thione and L-arginine.
One of skill would r~cogni~ that m~lifir~tionc can be made to the single
chain Fv region and fusion proteins comprising the single chain Fv region without
~iminiS1ling their birlcgjr~l activity. Some m~ifiration~ may be made to f:~~ilit~te the
cloning, e~ression, or incol~o.dtion of the single chain Fv region into a fusion protein.
Such m~ifi~tionc are well known to those of skill in the art and incllld~, for eY~mple, a
methionine added at the amino ~~ inl~s to provide an initi~tion site, or ~ition~l amino
acids placed on either t~ c to create conveniently located restrir,tion sites ort ....;n~;on co~n~ For eY~mp~e, in a ple~e~led embo~liment~ the primers used to
construct B5(Fv) will introduce a sequence encoding an iniLi~ mP~ onin~ for
.sion in E. coli and an Ndel restrirtion site to f~rilit~t~ cloning.
One of skill will recognize that other mo~ifi~tions may be made. Thus,
for e~mple, amino acid substitutio~c may be made that increase sre~-ificity or binding
affinity of single chain Fv region and fusion proteins compri~ing the single chain Fv
region, etc. ~ /ely~ non es~n~;~l regions of the mol~ule may be shortened or
elimin~t~Pd entirely. Thus, where there are regions of the moloculP that are nott~l~m~Plves involved in the activity of the mol~ule, they may be eli~ n~fi or repl~ Pd
with shorter sçgment~ that serve to ...~in~.~ the correct spatial relationships between the
active co",ponents of the molecule. ~It~Prn~tively more flexible seg...~nl~i may be placed
in interdomain regions which then can farilit~tp folding or production of the molecule
(Rrinkm~nn, et al. Proc. Natl. Acad. Sci. USA, 89: 3075-3079 (1992).
- 30
Di~pnostic Assays
In ~dition to the ~-~e~ing of immunotoxins to tumors in a cancer patient,
the recombinant antibodies of the present invention also recognize m~teri~ls such as
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28
surface mucins on tumor cells that would be e~ ed to be shed into the ~Ulluu~d~
tissues, picked up by the blood stream, and det~P~t-qhle in blood ~mpl~~ taken from
distant sites. Such shed ~qnti~enc have proven to be useful in the diqg~ncic of primary
and lecull~nt cancers using qntiho~lips that react to these shed ~ntigPnc A cull. nlly
useful ~ E~ of this is the CA125 antigen that can be assayed in sera from pqtiPntc
with ovarian cancer to predict lccwl~ nce or to confirm a primary ~iiqgnocic of tumor.
Similarly, Bl, B3 and B5 may be useful in the di ~ no,cic of tumors.
Also, the selective reactivity of these antibodies with certain types of
tumor cells may be exploited for anatomic pathological ~ qgnosic of t!~mors, cl~iryii g
the type and origin of tumors, and whether a particular group of cells ~ep~senl~ a
~cull~,~ce of a previous tumor or the development of another primary tumor els~ c~c.
Such a diagnostic dct~ q-t;o~ can be useful for the ~se~uent plqnning of anti-tumor
therapy in each particular patient. In particular, immunohictochemi~-ql pathologic
di~nQ~i~ in tissue SeCtions (e.g., biopsies) or cytological prepqration~ (e.g., Pap smears,
effusions) can be ~lr~ led using the monoclon"l antibodies of the present invention.
Another potential use of such targeting qntiho~i~c could be in the ~li~nn~ic
of macluscopic foci of a tumor using antibodies Bl, B3 or B5 coupled to r,qdioi-cotopes
that could be d~l~cl~ either by eYt~rnql body SC~qnning (im a~ing di~gnosis) or by
loc~li7~ti~ n using r~di~tion ~e~tor probes at the time of eYplor~tory Sl~lgcl~
In general, the r~i ~ nostic m eth ~ s described above involve co~t~cting a
Bl(Fv), B3(Fv), B5(Fv) or chim ~.ric Fv region with a biological sample either in vivo or
ex vivo and s~s~u~ nlly ~et~ting the binAing of that antibody to the target tissue. In a
prcf~lcd embodh--cnt a ~li~nostic method comprises the steps of (a) removing a tissue
or fluid sample from a patient; (b) adding an antibody which includes the Fv region of a
heavy chain of a first antibody and the Fv region of a light chain of a second antibody,
where the Fv regions are recombinantly fused to form a single molecule that specific~lly
bind a LewisY-related c~l,ohydldte ~ntigPn; and (c) detectin~ for the presence or absence
of the antibody in the ~ mI'I~-
In a prefelled embodiment, detection is by the dete~tion of a label bound
to the antibody. Means of labeling antibodies are well known to those of skill in the art.
Labels may be direclly linked through a covalent bond or covalently through a linking
mol~cnle which typically bears reactive sites capable of forming covalent bonds with the
label and the antibody le~ rely. A common approach is to label the antibody and the
CA 02203236 1997-04-21
WO 96/13594 PCTIUS95/13811
29
hbel with either avidin or streptavidin or biotin which, in turn, bind irreversibly with
each other.
Suitable labels are well known to those of skill in the art. The term
"label", as used herein, refers to a co",~si~ion dett ~t~hle by s~ectluscopic,
S phot~hc...ir~l, bioç~ ~h~mir~l, or c~ mir~l means. For e~ample, useful
labels include Pdioa~tive mol~cules such as 32p, 14C, l25I, 3H, and 35S, fluor~scellt dyes
such as fluo,~3~in or rho~A...;..ç, electron-dense reagents, enzymes (as commonly used
in an ELISA), lumin~scent enzymes such as luciferase and the like.
10 Phalmac~ l Compositions
The ~ecomhinAnt fusion proteins and pharm~c~uti~l compocitions of this
invendon are useful for p~ e-~l, topical, oral, or local, ~minictration, such as by
aerosol or tr~ncderm~lly~ for prophylactic and/or th~,a~eulic ~ ..rnt The
p~ Aceutir~l compositionc can be ~iministered in a variety of unit dosage forms
15 ~n~ing upon the method of ~minict~tiQn. For example, unit dosage forms suitable
for oral ~~mini~trati~n include powder, tablets, pills, c~ps~les and lo,~nges. It is
1~'4~,r~ that the fusion proteins and ph~rm~eutir~l compositions of this invention,
when ~lmini~t~ored orally, must be plo~c~d from digestion. This is typically
acco-.-~ lich~ either by complexing the protein with a co-~ ilion to render it reci~t~nt to
20 acidic and ~ lllalic hydrolysis or by p~rl~ing the protein in an ap~lo~,iately resistant
carrier such as a liposo...e. Means of protecting proteins from digestion are well known
in the art.
The recombinant fusion proteins and pharm~c~utir~l co~ )oc;linns of this
invention are particularly useful for ~alt;nte~ dminist~tion, such as intravenous
25 ~ministration or ~mini~tr~tion into a body cavity or lumen of an organ. The
co---posil;~n~ for ~-imini~tr~ti-~n will commonly comprise a solutinn of the single chain
antibody or a fusion protein compri~ing the single chain antibody dissolved in apharm~euti~lly ~ccept~hle carrier, preferably an aqueous carrier. A variety of aqueous
c~,ie~ can be used, e.g., buffered saline and the like. These solutions are sterile and
- 30 gene~lly free of unde~ hle matter. These compositionc may be sterili7Pd by
convenLional, well known st~rili7~tion techniques. The co."~;Lions may contain
pharmac~uti~lly ~ept~hle ~llxili~ry substances as required to appf~im~te physiological
co~1ition~ such as pH adjusting and burr~ing agents, toxicity adjusting agents and the
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like, for eY~mple, sodium ~Rt~te, sodium chl- ri~e, pot~inm chloriAe~ c~lrillm
chlori~e, sodium lactate and the like. The concentration of single chain antibody, fusion
protein, or labeled single chain antibody in these formulations can vary widely, and will
be ~l~t~ prim~rily based on fluid volllm~s, vi~co~itip~ body weight and the like in
5 accor~ce with the particular mode of ~~mini~tr~tion s~l~te~ and the patient's needs.
Thus, a typical pharmAreutirAl col.-posilion for intravenous ? imini~tr~tirJn
would be about 0.01 to 100 mg per patient per day. Dosages from 0.1 up to about 1000
mg per patient per day may be used, particularly when the drug is ~ iminict~red to a
s~lud~d site and not into the blood stream, such as into a body cavity or into a lumen of
10 an organ. Actual metho~$ for pl~aling pa,enlelally ~-1minictr~hle colllpos;L;ons will be
known or ap~arent to those skilled in the art and are described in more detail in such
publirAti-n~ as Remington's Phann~7cel~ic~77 Science, 15th ed., Mack Publiching
Co.llpa,ly, Easton, Pennsylvania (1980).
The co...~ nc conl~ining the present fusion proteins or a coc~t~il
thereof (i.e., with other proteins) can be ~lministpred for the~Al eulic tre~tmpnt~ In
thi ~l eulir applir~tirJn~, colllpoS;~;rJn~ are ~~lmini~tered to a patient ~urr~ g from a
~ii~, in an Amount s~ffiri~-nt to cure or at least partially arrest the disease and its
complir~tinn~. An amount adequate to accomplish this is defined as a "th~.Al~eulir~lly
effective dose. " Amounts effective for this use will depend upon the severity of the
disease and the general state of the patient's health.
Single or multiple ~-lminict~Ation~ of the coll,~iLions may be ~-lmini~tPred
depen-ling on the dosage and fic~luellcy as re~uiled and tolerated by the patient. In any
event, the colllposilion should provide a sllffici~nt ~luanlil~ of the proteins of this
invention to effectively treat the patient.
Among various uses of the ~loloAic fusion proteins of the present
invention are included a variety of disease con~iitions caused by spe~ific human cells that
may be Pl;...;n~Pd by the toxic action of the protein. One prer~llcd application is the
eAt~..ent of cancers, in particular cancers in which the tumor cells express carbohydrate
~ntigen~ that are members of the LewisY family. Such cancers incltlde, but are not
30 limited to colon, breast, esophagus, bladder, gastric, head and neck, lung and ovarian
c~r.~o",as. The fusion proteins may also be used in vitro, for example, in the
e]imin~tion of harmful cells from bone marrow before trAn~plAnt where those cells
express LewisY-related antigens.
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31
Kits
This invention also embraces kits for .~ h or ~liq.~nQstic purposes.
Research kits typically include one or more co~t~inprs cont~ining the single chain
antibodies of the present invention. In a ~ef~l-ed embo-limpnt~ ~~sea-~ch kits comprise
S co~ ;n~ ~ cor.~inin,~ single chain Bl(Fv), B3(Fv), B5(Fv), chimPric Fv, ...I~ Pd Fv or
h.. ~n.,~d Fv antibodies in a form sllit-qhlP for derivatizing with a second mol~cl-lP, e.g.
a label, a drug, a ~;y~oto~i~-, and the like. In another e ..ho~;...~nt~ the .~sed-.;ll kits may
contain DNA sequences enco lin~ these antibodies. P.t;f~ dbly the DNA sequences
e ~in~ these antibodies are provided in a plasmid suitable for trqn~fP~tion into and
10 ~A~ sion by a host cell. The plq~mid may contain a promoter (often an inducible
promoter) to regulate eA~less;on of the DNA in the host cell. The plq~mi~ may also
contain a~prop~iate restriction sites to fq~ilitqtP the insertion of other DNA sequences
into the plq~mi-l to produce various fusion proteins. The plq~mi~$ may also contain
nu~cr~nls other cl~ to fql ilitqte cloning and t;Ay-ession of the en~ed proteins.
15 Such elPmpnt~ are well known to those of skill in the art and in~ de, for example,
S~plprt-qhle~ nitiqti~n CO~Q~ on codon~, and the ~e.
Diqgnostic kits typically comprise cont~;n~ conti~ining the antibodies
d~sc. ;hed above. The q-ntihodiPs are thPm~PlveS derivatized with a label or, ql~ ;v~ly,
they may be bound with a secondary label to provide subsequent dete~tion. As descrih~Pd
20 above, such labels may include r~q~ lqhel~, flu.~ ~scel~t labels, el~y--,atic labels, i.e.,
horseradish pero~ q-~e (HRP), or the like. The kit may also contain app-ol) iates~nd~. y labels (e.g. a sheep anti- mouse-HRP, or the like). The kit may also contain
various .cagen~ to fq~ilitqte the binding of the antibodies, the removal of non-s~ific
binding antibodies, and the detection of the bound labels. Such reagents are well known
25 to those of skill in the art.
Methods for using the research and diagnostic kits described above are
generally well known, and will generally be provided in an instluction manual for use of
the kit.
EXAMPLES
The following eYq~mples are offered by way of ill~lstr.qtion, not by way of
limitqtion.
Example 1
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32
Clnnin~ of DNA Fr~nPrlts Encorlin~p the Heavy and ~ ht Fv I~P~ion of MAb B3
B3 cloning ~ ;.,.entc and propagation of p!~cmiric were carried out
initially in E. coli HB101 (Boyer et al., J. Molec. Biol. 41: 459-72 (1969)). DNA
mPntc enr~linE the Fv portions of heavy and light chain of monoclc)n~l ~ntihody
5 (MAb) B3 were obt~ined by (PCR~) amplifir~tion of single st~nd~d DNA which wassynthPci7~ by random primed reverse t~nccription of mRNA from a B3 mnnoclon~l
antibody producing hybridoma cell line. Polymerase chain reaction (Saiki et al.,Science, 239: 487-91 (1988)) was pe.ro~ d using the Perkin Elmer GeneAmp kit andan Perkin Elmer/Cetus thc~ ~ycler, under conditi~nc as dçscnhed in Ch~l-rlh~ry et al.,
Proc. Natl. Acad. Sci. USA 87: 1066-70 (1990).
The primer pair B3-Hl and B3-H2 was used for amplifir~tion of the heavy
chain Fv coding region, while the primer pair B3-Ll and B3-L2 was used for
amplifir~tion of the light chain Fv coding region (see Table 1 and Sequence ID Nos. 1,
2, 7, and 9). These oligonuclçQtides have at their 3' ends con~ t sequences that occur
15 at the beginni~g and end of mouse Fv DNA. At their 5~ ends are restrirtirJn
endomlrl~ce ~ecognilion sites (NdeI, R~mT-TT,T-Tin-lTTT) for cloning of the PCR products
as shown in Fig. la. The products of the ~mplifir~tions of heavy- and light chain Fv
DNA fr~mPntc were idPntified by agarose gel electrophoresis to be DNA fr~gm~nt~
~t~o~n 350 and 400 bp. They were purified from gels, cut with BamHI or T~Tin-lm
20 (Fig. la) and, after pllrifir~tion on a second gel, ligated with T-Tin~lm- or BamHI
lin~ri7f~d and dephosphorylated pBR322 vector (Bolivar et al., Gene, 2: 95-113 (1977)).
The nurl~otide sequence of the light- and heavy chain Fv coding region of monoclonal
antibody B3 was de~ "~ined from double stranded pl~cmid DNA using sequenring
plilllel~ (New F.ngl~nd Riol~hs, Beverly, lU~cc~cl.~ c, USA) ~jar~nt to the BamHI or
25 ~in~m site of pBR322 and a T7 polymerase sequencing reagent kit (United States
BiochPmic~ls, Cleveland, Ohio, USA).
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33
Tabl- 1. PCR primer~ u~ed to amplify Fv heavy and light chain6. The Frl
primer~ were de~igned according to the amino acid ~eguence~ which were
dete i n~ by Edman degradation, and are indicated in oingle letter code
above the primer ~eguences. Underlined Met are initiator methion;n~
S codons. Other underlined amino acid~ in the light chai~ primers are
~ of the peptide linker that fu~e~ VH to VL in the ~inqle chain
configuration. Underlined nucleotides in BlHFrl and B5HFrl encode the
initiator me~hioni ne for expres~ion in E. coli, and include an NdeI
re~triction site. Underlined nucleotide~ in BlHFr4 and B5HFr4 are
complementary to the coding sequence for sc, --Ls of the peptide linker
that fu~es VH to VL in the single-chain configuration. Underlined
nucleotides in BlLFr4 B5LFr4 are complem~ntary to the coding ~equence of
the junction between the Fv and PE38 and include a Hi~dIII restriction
~ite.
Sequence
Seq.
Name 5'-3' ID
Heavy chain primers
B3-Hl TAACTAGGATCCGTCCATATGGATGTGAAGCTGGTGGAG-
TCTGG
25 B3-H2 TGGATAGACTGATGGGGATCCGCCTCCGCCTGAGGAGAC 2
M E V Q L V E S G G 40
BlHFrl ~ATATACATATGGAGGTGCAGCTGGTGGAATCTGGAGGA 3
_ E V K L V E S G G 41
BSHFrl GATATACATATGGAGGTGAAGCTGGTGGAATCTGGAGGA 4
GammaCHl AGCAGATCCAGGGGCCAGTGGATA 5
BlHFr4 ACCGGATCCGCCTGCAGAGACAGTGAC 6
B5HFr4 ACCGGATCCGCCTCCGCCTGAGGAGACAGTGAC/G 7
L iqh t ch a in D r im er s
B3-Ll GTCTCCAAGCTTGGGGATCCGGTGGTGGCGGATCTGGAGG- 8
TGGCGGAAGCGATGTGCTGACCCAGTCTCC
B3-L2 AGTTGGTGCAGCATCAAAAGCTTT[G/T]A[G/T][T/C]- 9
TCCAGCTT[T/G]GT[G/C]CC
B3-L3 TTGGGGATCCGGTGGTGGCGGATCTGGA 10
45 B3-L4 AGCGGGAATTCATTATTTAATTTCCAG~lll~lCCCCGAC 11
G G G S D V V M T Q 42
BlLFrl GGTGGCGGAAGCGATGTTGTGATGACCCAA 12
G G G S D V L L T Q 43
B5LFrl GGTGGCGGAAGCGAl~llllGTTGACCCAA 13
C-kappa TGGTGGGAAGATGGATACAGTTGG 14
BlLFr4 GGAAGCTTTCAGCTCCAGCTTGGT lS
SS B5LFr4 GGAAGCTTTATTTCCAACTTTGT 16
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34
Example 2
Cloning of DNA Fragments Fnro~;n~ the Hea~y and Light Fv Segments of MAbs Bl
and B5
To obtain DNA en~-~in~ the variable regions of the heavy and light chains
S of Bl and BS, Poly(A)+ mRNA was pr~pafed from 105 hybridoma cells and
rcv~ t~nc~ribed using random hPY~mPrs as primers to yield first strand cDNA.
SeF~r~tP PCR~ re~tinns were carried out to amplify fragmPnt~ encomp~ing heavy
chain variable through part of CHl domains, and light chain variable through part of
C-kappa. The Bl and BS VN sequences were ~mplifiPd using S' end primers de~ignPdaccording to the amino-te~nlinal protein sequence of the Bl and B5 heavy chains and 3'
end primers ~ecignPd according to conc~pncus immunoglobulin conct~nt region sequences
(Kabat et al. (1991) supra.). In particular, Bl VH was ~mplifiPd using 5' end primer
BlHFrl and 3' end primer ~mm~CHl (Table 1, Sequence ID Nos. 3 and S
l~;livdy). BS VH was ~mp1ified using S' end primer BSHFrl and 3' end primer
B5HFr4 (Table 1, Sequence ID Nos. 4 and 7 ~ ively). Primer ~.~mm~CHl was
designPd according to con~nc~s IgGl CHl region codons 122-129 while primer BlHFr4
was d~P-~ignP~ according to the del~lllined nucleotide equence of codons 109-113 of Bl
VH (Kabat et al., (1991) supra.).
The Bl and B5 VL sequences were ~mplifiP~i using 5' end primers BlLFrl
and B5LFrl (Table 1 and Sequence ID Nos. 12 and 13) which were d~Psi~nP11 according
to the amino-l~-,-inal protein sequence of Bl and B5 light chains ~espe~;liv-ely (Sequence
ID Nos. 20 and 22) in combination with the primer C-kappa (Table 1, Sequence ID No.
14). Primer C-kappa was decignP~ according to concpncus kappa light chain codons113-120 (Id.).
PCR was pelrol........ ed as described by Rrinkm~nn et al., Proc. Nat. Acad.
Sci. USA 88: 8616-8620 (1991).
The crude PCR products were subcloned into a PCR~ cloning vector
(Invillogen, San Diego, California, USA) employing blue/white selection. Clones
co~t~ining the correct size insert by DNA restriction analysis were identified. The
30 nucleotide sequence of the heavy or light chain coding regions was determined from
double stranded plasmid DNA using sequen~ing primers (Invitrogen) ~ epnt to the
PCR~ EcoRI cloning site and the SequenaseTM kit (United States Biochpmir~l Corp).
Three to five independent clones were sequenced for each amplified DNA segment. The
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nucleotide sequences of the Bl VH and VL are shown in Sequence ID Nos. 19 and 20les~ecliv~ly, while the nucleotide sequences of the BS VH and VL are shown in Sequence
ID Nos. 21 and 22 ~ ectiv-ely. BS could not be ?~mplifi~l using a mu chain CHl
primer, so the c~n~ns-~c heavy chain BSHFr4 primer (Table 1, Sequence ID No. 7) was
S used instead. VH Primer BSHFr4 was decignPd according to concPn~s IgGl Fr4 region
codons 109-113 (Kabat et al., (1991) supra.)
~ lipnmPnt of Bl, B3 (Rrinkm~nn et al., Proc. Nat. Acad. Sci. USA 88:
8616-8620 (1991)), and B5 Fv sequences revealed that B5 is highly homologous to B3
(91.6% identity in VH and 94.9% identity in VL coding sequence) and to the anti-LewisY
antibody H18A (l~nP~ et al., J. Biochem., 113: 114-117 (1993)) (93.3% identity in VH
and 97.6% identity in VL coding sequence). Bl differs concide~hly both in framework
and in CDR sequence from both B3 (83.9% identity in VH and 88.9% identity in VL
coding sequence) and B5 (86.3% identity in VH and 91.2% identity in VL coding
sequence), and does not show high sequence identity to any known anti-carbohydrate
antibod~ in a d~h~ce search (Dc~re,~.lx et al., Nuckic Acids Res., 12: 387-395 (1984)).
All three ~nt~ os have a mouse class m heavy chain and a kappa II light chain (Kabat
et al., supra). The di~r~iences in sequence belween Bl and B3 may explain why they
recQgJli7~ dirr~c"l epilopes of otherwise similar antigens (see, Pastan et al., Cancer
Res., Sl: 3781-3787 (1991) and U.S. Patent No. 5,242,813).
Example 3
Construction of Plasmi~c for Expr~ccion of B3(Fv) and B3(F~ lmmunoto~inc
A) Construction of B3(Fv) and B3(Fv)-PE40
The ~Ap~ss;on pl~cmi~ pVC38H con~ ;nc the gene from the immlmQtoxin
25 TGFa-PE40 under control of the T7 promoter (Ch~-)dh~ry et al., Proc. Natl. Acad. Sci.
USA 87: 1066 70 (1990)), the Tc ~nc~ription termin~tor at the 3' end of the PE40coding region and the single strand replication origin, F+, to generate single st~nded
phage DNA by cotransfection with (M13) helper phages, if desired, to create derivatives
of the p~mid by site dilcc~ed mutagenesis. The TGF~x coding region in pVC38H has30 an NdeI ~ecognilion site at the 5' end and a ~in-lTTT site at the point of conn~tion to the
DNA e-nco~ling PE40.
To create a pl~mid for ~Aplcssion of the immunotoxin B3 (Fv)-PE40
(pULEE3), the TGFa gene was removed and reE~l~re~ by the B3(Fv) gene in a 3 -
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36
fragment li~ti~n, using an NdeI/BamHI fr~mPnt of the heavy chain coding region and
the T~m~Tl~Tin~m fr~m~n~ encoding the light chain Fv (Fig. la). R~u~ sequence
analysis showed a mut~tion (dPletion and fr~mP~hift) at the 5' end of the light chain Fv
gene due to a sc~luence repetition in the PCR primer ~nn.o~lin~ region, site-directed
mutagenesis was pe,~l."ed (Kllnk~l, Proc. Natl. Acad. Sci. USA, 82: 488-92 (1985)),
using uridine incol~ldted single st~nded phagemid DNA (pULEE3) as the mut~en~sistçmpl~te. In the res--ltin~ pl~mid (pULIl), the correct amino end of the B3 light chain
established by partial protein sequçn~in~ of mnnoclon~l antibody B3, was reconstructed.
To make another B3(Fv) im",u,lo~o~ , B3(Pv)-PE38DKEL, the PE40
coding region was removed from pULIl from the T-TinATTT site to an EcoRI site po~itiol-çd
just beyond the PE40 gene, and re~l~ce~ by a ~indmlEcoRI fr~mt-nt from pRK79K
çncodin~ the PE variant PE38KDEL which lacks domain Ia (amino acids 1-252) and part
of domain Ib (amino acids 365-380), and also cont~ins an altered carboxyl tel" inal
s~u~nce KDEL (C'h~udh~ry et al., Proc. Natl. Acad. Sci., 87: 308-12 (1990)). TheeA~,iession pl~miA pULI4 for production of B3(Pv) was constructed by removal of the
light chain and PE40 coding region from pULI1 from BamHI to EcoRI which was
replaced by a PCR f~ment obtained by ~mplifi~tio~ of the light chain Fv coding
sequence with the primer-pair B3-L3 + B3-L4. The primer B3-L3 (Table 1) is similar
to B3Ll,used for cloning of light chain Fv from cDNA and B3-L4 (Table 1) is, in the 3'
part for primin~ the PCR, identic~l to B3-L2, but, at the 5' end, the ~intlm site for
fusion to PE-sequences is repl~ced by t~n~l~ti-)n stop codons followed by an EcoRI
,~gn~lion sequence.
B) Construction of pULI7. the ~ ",il,d for Exl~.~ on of LMB7. (B3(Fv)-PE38
with C~Connector
B3(Fv)-PE38, also called LMB7 is one recombinant B3(Fv)-imm--notoxin
of this invention preferred for use as a cancer the~apeulic. The pl~mi~ pULI7 for
.;A~ ,s~ion of B3(Fv)PE38 was constructed as follows: Plasmid pULIl contains the Fv
region of monoclonal antibody B3 in the form of a single chain Fv co~ ining a
(Gly4-Ser)3 peptide linker between VH and VL, fused to PE~0, a trun~ ~ted form of
Pseudomonas exotoxin (see Rrinkm~nn et al., Proc. Nat. Aca~. Sci. USA 88: 8616-8620
(1991) and Fig. lb). To improve folding and production of this molecule, a flexible
"connector" peptide, de~ign~ted C3, was added between the Fv and the toxin moiety by
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37
PCR mutagenesis (Rrinkmqnn, et al. Proc. Natl. Acad. Sci. USA 89: 3075-3079 (1992))
to result in pULI6. Pinally, part of the toxin portion of pULI6 was re~ d with ashorter mo~ le with the same activity, PE38 (lacking domain Ib of PE), repl-q~ ng by
subt~l~ning a SalI-EcoRI toxin fragment of pULI6 with the PE38 coding SalI-EcoRIfragment of pCS10 (Siegall et al. J. Biol. Chem., 264: 14256-14261 (1989)). The
res~-lting ~ s~ion plq~mi-l, which codes for the immunotoxin B3(Fv)-PE38 is pULI7
(Fig. lb).
FY~mple 4
Construction of 1~ 1$ for EA~ ~ On Of B~ and B5(IiO-lmmunotoYAins
For c~n~ssion as single-chain immllnotoxins, Bl and B5 Fv fra~mPn
re~l~~~ B3Fv sequences in the e.~ ssion plq~mid pULI7 which e-n-~,odes the
B3(Fv)-PE38 i~ unotoxin (Benhar et al. Bioconjug. Chem., 5:321-326 (1994)). For
each Fv, the VH sequences were PCR ~mplifi~d using the heavy chain clones in PCR~
plq~mi~1s as t~n plqtçs. Primers BlHFrl and 5~-phosphorylated BlHFr4 were used to
a",l~liry B1VH, while p,i",el~ B5HFrl and 5~-phQsrhorylated B5HFr4 were used to
amplify BSVH. The VL sequences were qmplifi~pd using the light chain clones in pCR~
plqcmids . S l,~,"~ t,5 ~il~ BlLFrl and 5~-phosphorylated BlLFr4 were used to
amplify B1VL, while plilll~ BSLFrl and 5'-phosphor,vlated BSLFr4 were used to
a"l~liry B5VL. The primPrs had at their ends sequences that are comp]A-nent~ry to the
tr-qnCl-q-tion initiqti~n, peptide linker and Fv-toxin junction (col~n~lQr) which are collllllo
to the single-chain Fv-immunotoxin e ~ression vectors. Primers BlLFr4 and B5LFr4were de-signed according to the detPrmined mlclPQtide sequence of codons 102-107 of Bl
and B5 VL respectively. The PCR ~mplifi~ ti~A~ns were ~lrul,l-ed as described inExample 2.
The PCR pludu-;ls were purified using spin col~mn~, combined and
~nnP~lPd to a uracil-co~.lAinin~ single-stranded DNA phagemid pULI7 which encodes the
single-chain immllnotoYin B3(Fv)-PE38. The phagemid was plcl~cd by rescue of
pULI7 phagemid with an M13MK07 helper phage (Bio-Rad, Hercules, California, USA).
The DNA was eYtPnded and ligated according to the MUTA-GENE~ mutagenesis kit
p~locol (Bi~Rad).
Since the ~nne~ling efficiency of the PCR fr~gm.ont~ to the single-st-~nd~d
template, and hence the mutagenesis effici~Pncy was relatively low (about 10%), an
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38
ad-litir~n~l step was added. Plasmid DNA obtained from a pool of transro""anls from
the mut~PnP~i~ reaction was digested with a restriction endon~ P~e which had a unique
site in the B3Fv t~Pmrl~t-p~ but whose site was absent from both Bl and B5. The digested
DNA was used to re-transform E. coli cells. Following this extra step ~n~u~n~ were
S ob~in~d with an çffil~iPncy greater than 80%. Correct clones were i~ipntifipd by DNA
restriction analysis and verified by DNA sequPn~ing. The res~llting i~ lnotoxin clones
were narned pBl(Fv)-PE38 and pB5(Fv)-PE38.
le S
Expression and Purification of Recombinant B3(Fv)-Immunotoxins
pl~mi~s were lldn~rol",ed in the ~y~ssion-host E. coli BL21 (~DE3)
(Studier et al. J. Mol. Biol. 189: 113-30 (1986)). The b~l~t~ri~ were grown in
~up~ conl~ining 0.2~.4% glllc~se, 0.05 % MgSO4, and 100 ~g/ml ~mpicillin,
induc~d in the log phase at OD600 of 3.0 with 1 mM isop,uy~l-B-D-thiogala~;~y~ nos;~le
(IPTG) and harvested 90 min later. About 30% of the total protein of the induc~dcultures was the leco",binant ~ yress;on product which was depo~iled in inclucir)n
bodies. The purified in~ inll bodies cont~in~ almost pure recombinant protein, which
had the ~ l e.c~d size of about 67 kDa for a single chain imml-notoxin. The recombinant
i"""unotoxin mol~ules were solubilized, refolded, purified, and the protein was
analyzed as previously desçrihed (ch~ h~ry et al., Nature 339: 394-97 (1989) & Batra
et al., J. Biol. Chem. 265: 15198-202 (1990)). Protein conc~ntr~tions were dele~,inf~d
by Bradford assay (Bradford, Anal. Biochem. 72: 848-54 (1976)).
Example 6
E~ on and Purification of Bl(Fv)-PE38 and B5(Fv)-PE38
Cultures of E. coli (BL21~DE3, see Studier, et al. J. Mol. Biol. 189:
113-130 (1986)) were transrol",ed with each ~A~lession pl~cmi~ to produce Bl(Fv)- and
B5(Fv)- immunotoxins. Following IPTG induction, the overproduced fusion proteinsaccumulated in in~ ion bodies. These were i~ol~t~d by solubilization and refolding of
incl~ on body protein using redox-shuffle as described (Buchner et al., Anal. Biochem.
205, 267-270 (1992)). BAefly, inclu~ion bodies were dissolved in 6 M gll~ni~1ine(HCl)/0.1 M Tris(HCl) Ph 8.0/2 mM EDTA and reduced by the addition of solid DTE
to a final conc~nt ation of 65 Mm at a protein concentration of 10 mg/ml. The
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solubilized and reduced inchlsion body proteins were diluted x 100 into 0.1 M Tris (Hcl)
Ph 8.0/0.5 M L-arginine/0.9 Mm o~riAi7.ed glut~thione/2 Mm EDTA and were allowed to
refold for 36 hr at 10~C. The refolded proteins were extensively dialyzed against 20
Mm Tris (Hcl) Ph 7.4/2 Mm EDTA/0. l M Urea. P u~lly refolded proteins were
S sep~aled from cont~min~ting proteins and aggr~gates by sequential ion-eYch~ngecl;lolnatography on Q ~,harose and Mono Q columns (Pharmacia, Piscalawdy, New
Jersey, USA) followed by size eYclu~ion chromatography on a TSK G3000SW
(Toso~7 Montgomeryville, Pennsylvania, USA) column. Typically, purified
monomeric proteins were over 95 % pure.
Example 7
Cytotoxic Activity of Ch~ ;rPlly ~.;nlr~3 and Recombinant B3(Fv)-Immunotoxins
Assays ...~ ;"g inhibition of protein synthesis were p~lrol.,.ed as
previously ~les~ribeA. (~h~u-lh~ry et al., Nature, 339: 394-97 (1989) and Batra et al., J.
Biol. Chem. 265: 15198-202 (1990)). All assays were performed in 96 well plates each
well co~ ing 1.6 x 104 cells in 200 /11 meAium. For co-,lpt;Lilion assays ~e~ign~A to
prove the ~cificity of the recombinant immlmotoxins~ the meAillm was changc~ and 50
g/well of ~ntibo~y was added 15 min prior to the addition of the illllllllt~o~
As shown in Figure 3 and in Table 1, the recombin~nt single chain
20 immlln~toxins inhibited protein synthesis in cells t;Ap~cssing the B3 antigen but not in
non e,~lcs~ing cells, simil~rly to the previously described results with cllP-m~conjugate of B3 with a trunc~t~d form of PE (Pai et al., Proc. Natl. Acad. Sci. USA, 88:
3358-62 (1991)). The relative potencies of the c~emic~l conjugate and the single chain
i"",..inot~uns were about the same on the four antigen positive cell lines MCF7,CRL1739, A431 and LNCaP. The most active agent was B3(Fv)-PE38KDEL.
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Tabl- 1. Activitie~ of B3 immunotoxins on different cell line~.
Cytotoxicity (ID~) in ngtml (pM).
Cell Cancer Type B3 B3(Fv)- B3(Fv)- B3-Ly~PE40
Line antigen PE40KDEL PE38
MCF7 brea~t ++ 3 (50) 0.2 (3.2) 3 (16)
CRL173 ga~tric ++ 3 (50) 0.3 (5) 3 (16
A431 epidermoid + 3 ~50) 0.8 (13) 8 (42)
vulva
LNCaP pro~tate + 40 20 (325) 85 (460
(1330)
KB3-l epidermoid - ~1000 >1000 >1000
cervix
HUT102 adult T cell - ~1000 >1000 >1000
leukemia
The recombinant single chain B3-Fv immllnQtoxins did not affect B3
antigen-negative control cells. The cytotoxicity of the recombinant B3(Fv)-PE40 (ID50 =
50 pM; 3.0 ng/ml) was similar to the chPmir~lly linked B3-immunoconjugate (ID50 = 42
pM; 8 ng/ml), whereas B3(Fv)-PE38KDEL was much more active than the chPmi~l
conjugate (ID50 = 13 pM; 0.8 ng/ml). This is despite the fact that the single chain
ot~Ains possess only one antigen binding site per mol~P~ulP and the ch~-mi
conjugate has two (see Table 2 below).
Table 2. Structure and Activity of B3 Immunoto~ins on A431 Cel 8.
ImmunotoxinToxin Part C-TermBinding ID~
B3 c~ ioa l PE40 REDLKbivalent 8.0 ng/ml
conjugate (42 pM)
25B3(Fv) PE40 REDLKmonovalent3.0 ng/ml
fu~ion (50 pM)
protein
B3(FV) PE38 KDELmonovalent0.8 ng/ml
fusion (13 pM)
30protein
B3(Fv)-PE38KDEL has two features that distinguish it from B3(Fv)-PE40.
One is that a portion of domain Ib encomp~ing amino acids 365-380 is deleted. This
removes the di~ulfi~e bond formed between cysteine residues at positions 372 and 379,
35 which might form ~ ulfide bonds with other cysteines during the ~ alu~tion process
and thereby result in the creation of inactive chimP-ric toxins. The second feature is that
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41
the c~l u~yl ~- ...in~s of the toxin is changed from the origin~l sequence REDLK to
KDEL. When the ~i~ulfide bond was removed in other mol~culPs, the increase in
activity was small. For eY~mplr, TGFa-PE38 is only 50% more active than
TGF~-PE40 (see Siegall et al., J. of Biol. Chem. 264: 14256-14261 (1989)). IL6PE38
5 is no more active than IL6 PE40. Ch~nging REDLK to KDEL usually only produces a
two to three fold increase in activity of chimP-~ic toxins.
To analyze whether the ~;yloto~icity of B3(Fv)-i.. ~ olo~ins was specific,
co...pe~ n eY~rim~pnt~ were carried out with an excess of monocl( n~l antibody B3.
The data in Figure 3(b) shows that the intoxi~tion of A431 carcinoma cells by B3(Fv)-
PE38KDEL is due to the specific binding to the B3 antigen, since its CylOtO~iCity was
blocked by excess B3 but not by MAb HB21 which recogni7es the transferrin rece~lor on
these cells (Haynes et al., J. Immunol., 127: 347-51 (1981)). A large excess of
monorlo~l~l antibody B3 is n~c~A.y for reversal of cytotoxicity, probably because there
is a large amount of the B3 antigen on the surface of A431 cells (Pai et al., supra.)
F~h~ le 8
Anti~en Bin-li~. ADP-Ribosylation and Specific Cytot~Yici~y of Recombinant
Imll~uL~toxin~
A) ADP-Ribosylation Activity
The ADP-ribosylation activity of edch of the immunQtoxins was to tested
to verify that they were of equal en~y~l-alic activity. ADP-ribosylation activity was
de~~ ed by the inco.~ldLion on ['4C]-NAD into acid-pre~ip;L~hlp m~Pri~l using
elongation factor 2 enric~p~ wheat-germ extract (Collier and K~n~Pl, J. Biol. Chem.,
246: 1496-1503 (1971)). As shown in Fig. 4(A), B3(Fv)-PE38, which was used as a
reference molecule, Bl(Fv)-PE38 and B5(Fv)-PE38 had similar ADP-ribosylation
activities.
B) Specific C~totoxicity
Cytotoxicity towards A431 cells was measured by the inhibition of
inccl~ld~on of [3Hl-leucine into cell protein, following 2 hours or 20 hours of
inrubation of the cells with serial dilutions of immnnotoxin~ in PBS + 0.2% BSA (see
Rrinkm~nn. et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620 (1991)). As shown in
Fig. 4(B), when tested on A431 cells which strongly bind the B3 and the Bl MAbs,
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42
B3(Fv)-PE38 has an IC50 of 2.8 ng/ml and 2.0 ng/ml following 2 or 20 hours in~ub~tion
ly. Bl(Fv)-PE38 has an IC50 of 0.6 ng/ml and 0.3 ng/ml following 2 or 20
hours incubation ~spec~ ely. B5(Fv)-PE38 has an IC-50 of 120 ng/ml and 20 ng/ml
following 2 or 20 hours in--ub~tion respectively.
S To check the speçificity of the immunotQ~in~' the same ~;~lolu~ic assay
(Rrinkm~nn et al. supra.) was done on ~ liti~n~l cell lines. As shown in Table 3,
B3(Fv)-PE38, Bl(Fv)-PE38, and B5(Fv)-PE38 had the same spectrum of recognilion of
the cancer cell lines tested albeit having dirrer~i~t levels of ~i~loto~ic activity toward the
antigen-positive cells, which correlates with the binding affinity of each immlmotoxin
toward its cellular binding site. These cell lines differ in their level of B3 or Bl antigen
.sion (Pastan et al., 1991; Rrinkm~nn et al., 1993; see Table 3).
Table 3. Cytotoxicity of Bl(Fv)PE38, B3(Fv)PE38, and B5(Fv)PE38 toward various cell
lines.
Cell Source Bl or B3 Bl(Fv)- B3(Fv)-B5(Fv)-
Line antigen PE38 PE38 PE38
expression
A431 Epidermoid +++ 0.3 2.0 20
carcinoma
MCF7 Breast +++ 0.6 4.0 22
carcinoma
LnCap Prostate + 2.7 21 210
carcinoma
KB 3-1 Cervical - ~lOOO >lO00~lOO0
carcinoma
HUT102 T-cell - >1000 >1000>1000
leu~
L929 Mouse - >1000 >lO00>1000
fibroblast
C) ~nti~en Bintlin~ Affinity of Bl(Fv)-PE38 and B5(Fv)-PE38
The specific antigen binding of the immunotoxins was further analyzed by
de~ llination of their binding affinity to antigen positive cells by colll~tition assays, in
which increasing co~ n~ ions of each immllnotoxin were used to colllpele the binding
of iotlin~tçd B3 IgG or Bl IgG to A431 ~lpnoc~rcinoma cells at 4~C as described by
Benhar et al., Bioconjug. Chem., (1994) supra). As shown in Figure 5, Bl(Fv)-PE38
coll,pe~d for the binding of [l25I]-Bl IgG to A431 cells by 50% at 1.3 ~M, and for the
binding of [~ -B3 IgG by 50% at 1.7 ~M. B3(Fv)-PE38 colll~led for the binding of
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43
[~25Il-Bl IgG to A431 cells by 50% at 2.7 ~M, and for the bin~ling of [l25Il-B3 IgG by
50% at 2.5 ~M. B5(Fv)-PE38 col,-peted for the binding of ['25Il-Bl IgG to A431 cells
by 50% at about 50-100 ~M~ and for the binding of [l25Il-B3 IgG by 50% at 50 ~M. Bl
IgG c~lll~t~d by 50% for tne binding of '25I labeled Bl IgG at 110 nM and B3 IgGS co.. ~ ed by 50% for the binding of l25I labeled B3 IgG at 200 nM (not shown).
The analyses of tne Bl(Fv)-PE38 and B5(Fv)-PE38 and their co~ on
with B3(Fv)-PE38 showed that all tnree had similar ADP-ribosylation activities (Fig
4(A)), inrlir~ting that cytotoxic activity dirrel~nces belween the imm~mntoyin~ did not
result from dirr~re, t enzymatic activity, but instead reflect relative antigen binding
10 ~ffiniti~s. The CY~ UA1C assays (Fig. 4(B) and Table 3), show that the ~;ylvlOAiC activity
of Bl(Fv)-PE38, B3(Fv)-PE38, and B5(Fv)-PE38, is spec-ific~ as they all kill antigen
positive cells, whose sensitivity to into~yic~tion is p~po,lional to the level of antigen
l_A~l~si,ion, while antigen-negative cells are spared. The activities of the immllnotoYin~
varied with Bl(Fv)-PE38 being the most potent. In a 20 hr assay Bl(Fv)-PE38 had an
IC50 of 0.3 ng/ml on A431 cells, and B5(Fv)-PE38 was the least potent, with an IC50 of
20 ng/ml on A431 cells.
The antigen binding assays (Fig. 5) showed that appa~nlly Bl and B5
rec~gni7~ the same antigen as B3, because all three immunotoxins collll~ete for the
binding of l25I labeled Bl IgG and B3 IgG. However, the possibility of each recognizing
a different epitope of a mutual antigen can not be eYcl~ded. A clear co.lelalion was
observed ~lween each il.,.,.u.~otûAins' antigen binding affinity and its Cylot~Aic potency.
The relative low affinity of B5(Fv)-PE38 is conci~tent with its being derived from an
IgM.
~ 9
Stability of ~nunotoxins
The stability of the Bl(Fv)-, B3(Fv)- and B5(Fv) immunotoxins following
heat llM~ 'nt was det~ ned by inc~lb~ti~n at 0.1 mg/ml in PBS at 37~C for 4 hours,
followed by analytical cl~l-,atography on a TSK G3000SW (TosoHaas) column, to
5 the monolll~.~ from the aggregates. Cytotoxic activities of aliquots of heat
treated immunotoxins were determined as described above, and colllpalcd to the activities
of the untreated immunotoxins.
As shown in Fig. 6(A), all three immunotoxins were monomeric before
the in~lb~tion (Fig. 6(A), broken lines), whereas after 4 hours of incubation in PBS at
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44
37~C, about half Bl(Fv)-PE38 and B5(Fv)-PE38 were aggl~galed, and B3(Fv)-PE38 was
comp'etely agg~egdted (Fig. 6(A), solid lines). As shown in Fig. 6(B), following the 4
hours 37~C ll~n~ent~ Bl(Fv)PE38 had an IC50 of 1.8 ng/ml which is 25% of its
cylo~c activity before tre~tmP-nt B5(Fv)-PE38 had an IC50 of 30 ng/ml (Fig. 6(B))
5 which is 66% of its cylotoAic activity before ll~~n~ent No ~;y~O~OAiC activity could be
detectP~ after tre~tm~Pnt of B3(Fv)-PE38 for 4 hours at 37~C.
These stability assays reveal differences in stability among the single-chain
Fv-immunQtoxins tested here. This is evident both from the ~;yloto~.iCity assay (Fig.
6(B)) and the from the stability assay (Fig (6)). The B3(Fv)-PE38 is so--lewl,at unstable
at 37~C (Benhar et al., 1994 supra; Rnnkm~nn et al., Proc. Na~l. Acad. Sci. USA, 90:
7538-7542 (1993)) and undergoes inactivation mainly by agg,t;ga~ion. As a cons~uence
it shows little dirr~rence in CylOtO~iC activity when incub~t~ 2 or 20 hours on A431
cells, because most of the immunotoxin is inactivated after 2 hours. Bl(Fv)-PE38 is
more stable, as in-lir~ted by the fact that its cytotoxic activity following 20 hours
incub~linn on A431 cells is twice the activity following a 2 hour incub~ti-m, and by its
reduced aggl~gation and inactivation following incub~tion at 37~C. B5(Fv)-PE38 may be
the most stable as its CylOtOAiC activity following 20 hours incub~tion on A431 cells is
six fold higher than the activity after a 2 hour incub~tion. B5(Fv)-PE38 seems to
agg~egate as much as Bl(Fv)-PE38 in the absence of antigen, but, when in~jul~t~d with
cells, it a~ to be more recict~nt than both Bl(Fv)-PE38 and B3(Fv)-PE38 to
inactivation following inc~lb~tion at 37~C. Since the antigen binding studies were done
at 4~C for three hours, con~itiollc under which all three immllnotoxins are stable, the
relative binding ~ffiniti.os of the immunotQxins best correlate with their relative ~;ylO~O~iC
activities following a 2 hour incubation period.
F~;....l,lc 10
Assay of Blood Levels of B3(Fv)-PE38KDEL in Mice
Six week old (19-20 gm) female Balb/c mice were injected with 10 ~g of
B3(Fv)-PE38KDEL in the tail vein. Blood was drawn at various time intervals and the
30 level of the immlmotoxin measured by incubating serum with A431 cells and me~Cllring
inhibition of protein synthesis. A standard curve was made with pure
B3(Fv)-PE38KDEL and the blood level of immunotoxin (which is shown in Figure 7)
c?l~ul~ted using this curve.
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F.Y~mple 11
Anti-tumor Activity of B3(Fv)-PE38KDEL in Nude Mice Bearinp a ~u
Epide~noid C~ "ua
A431 cells (3 x 106) were injected subcutaneously on day 0 into female
S nude mice (4-6 weeks old, 18-20 gm). Mice with S mm by S mm tumors, that usually
developed by day 4, were treated with B3(Fv)-PE38KDEL or, as a control, with MAbB3
or antiTac(Fv)-PE38KDEL (ch~ 1h~ry e~ al., Nature 339: 39497 (1989)). R~--se thelifetime of B3(Fv)-PE38KDEL in the circulation of the mice was observed to be only
15-20 min (Figure 7), six injections were given at 12 hour intervals into the tail vein,
10 star~ng 4 days after tumor i~ n~ ;Qn Each h~~l"c.-t group con~ist~ of five ~nim~
The volume of the tumor was c~lcul~ by (tumor volume in cm3=length x width2 x
0.4).
As shown in Figure 8, injection of either 2.5, 5 or 10 ~lg twice daily produced
complete tumor r~_g,~ssion despite the fact that B3(Fv)-PE38KDEL has a short lifetimP
(15-20 min) in the circul~tion Partial l~E;~s~ion was observed when only 0.5 ~g was
nie~t~. No toxicity was observed at these doses. In ~ltlition, when mice with large
tumors about 1 cm in ~ were treated with 5 ,ug twice a day for 4 days, complete
egn ssion of these large tumors con~inil~ about 5 x 104 cells rapidly oc ;u"~d (Figure
8(D)). Previously, it was found that even the ~mini~tration of 75 ~ug per day for 5 days
20 of a ~emi~l conjuga~e CGIllpO~d of B3 and PE40 (see Table 2) only produced partial
,egn_ssion of large tumors despite the fact that the chPmi~-~l conjugate has a much longer
lifetime in the blood (4 hours). The recombinant molecllle probably has a higher..... or activity in the mouse model because of its small size which allows better
access to tumor cells. Regression of MCF-7 tumors (breast carcinoma) also was
25 observed with 5 ~g twice daily of B3(Fv)-PE38KDEL.
Example 12
Chim~ric Fv l~ion ~.. un~otoxins and l~ t~ted ~ u~ otoxins Show I~ ased
Stability
In order to investig~te the mPrh~ni~m contributing to the greater stability
of B5(Pv)-PE38 as co,l,pared to B3(Fv)-PE38 immunotoxins comprising chimPric Fv
regions in which the light and heavy chains were derived from different antibodies were
constructed as ~escribed below. In addition, B3(Fv)-PE38 immunotoxins carrying VL
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46
mut~tionc M4L (in which methionine 4 is replaced with leucine) and S7T (in whichserine 7 is repl~~ed with t~llconine) in combination or Sep~halely were ~ ed. The
~ loLoA,city and stability of the chimPric Fv and mut~tPd fusion proteins was then
de~l ",in~d.
s
A) Clonin~p and Expression of Chimeric Fv and ~ t~te~l Immunotoxins
To produce chimPric Fv immunotoxins, DNA encoding the variable
regions of the heavy and light chains of B5 was p~ ~cd from mRNA ob~illed from B5
hybridoma cells as described in Example 2. To generate single chain immlmotoxins with
Fvs of B5, the VH and VL fr~gmPnt~ were PCR ~mplifiPd using phosphorylated primers
to enable the lig~tion of eYtended PCR products (see Ex~mple 2). The res-llting PCR
products were used as "primers" in a "domain chllMing" scheme where they re~l~~ed the
coll~ponding B3(Fv) VH or VL regions or both, generating single chain Fv-toYin
l_A~ ion pl~cmit~ having B3VH-B5VL, B5VH-B3VL, and B5Fv (Figure 9). The
eYtpncinn of templ~tP-primer~ lig~tion, tran~roll.lation and analysis of clones are
dPsçribed in F-~mpl-- 2 and 4. This pr~lu~e resulted in the generation of pl~cmi~s
for eAp~cssion in E. coli in which either the VH or the VL domains of B3 were replaced
by the colles~onding domains from B5 (Figure 9).
In addition, pl~mi~s eA~lessiilg B3(Fv)-PE38 derivatives in c~l~ g VL
mut~tion~ M4L and S7T in combination or sep~ely, were ~l~aled by site-spe~-ific
mutagenesis (Kl-nlcP.l, et al. Proc. Natl. Acad. Sci. USA, 82: 488-492 (1985)). In
~d~lition, similar pl~mj~lc CA~l~ ssing B3(Fv)-PE38 derivatives carrying VL mut~tinn~
M4L and S7r together or sep~ely were plel~ed by site-specific mutagenesis using
oligonl~cl~tide primers.
pl~mi~s enco~ing B3(Fv)-PE38, B5VH-B5VL-PE38, B5VH-B3VL-PE38,
B5(Fv)-PE38 or mutated B3(Fv)-PE38 were eAl.lessed and the fusion protein purified as
described in Example 6. Typically, monomeric proteins were recovered that were over
95% pure.
B) Specific cytotoY;c;ty of recombinant immunotoxins.
The CylO~OAiC activity of B3(Fv)-PE38 and its derivatives was ~e~
according to the method of Rrinkm~nn et al., Proc. Nat. Acad. Sci. USA 88: 8616-8620
(1991), by mP~ rin~ the inco~ ion of [3Hl-leucine by various human carcinoma cell
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47
lines after tre~tment with serial dilutions of the immnnotoxin in phosph~tP l~urL.~d saline
(PBS) con~ -ing 0.2% BSA as de~rihed in FY~mrle 8.
When tested on A431 cells, which strongly bind monoclnn~l antibody B3,
B3(Fv)-PE38 has an IC50 of 2.8 ng/ml and 2.0 ng/ml following 2 or 20 hours incub~tion
S l~ s~clively. B3VH-BSVL-PE38 and B3(Fv)-PE38 VL: M4L S7T are more active and
have idlo-nti~l IC50s of 0.6 ng/ml and 0.3 ng/ml following 2 or 20 hours incl~b~tion
f~s~:lively. B5(Pv)-PE38 is much less active with an IC50 of 120 ng/ml and 20 ng/ml
following 2 or 20 hours incub~ti~n l~ s~;lively. B5VH-B3VL-PE38 has an IC50 of 200
ng/ml and 120 ng/ml following 2 or 20 hours inc~lb~;nn respectively (data not shown).
C) Stability of the recombinant immunotoxins.
The stability of B3VH-B5VL-PE38, and B3(Fv)PE38;VL M4L S7T were
tested and cG-Ilp~d to that of B3(Fv)-PE38 by de~,...il-Ati~ n of their rc~l,ecliv-e levels of
aggrcgation and inactivation at 37~C as described in FY~mple 9. All three imm~-notoxin~
15 were ~.;n~;pAlly ono~.ic before incub~tion at 37~C. After one hour of incub~tion in
PBS at 37~C, about half of B3VH-BSVL-P_38 and B3(Fv)PE38 VL: M4L S7T were
aggl~ated, whereas B3(Fv)-PE38 was about 75% aggr~ated. After 2 hours of
ineub~tion in PBS at 37~C, 60% of B3VH-BSVL-PE38 and B3(Pv)-PE38 VL: M4L S7T
were agglcgated, and B3(Fv)-PE38 was ~ 80% aggregated. After 4 hours of in~ub~tion
in PBS at 37~C, about 80% B3VH-BSVL-PE38 and B3(Fv)-PE38 VL: M4L S7T had
agg~egated, whereas B3(Fv)PE38 was almost completely agg~galed.
The .;~lotoAic activities of these i~ notoAins are shown in Figure 10.
Following the 1 hour inc~b~ n at 37~C in PBS, B3(Fv)-PE38 had an IC50 of 8 ng/mlwhich is 25% of its c~lot(jAic activity before tre~tmPnt After 2 hours at 37~C, it had an
IC50 of 200 ng/ml which is about 1 % of its ~;ylotu~Lic activity before lr~ nt Both
B3VH-BSVL-PE38 and B3(Fv)-PE38: VL M4L S7T cytotoxic activities after one hour at
37~C in PBS were similar to their ~letlç~ Pnt activities. After 2 hours they showed an
IC50 of 3.5 ng/ml which is 12% of their ~ otoAic activity before tre~tmpnt~
The lower IC50 of the chimera and the mutant can be explained by the fact
that both are more stable that the wild type B3(Fv)-PE38. This i,l,pruv~d stability was
evident from their slower agglegalion and loss of CylOtOAiC activity upon incub~tion in
PBS at 37~C. Very little B3(Fv)-PE38 monomer survives a 2 hours incubation, whereas
the stabilized variants survive for a longer time. This correlates with the fact that while
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48
B3(Fv)-PE38 ~;~lO~iC activity is only slightly increased if A431 cells are l_~essed to it
for 20 hours instead of two hours, whereas the stabilized variants show a 3 fold increase
upon a 20 hour inl ub~lion when cG.~Ipa~cd to a 2 hour incub~tion on A431 cells.Site specific mutagenesis was used to identify which of the three VL
S residues that differ between B3 and B5 was previously responsible for the stabilizing
effect. Since B3VH-B5VL-PE38 and B3(Fv)-PE38: VL M4L S7T (which differs from thechimera only at the fourth CDRl residue) had identir~l ch~ t~Pri~tics in the assays, the
CDR residue is not the stabilizing one. Analysis of B3(Fv)-PE38 derivatives carrying
mutations VL M4L or VL S7T separately showed that repl~ing VL mPthioninP 4 with
leucine stabilized the immunotoxin as much as the B3VH-B5VL-PE38 combin~tion,
whereas replacing VL serine 7 with threonine had no stabilizing effect (data not shown).
A binding study using a BioCore instrument intlir~tP~ that B3(Fv)-PE38:
VL M4L S7T has a similar off rate to that of B3(Fv)-PE38 (0.0023 and 0.0021,
ely) whereas the on rates diffa (1150 and 984, ~cs~ ely). The app~ru~t Kd
is 2.33 x 104 for B3(Fv)-PE38 and 1.84 x 10~ for B3(Fv)-PE38: VL M4L S7T. This
data CO~l~ lates with binding data obtained by c~.--l~lition with l25I B3 IgG.
Example 13
Hum~ni7~t;on of the B3(Fv) Antibody
B3(Fv) was hum~ni7~d by a process of "framework eYch~ngen. As will be
explained in detail below, the variable domains of the heavy and light chains were
aligned with human antibody sequences and, by co...p~.icon of each domain with its best
human homolog, rl~llewo,h residues that differed between the mouse B3 and its human
homolog were identifiP~. Eleven framework residues in VH and eight in VL were
25 changed by site-specific mutagenesis to human residues and introduced simult~nP~usly
into a pre-assembled single-chain Fv (scFv) t;~pres~ion C~ P,~t~P..
A) Identification of Residues for H~-m~ni7~tion
A structural model of B3(Fv) was constructed based on the crystal
30 structure of the variable domains of monoclonal antibody McPC603 (Satow et al.,J. Mol.
Biol., 190: 593-604 (1986); Abola et al., pp. 107-132 in Crystallographic
d~abases-inforrna~ion content, Software Systems, SçiPntific Application, eds. Allen,
Bergerhoff, & Sievers, Data Comm. of the Intl. Union of Crystallogr., Bonn (1987);
CA 02203236 1997-04-21
WO 96/13594 PCTIUS95/13811
49
Protein Data bank Entry IMCP), which was mo~ifiPA and refined by an energy
",;n;...;,~l;on ~lg~rithm using the pfogl~.. CHARMM (Brooks et al., J. Comput. Chem.,
4: 187-217 (1983)) version 22. The construction of this refined model in described in
detail els~ e aung et al., Protein Structure Function and Genetics, 19: 35-47
5 (1994)). The amino acid sequences of B3 VH and VL were in~ependently aligned with all
the human antibody sequences contained in the SWISS-PROT Data Base using the
FASTA ~ro~l~ll (Release 27.0 10/93) (Devereux et al., Nucleic Acids Res. 12: 387-395
(1984)).
The VH of the human fetal immllnoglobulin 56Pl'CL (Schroeder et al.,
Science, 238: 791-793 (1987)) had the highest overall se~luence identity and had the
highest identity in the framework regions. The ~lignment of B3 VH with 59Pl'CL VH is
shown in Figure ll(A). The VL of the human IgM GM607 (Klob~ et al., Nature, 309,73-76 (1984)) (SWISSPROT file sw:kv2e-human) scored fourth in overall sequence
identity (77.7%), but had the highest identity in the framework regions. The ~lignmpnt
15 of B3 VL with GM603 VL is shown in Figure l l(B). The amino acid residues that differ
are idPntifiPA in Figure 11 by vertical lines above the sequence. Based on eypprim~pntc
with B3(Fv)-PE38 n.~,t;..~l~ (Benhar, unpublished), and on the analysis of B3Fv using the
structural model, it was decid~P~ to preserve the mouse residues at VH positions 1, 3, 19,
24, 89, and 91 and VL po~itionC 2, 3 and 41 (Kabat, et al. Sequences of proteins of
i~rununological interest. 5th eAition. U.S. (1991); Figure 11). These residues are
i-lPntifieA by ~cteric~c in Figure ll(A) and ll(B), some of them are inter-domain
residues and others are buried and therefore are not expected to be part of an
ogel~ic epitope (Padlan, Mol. Irrununol. 28: 489-498 (1991) and Roguska et al.
Proc. Natl. Acad. Sci. USA, 91: 969-973 (1994)).
The human antibodies chosen also had simil~nty to B3(Pv) in the sequence
of the co",ple "Pn~ det~ ining region loops (CDRs), and had the same CDR length
which further in~ir~teS that they belong to a similar structural group, and possibly have a
similar canonical structure of the CDR loops (Chothia et al., J. Biol. Chem. 227:
799-817 (1992); Williams et al., Eur. J. Immunol. 23: 1456-1461 (1993)).
B) Construction of plasmids CA~ hl~m~n;7ed variants of B3(Fv)PE38.
B3 VH and VL gene segmrnt~ in pl~mi~l pULI7 (Fig. 13(A)) enco~ing
wild type B3(Fv)-PE38 was sele~ted for modification via site-directed mutagenesis.
CA 02203236 1997-04-21
W 096/13594 PCTrUS95/13811
Uracil con~ ;ng single-stranded DNA was y~ d by rescue of our F+ origin
co~ ;ng pl~cmids with an M13KO7 helper phage and was used as a tPmpl~tP for
site-specific mutagenesis (Kllnkel, Proc. Natl. Acad. Sci. USA, 82: 488-492 (1985)).
The complete nucleotide sequence of the gene encoding B3(Fv)-PE38 has been described
(~rinkm~nn, et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620 (1991)). Mutagenicoligon~]rlP4tides used and the mutation, they are listed in Table 4.
B3 VH and VL gene seg"lents in pl~cmi~ pULI7 (Fig. 12(A)), enr~ing
wild type B3(Fv)-PE38, were indepen~ently hum~ni7ed by site specific mutagenesis. A
set of four oligonurlPotides was used to cimult~n~p4usly introduce the mutations into each
10 sP~P ~1, with most of the oligonucleotides ch~nging more than one mouse to human
codon. In B3 VH~ the mut~tionc introduced were LllV and G16R, T40A, E42G and
R44G, A74S and R75K, S82aN, R82bS, K83R and S84A. The res~llting pl~cmid wac
pB3HUMVH-VL-PE38 (Fig. 12(B)). In B3 VL, the mutations introduced were S14T,
L15P, D17E and Q18P, K45Q, L83V, SLOOQ and L104V. The res--lting pl~cmi~,
pB3HUMVH-VL-PE38 (Fig. 12(C)) was used as a ten pl~tP for a second mutagenesis with
the combined heavy chain mutagenic oligonucleotides generating pl~cmi~
p~UMVH-HUMVL-PE38 (Fig. 12(D)), which encodes the hum~ni7ed B3(Fv) single-chain
i.. u.. ,luAin. The residues that were .. ul~PA are identified in Fig. 11 by their numbers.
20 C) Expl~ stcn and Purification of Recombinant ~otei-.s
Expression pl~cmitls encoding B3(Fv)-PE38 or its hllm~ni7~d derivatives
were introduced into E. coli strain BL21 (~DE3) (Studier et al. J. Mol. Biol. 189,
113-130 (1986)) and the recombinant proteins were c;A~r~ss~ as in~lllcion bodies as
described in Example 6. The single-chain immunotoxins were obtained by solubilization
25 and refolding of inclusion body protein as described and subsequently purified as
described in FY~mple 6. Typically, the monomeric proteins were obtained at over 95%
purity, as determined by non-reduçing SDS-PAGE of the product.
CA 02203236 1997-04-21
WO 96/13594 PCT/US95t13811
51
Tabl- ~. Oligonucleotides utilized for site directed muta~n~ and the
mutation~ they introduced. Restriction site~ which were introduced into
the~e oli~on~lcleotide~ to facilitate identification of mutated clones are
underline~.
S PrimerSequence Mutation Seq.
ID
l5'-GGAGAGTTTCAGGGAGCGCCCGGGGTGCVH: LllV; Gl6R 23
ACGACGC~CCCCC-3,
25'-TGCGArCr~rTCCAGGCCCTTGCCCGGGVH: T40A; E42G; 24
GCCTGG cr-AAr~r~ATA-3 R44G
35'-GAGG~ GCTA~ , AGAG VH: A74S; R75R 25
ATGGTGAACCG--3'
45'-TATGG~ C~CGGCGCGCAGGCTG VH: S82aN; R82bS; 26
TTCATTTGCAGGTA-3' K83R; S84A
0 55 ' - GrP~r-~.ATGGAGGCCGG~CCCGGGVL: 514T; Ll5P; 27
GTGACAGGTAAACTCAA-3' Dl7E; Ql8P
65'-AA~---~-AGATCAGCAGCTGTGGAGAC VL: K45Q 28
TGGGCTGG-3'
75'-GCAGTAATAAArTCCGAC~C~ AGCC VL: L83V/ 29
TCCAC-3'
85'-GGAAGCTTTAATTTCGAC~--G~ACCC VL: SLOOQ; Ll04 30
TGGCC~ ArGTGAATGG-3'
15 D) Cytot~Y;r;ty and l~;rl;.,~ Amnity of Hl~mqn;7ed B3(Fv)-PE38
The ADP-ribosylation activity of each immunotoxin was tested, to verify
that they are of equal en~y,.lalic activity. As shown in Figure 13(A), B3(Fv)-PE38 and
the hum~ni7~ variants had similar ADP-ribosylation activities.
The CylotoAic activity of B3(Fv)-PE38 and of its hum~ni7~d variants was
measured by incub~ting various human carcinoma cell lines with serial dilutions of the
immunotoxin, and me~uring the inco~poration of [3Hl-leucine as ~escribe~ in FY~mpl~
8. As shown in Figure 13(B), B3(Fv)-PE38 had an ICso of 1.8 ng/ml on A431 cells
which express high levels of the B3 antigen. The variant B3VH-HUMVL-PE38 (HUML)
had a similar Cyl~tO~iC activity, while B3HUMVH-VL-PE38 (HUMH) and
B3-HUMVH-HUMVL-PE38 (HUMH+L) had ICsos of about 4.4 ng/rnl.
To check whether hum~ni~ing B3(Fv)-PE38 caused a change in antigen
specificity, the same cylolo~-ic assay was done on additional cell lines. As shown in
CA 02203236 1997-04-21
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52
Table 5, B3(Fv)-PE38 and all the hllm~ni7P4 variants had the same spectrum of
ccog~ n of the cell lines used. These cell lines differ in their level of B3 antigen
t;A~ssion (Rrinkm~nn et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542 (1993). This
result in~ir~tPs that the antigen binding sperifiçity of the B3(Fv) was not altered by the
S h,....~n;~;ng process.
The spefific antigen binding affinity of the B3(Fv) immunotoAins was further
analy_ed by de~~ in~;on of the binding affinity of B3(Pv)-PE38 and the hll...~ni~
variants to B3 antigen bearing cells by a co",~!;l;on assay, in which increasingc~nc~Pnt~tion~ of each imml~noto~rin were used to col"~ele for the binding of [l25IJ-B3
IgG to A431 cells at 4~C. As shown in Fig. 13(C), both B3(Fv)-PE38 and HUML
blocked the binding of t'25Il-B3 antibody to A431 cells by 50% at 1-2 ~M. However,
HUMH and HUM2H+L had a lower affinity, and colllpe~ed by 50% at 4-5 ~M. The
results from the cylolo,.icity and the binding assays in(lic~te that the hum~ni7~d B3(Fv)
sufr~td a 2-3 fold loss in antigen binding affinity and that the ~l~m~ging mutation
15 probably resides in the VH S~gmP-nt
CA 02203236 1997-04-21
W O96/13594 53 c PCTrUS95/13811
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CA 02203236 1997-04-21
W O96/13594 PCTrUS95/13811
E) Back-M~ VT~ Residue 82b to Restore Activity
To restore the reduced activity of HUMH+L, some mutated forms which
were partially hum~ni7~d in the VH region were tested for cytotoxic activity. It was
found that a mutant of B3(Fv)-PE38 in which the VL was wild type and VH residues at
S positions 74, 75 or 83 were hllm~ni7Pd, did not lose cyloto,~ic activity, while a d~livdlive
in which residues 74, 75 and 82a, 82b, 83, and 84 were hum~ni7~d was about 5-6 fold
less active. In the B3 VH structural group (mouse III(A)) residue 82a is most commonly
asparagine, as it is in HUMH+L. However, serine is never found at position 82b.
Furthermore, the origin~l residue at position 82b, arginine, is ~cept~hle in human IgGs.
T~ fol~, residue 82b in HUMH+L was mut~te~l to arginine by site-spe~ific mutagenesis.
The resulting mol~Pcule is narned HUMB3(Fv)-PE38. The protein was purified to near
homogeneity and was subjected to activity and binding assays. As shown in Figure13(A), its ADP-ribosylation activity did not differ si~nific~ntly from that of the other
immunotoxins. Moreover, as shown in Figures 13(B), 13(C), and Table 5, its .;~lot~Aic
and antigen binding activities were improved relative to HUMH+L and were similar to
those of the original B3(Fv)-PE38 immunotoxin.
Example 17
Reacti~ity with Sera from Monkeys ~ J..i~ with B3(Fv)-PE38
Sera obtained from monkeys that had been immuni7~ with B3(Fv)-PE38
contain antibodies to PE38 as well as a lower reactivity with B3Fv. To assess the
success of h.. ~ni,;n~ B3Fv, sera from four Cynomolgus monkeys conl~;nil-~ sp~Pcific
anti B3(Fv)-PE38 titers (Id.) were pooled and used in an ELISA assay on plat_s that
were coated with B3(Fv)-PE38, HUMH+L, or HUMFv (B3HUMFv). An excess of PE38
25 was includ~P~I as a cG~ itor to preadsorb reactivity which is directed against the toxin
moiety of the molecule. As shown in Figure 14, the anti B3(Fv)-PE38 sera had a
weaker reaction against both HUMH+L and HUMFv than with the wild-type B3(Fv)PE38,
indi~ting that primate B3(Fv) e~ilope(s) were mi~ing in the hu...~ni7~d variants.
The above e-~mrlPs are provided to illustrate the invention but not to limit
30 its scope. Other v~iants of the invention will be readily appalent to one of ordin~y
skill in the art and are encomp~sPvd by the appended claims. All publications, p~t~nt~,
and patent applications cited herein are hereby inco~ ted by reference.
-
CA 02203236 1997-04-21
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Exam~le 18
The Vv Tyrosine to Serine Mutation Improves the Aff~mib of Anti-LeY Carbohydrate Antibodies
When co..,p~. ;ng the arnino acid sequence of B3 to the conserve~d
S f ~I-CWO~ sequences of other antibodies (Kabat et al. (1991) supra.) it was oPtserved
that a lylosine at position VH 95 was eY~-h~n~ed to serine, which is very lmll~u~l. To
investi~tP the sig~ifi~nce of this eY~h~nge, this residue was mut~t~d to tyrosine or to
phenyl~l~nin~, which are the arnino acids that are mostly present at this position in other
antibodies. It was found, that both eychanges reduced the affinity of B3(Fv) co"l~ ed to
the moleclllP with the original serine al~pro,~im~tely 10 fold. Thus, the serine is
in,~.~lt for high affinit,v binding. This was uneA~cled and surprising, because
position VH 95 is in the VH VL intprfa~e~ and thus neither in the binding region nor close
to it (see, commonly ~c~igned U.S. Patent Application Serial No. 08/077,252 filed on
June 14, 1993). This eY~-ludes the possibility that a direct (contact) effect of the serine,
influ~n-~s binding. The most likely eYpl~n~tion for the effect of the serine is, that,
be~cause it is po~iti~ n~ in the VH VL intP.rf~e and slightly deshbili~s the interface
cont~ts it enables a movement of VH relative to VL, m~Ai~tin~ a so called "induc~d fit"
antibody binding mode (Rini et al., Science, 255: 959-965 (1992) and Stanfield et al.
Structure, 1: 83-93 (1993)). To analyze whether the serine mutation" at VH 95, which
was found in B3(FV) increases only the affinity of B3(Fv) or if it can (possibly by
mPAi~ting induc~.d fit) also increase the affinity of other LeY binding antibodies, we
introduced this mutation into B5(Fv). B5 Fv binds like B3 LeY but cont~in~ a dirÇerent
antibody binding site, and in addition contains the "conserved" tyrosine in position VH
95. FYch~nge of this tyrosine to serine was done by site directed mutagenesis (l~lmk~l,
et al. 1985) supra.). The reslllting B5(Fv)-ser VH 95 mutant molecule (de~ign~t~B5(Fv): VH Y95S) showed 4-fold increase in specific binding activity as analyzed by
Cy~t~Aicity assays, from which the (relative) ~ffinitiPs can be d~uced. Thus, the VH
lylosine to serine 95 mutation not only increases the affinity of B3(Fv) but also of one
other LeY binding antibody and probably others. Since the mPch~ni~m by which the ser
95 causes increased affinity is most likely f~cilit~tion of induced fit binding, it is
expected that this mutation can also increase the affinity of certain other antibodies, in
cases where inrluc~d fit will generate increased interactions bel~een the antibody and the
antigen.
CA 02203236 1997-04-21
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56
Table 5. Relative binding affinity of single chain fusion proteins with
~,ibsl;t~lt;Qnc at VH position 95.
Single ChainAmino Acid at Position Per~nlage Rin-ling
Antibody 95 of VH Aff1nitY-
S B3(Fv) tyrosine 10%
B3(Fv) serine 100%
B5(Fv) tyrosine 25 %
B5(Fv) serine 100%
Best binding afrmity was set to 100%.
CA 02203236 1997-04-21
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57
S~OUENCE LISTING
( I ) GT~'NFR~L INFORMATION:
(i) APPLICANT:
(A) NA~SE: The United States of America,
as represented by
The Secretary of the Department
of Health and Human Services
'B' STREET: 6011 Executive Blvd., Suite 325
,C CITY: Rockville
D STATE: Maryland
El CODh.nY: U.S.A.
F POSTAL CODE (ZIP): 20852
G TELEPHONE: (301) 496-7056
H TELEFAX: (301) 402-0220
I TELEX:
(ii) TITLE OF lNvh~,lON: TUMOR-SPECIFIC ANTIBODY FRAGMENTS,
FUSION PROTEINS, AND USES ~n~Or
(iii) NUMBER OF S~yur;N~r;S: 53
(iv) CORRESPONDENCE ADDRESS:
,'A) ADDRESSEE: Townsend and Townsend and Crew
B STREET: Steuart Street Tower, One Market Plaza
C, CITY: San Francisco
D STATE: California
E c~uh~r: us
FJ ZIP: 94105-1493
(V) COIIrU~K pT~npRT-T' FORM:
(A' ~EDIUM TYPE: Floppy disk
(B ~CI~u.~: IBM PC compatible
(C, OPERATING SYSTEM: PC-DOS/MS-DOS
(D SOFTWARE: PatentIn Release #1.0, Version ~1.30
(vi) ~unK~- APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/767,331
(B) FILING DATE: 30-SEP-l99l
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/596,289
(B) FILING DATE: 12-OCT-1990
(viii) A..O~/AGENT INFORMATION:
(A) NAME: Weber, Ellen Lauver
(B) REGISTRATION NUMBER: 32,762
(C) Rr;rr;K~-lCE/DOCRET NUMBER: 15280-126-1-lPC
(ix) TT~T~CQ1s1~JNlcATIoN INFOR~sATIoN:
(A) TELEPHONE: (415) 543-9600
(B) TELEFAX: (415) 543-5043
(2) INFORMATION FOR SEQ ID NO:l:
u~ r; CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02203236 1997-04-21
W O96/13594 PCTrUS95/13811
58
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: 1..44
(D) OTHER lNrORMATION: /standard name= "Heavy chain
primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:I:
TAACTAGGAT CCG~C~ATAT GGATGTGAAG CTGGTGGAGT CTGG 44
(2) INFORMATION FOR SEQ ID NO:2:
(i) s~yurh ri CHARACTERISTICS:
,'A' LENGTH: 39 base pairs
BI TYPE: nucleic acid
C STRANDEDNESS: ~ingle
~D TOPOLOGY: linear
( ii ) ~OT FCU~-~ TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: l..3~
(D) OTHER INFORMATION: /standard name= "Heavy chain
primer~ ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TGr-~T~r-ACT GATGGGGATC CGC~ICCGCC T~-~Gr-~r,AC 39
(2) INFORMATION FOR SEQ ID NO:3:
(i) S~ NCF CHARACTERISTICS:
'A' LENGTH: 39 ba~e pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: 1..39
(D) OTHER lNrORMATION: /standard name= "Heavy chain
primern
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
~AT~TA~AT~ TGGAGGTGCA GCTGGTGGAA TCTGGAGGA 39
(2) INFORMATION FOR SEQ ID NO:4:
(i) s~yur;~ri CHARACTERISTICS:
~A'I LENGT~: 39 ba~e pairs
B TYPE: nucleic acid
Cl STRANDEDNESS: ~ingle
D) TOPOLOGY: linear
CA 02203236 1997-04-21
W O96/13594 PCTrUS95/13811
59
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: l..3~
(D) OTHER lNr-RMATION: /~tandard_name= "Heavy chain
primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
rpTA TGGAGGTGAA GCTGGTGGAA TCTGGAGG............................. 39
(2) INFORMATION FOR SEQ ID NO:5:
( i ) ~hyUh~_h CHARACTERISTICS:
(A~ LENGTH: 24 base pairs
(B TYPE: nucleic acid
(C STRANDEDNESS: single
(D~ TOPOLOGY: linear
(ii) MOTECUTE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: l..24
(D) OTHER INFORMATION: /~tandard name= "Heavy chain
primer~
(Xi) ~hyUL.._h DESCRIPTION: SEQ ID NO:S:
AGCAGATCCA GGGGCCAGTG GATA 24
(2) INrO~ATION FOR SEQ ID NO:6:
( i ) ~UL.._~ CHARACTERISTICS:
'A' LENGTH: 27 ba~e pair~
B TYPE: nucleic acid
C, STR~NQFnY~SS: single
~Dl TOPOLOGY: linear
( ii ) ~nT ~CUT ~ TYPE: DNA (primer)
(ix) FEATURE:
(A) NA~E/KEY: misc feature
(B) LOCATION: l..27
(D) OTHER INFORMATION: /~tandard name= "Heavy chain
primer"
(xi) S~uL..CTC DESCRIPTION: SEQ ID NO.6:
ACCGGATCCG CCTGCAGAGA CAGTGAC 27
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A~l LENGTH: 33 ba~e pairo
(B TYPE: nucleic acid
(C ST~ANnTCDNESS: ~ingle
(D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
CA 02203236 1997-04-21
W O96/13594 ~CTrUS95/13811
(ix) FEATURE:
(A) NAME/KEY: mi~c feature
(B) LOCATION: l..33
(D) OTHER INFORMATION: /6tandard_name= "Heavy chain primer
B5HRr4"
(xi) -~yu~ DESCRIPTION: SEQ ID NO:7:
ACCG~-ATCCG CCTCCGCCTG A~-G~G~C~GT GAS 33
(2) INFORMATION FOR SEQ ID NO:8:
( i ) ShgUh~ _ r CHARACTERISTICS:
'A' LENGTH: 70 ba~e pairs
B TYPE: nucleic acid
Cl STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: l..70
(D) OTHER INFORMATION: /~tandard_name= Light chain
primer"
( Xi ) ~hgUh.._~ DESCRIPTION: SEQ ID NO:8:
~C~AAGC TTGGGGATCC GG.GG,GGCG GATCTGGAGG TGGCGGAAGC GATGTGCTGA 60
CCCAGTCTCC 70
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 42 ba6e pair~
IB TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: mi~c feature
(B) LOCATION: l..42
(D) OTHER INFORMATION: /utandard name= "Light chain
primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
AG.. GG.GCA GCATCAAAAG CTTTKAKYTC CAGCTTKGTS CC 42
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 28 ba6e pair6
B TYPE: nucleic acid
,C STRANDEDNESS: 6ingle
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
CA 02203236 1997-04-21
W O 96/13594 PCTrUS95/13811
(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: l..28
(D) OTHER INFORMATION: /ctandard name= "Light chain
primer"
(Xi) ~h~UhN~h DESCRIPTION: SEQ ID NO:lO:
TTGGGGATCC GGTGGTGGCG GATCTGGA 28
(2) INFORMATION FOR SEQ ID NO:ll:
( i ) Sh~Uh~_h CHARACTERISTICS:
'A' LENGTH: 40 base pair~
~B TYPE: nucleic acid
C STRANnFDNFSS: ~ingle
,D~ TOPOLOGY: linear
~ii) ~nT-~CUT-E TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: l..40
(D) OTHER INFORMATION: /standard_name= "Light chain
primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
AGCGGGAATT CATTATTTAA TTTCCAGCTT ~-CCCCGAC 40
(2) lhrvhMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 30 ba~e pairu
~B TYPE: nucleic acid
C ST~A~D~nNFSS: ~ingle
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: micc feature
(B) LOCATION: l..30
(D) OTHER IhrvRMATION: /~tandard name= nLight chain
primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGTGGCGGAA GCGATGTTGT GAT~-A~CC~A 30
(2) INFORMATION FOR SEQ ID NO:13:
( i ) ~h~Ur;N~ri CHARACTERISTICS:
'A' LENGTH: 30 base pairs
IB, TYPE: nucleic acid
,C STRPNnFnN~SS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: mi~c feature
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(B) LOCATION: 1..30
(D) OTHER INroRMATIoN: /~tandard_name= "Light chain
primer"
(xi) Sr;Qur,~-r; DESCRI~TION: SEQ ID NO:13:
GGTGGCGGAA GCGATGTTTT GTTG~CrP~ 30
(2) IhrORMATION FOR SEQ ID NO:14:
(i) ~r;~u~._r; CHARACTERISTICS:
~A'l LENGTH: 24 ba~e pair~
B TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: miac_feature
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /ctandard name= "Light chain
primer"
(xi) ~r;~uL._r; DESCRIPTION: SEQ ID NO:14:
~G.GGGAAG ATGr-~T~r~r TTGG 24
(2) INFORMATION FOR SEQ ID NO:15:
(i) ~r;QDri~.Cr; CHARACTERISTICS:
~A' LENGTH: 24 ba~e pair~
B TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: mi~c_feature
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /~tandard_name= "Light chain
primer~
(xi) ~r;QuL~_~ DESCRIPTION: SEQ ID NO:15:
GGAAGCTTTC AGCTCCAGCT TGGT 24
(2) INFORMATION FOR SEQ ID NO:16:
(i) ~r;QurN~r; CHARACTERISTICS:
A) LENGTH: 23 ba~e pairs
B) TYPE: nucleic acid
C) STRANDEDNESS: single
,D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: misc_feature
(B) LOCATION: 1..23
(D) OTHER INFORMATION: /~tandard_name= ~Light chain
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primer"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GGAAGCTTTA TTTCCAACTT TCT 23
(2~ INFORMATION FOR SEQ ID NO:17:
(i) SEQ OE NCE CHARACTERISTICS:
A' LENGTH: 357 ba~e pair~
BI TYPE: nucleic acid
C ST~P~nFP~FSS ~ingle
D TOPOLOGY: ~lnkn~..A
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: 1..357
(D) OTHER INFORMATION: /~tandard name= "B3
variable heavy chain"
(xi) SEQ OE NCE D~SCRTPTION: SEQ ID NO:17:
GATGTGAAGC TGGTGGAGTC T ~ GGGAGGC TTAGTGCAGC CTGGAGGGTC CCTGAAACTC 60
.C~.~.GCAA C~.~.GGATT CACTTTCAGT GACTATTACA TGTATTGGGT TCGCCAGACT 120
CrAr~GAArA GGCTGGAGTG GGTCGCATAC ATTAGTAATG ATGATAGTTC CGCCGCTTAT 180
TCPr-ArACTG ~AAAr~GGccG GTTCACCATC TcrAr-AGArA ATGCCAGGAA CACCC.~-AC 240
CTGCAAATGA GCCG.~IGAA GTCTGAGGAC ACAGCCATAT A-.C~-G,GC AAr7AG~TG 300
GC~.GCGCAG C~.G~... GC TTACTGGGGC c~Ar7Gr-AcTc TGGTCACTGT ~.C~. A 357
(2) lNrOR~.TION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
'A', LENGTH: 336 ba~e pair~
B, TYPE: nucleic acid
C ST~AN~Fn~SS: single
,DI TOPOLOGY: I~nl---,.,,
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: 1..336
(D) OTHER INFORMATION: /~tandard_name= B3
variable light chain"
(Xi) ~:QU~I._~ DESCRIPTION: SEQ ID NO:18:
GATGTGCTGA Tr~AccrAr~Tc TCCATTGAGT TTACCTGTCA G.~.. GGAGA TCAAGCCTCC 60
A.~..GCA GATCTAGTCA GATCATTGTA CATAGTAATG GAAArAccTA TTTAGAATGG 120
TACCTGCAGA AArcAGGccA G~c~AAAG CTCCTGATCT ACAAAGTTTC rAArCGATTT 180
TCTGGGGTCC CAGPrAr7GTT CAGTGGCAGT GGATCAGGGA CAGATTTCAC ACTCAAGATC 240
AGCAGAGTGG AGGCTGAGGA TCTGGGAGTT TATTACTGCT TTCAAGGTTC ACA.~..C~A 300
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TTCACGTTCG GCTCGGGG~C AAAGCTGGAA ATTAAA 336
(2) lNru~ATION FOR SEQ ID NO:l9:
(i) ~uu~ _~ CHARACTERISTICS:
~A' LENGTH: 36; ba~e pairs
B TYPE: nucleic acid
,C, sTR~Nn~n-N~-cs: ~ingle
D~ TOPOLOGY: unk- "
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: 1..363
(D) OTHER lN~ORMATION: /~tandard name= nB1
variable heavy chain"
(xi) ~yu~rCE DESCRIPTION: SEQ ID NO:l9:
GAGGTGCAGC TGGTGGAATC TGGPr-G~GGC TTAGTGAAGC CTGGAGGGTC CCTGAAACTC 60
.C~,~GCAG CCTCTGGATT CATTTTCAGT GACAATTACA TGTATTGGGT TCGCCAGACT 120
CCG~-~G~r-~ GGCTGGAGTG GGTCGCAACC ATTAGTGATG GTGGCACTTA TATCGACTAT 180
Tr~r-~GTG Tr-~GGGGCG ATTCACCATC Tcr~r-~ c~ ATGccAAr-AA TAATCTGTAC 240
TTGCAAATGA GCAGTCTGAG GTCTGAGGAC ACAGGCATGT ATTATTGTGG AAGGAGTCCG 300
ATCTACTATG ATTACGCCCC GTTTACTTAC TGGGGCCAAG GGA~G~1 CA~.~.~. 360
GCA 363
(2) INFORMATION FOR SEQ ID NO:20:
(i) S~YU~N~ CHARACTERISTICS:
'A' LENGTH: 336 ba~e pairs
BI TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
D) TOPOLOGY: ~nkn9~"
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: mi~c feature
(B) LOCATION: 1 336
(D) OTHER INFORMATION: /~tandard name= nBl
variable light chain"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GA,~,,~,GA Tr-~r-cr~ c TCCACTCTCC CTGCCTGTCA GTCTTGGAGA TCAAGCCTCC 60
A,~,~,,GCA GATCTAGTCA AAAC~,,~,A CACAGTGATG GAAAAACCTA TTTACATTGG 120
TTCCTGCAGA AGCCTGGCCA G~C~AACG CTCCTGATCT ACAAAGTTTC ~Arcr7ATTT 180
TCTGGGGTCC cAr~r~GTT CAGTGGCAGT GGATCAGGGA CAGATTTCAT ACTCAAGATC 240
AGCAGAGTGG AGGCTGAGGA TCTGGGAGTT TATTTCTGCT CTCAAAGTAC ACA.~,.CCG 300
CTCACGTTCG GTGCTGGGAC CAAGCTGGAG CTGA~A 336
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(2) INFORMATION FOR SEQ ID NO:2l:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 357 base pairs
B TYPE: nucleic acid
Cl STRANDEDI~SS: single
,D, TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: 1..357
(D) OTHER INFORMATION: /standard name= nB5
variable heavv chain~
(Xi) S~QDh~h DESCRIPTION: SEQ ID NO:21:
GAGGTGAAGC TGGTGGAATC TGG~G~.AGGC TTAGTGCAGC CTGGAGGGTC CCTGAPACTC 60
.C~.~.GCAA CCTCTGGATT TACTTTCAGT GACTATTACA TGTATTGGGT TCGCCAGACT 120
CC~GAr-~P-~ GGCTGGAGTG G~.CGCATAC ATTAGTAATG GTGGTGGTAG CACCTATTAT 180
Cr~GAC~CTG TAAAr,GGCCG ATTCACCATC Tcr~G~r-~c~ ACGCCAA~-~A CACCC.G.AC 240
CTGCAGATGA GCC~.~.GAA GTCTGAGGAC ArPGCr~TGT ATTACTGTGC AAGGGGGCTC 300
TCTGATGGTT C~.G~... GC TTA~.GGGGC C~r-Gr-~TC TGGTCACTGT ~C~.~A 357
~2) INFORMATION FOR SEQ ID NO:22:
( i ) ShQDh~h CHARACTERISTICS:
~A' LENGTH: 336 ba~e pair~
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
,D, TOPOLOGY: unkn~,n
( ii ) MQT~F-CUT-~ TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: l..3~6
(D) OTHER INFORMATION: /~tandard name= ~B5
variable liyht chain~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GA.~l... ~ T~-~CC~PAP~ TCCACTCTCC CTGCC.~.~A GTCTTGGAGA TCAAGCCTCT 60
A~ 6~A GATCTAGTCA GAGCATTGTA CATAGTAATG ~A~r~CCTA TTTAGAATGG 120
TACCTGCAGA AACCAGGCCA 6.~.C~AAAG CTCCTGATCT ACAAAGTTTC CAACCGATTT 180
TCTGGGGTCC r~GPC~GGTT CAGTGGCAGT GGATCAGGGA CAGATTTCAC ACTCAAGATC 240
AGCAGAGTGG AGGCTGAGGA TCTGGGAGTT TATTACTGCT TTCAAGGTTC ACATGTTCCA 300
TTCACGTTCG GCTCG6GGAC AAAGTTGGAA ATTAAA 336
(2) INFORMATION FOR SEQ ID NO:23:
(i) sEQD~ CHARACTERISTICS:
(A) LENGTH: 42 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: uingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: miuc feature
(B) LOCATION: l..4~
(D) OTHER INFORMATION: /label= Primer l
(xi) Sr;yur;N~r; DESCRIPTION: SEQ ID NO:23:
GGAGAGTTTC AGGGAGCGCC CGGGGTGCAC GACGCCTCCC CC 42
(2) INrORMATION FOR SEQ ID NO:24:
(i) sr;yur,r_r; CHAR~CTERISTICS:
Al LENGTH: 45 base pairu
B TYPE: nucleic acid
C ST~ANDFnN~SS: uingle
~D,~ TOPOLOGY: linear
(ii) MOTT'CUTT~' TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/REY: mi~c feature
(B) LOCATION: 1..45
(D) OTHER lNrOR~ATION: /label= Primer 2
(xi) s~Qur, _r; DFSCRTPTION: SEQ ID NO:24:
TGCGAr,Cr~C TCCPGGCCCT TGCCCGGGGC CTGGCGAACC CAATA 45
(2) INFORMATION FOR SEQ ID NO:25:
(i) sr;~r;r_r; CHARACTERISTICS:
~A'I LENGTH: 39 base pairu
B TYPE: nucleic acid
C sTR~NnT''n~FSS: Qingle
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: mi~c feature
(B) LOCATION: l..39
(D) OTHER IN~ORMATION: /label= Primer 3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GAGG~.~.~C TTGCTATTGT CTCTAr-~r-AT GGTGAACCG 39
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
,'A) LENGTH: 42 baue pairs
B) TYPE: nucleic acid
C) STRANDEDNESS: uingle
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
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(ix) FEATURE:
(A) NAME/REY: misc feature
(B) LOCATION: 1..42
(D) OTHER INFORMATION: /label= Primer 4
(xi) s~yu~.._~ DESCRIPTION: SEQ ID NO:26:
TATGGCTGTG .C~,CGGCGC GCAGGCTGTT CATTTGCAGG TA 42
(2) INFORMATION FOR SEQ ID NO:27:
(i) s~yu~_~ CHARACTERISTICS:
'A' LENGTH: 45 ba~e pair~
B TYPE: nucleic acid
CI ST~P~ FSS: uingle
~D, TOPOLOGY: linear
(ii) Mn~FCUT~ TYPE: DNA (primer)
(ix) FEATURE:
(A) NANE/XEY: misc feature
(B) LOCATION: l..45
(D) OTHER INFORMATION: /label= Primer 5
(xi) ~yu_.._~ DFSCRTPTION: SEQ ID NO:27:
G~ ATG G~GGCCGG~. ~.CCCGGGG~ GACAGGTAAA CTCAA 45
(2) l~rOR~ATION FOR SEQ ID NO:28:
(i) s~yB~ CHARACTERISTICS:
'A' LENGTH: 36 ba~e pairs
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MnTFCUT~ TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/XEY: mi~c feature
(B) LOCATION: l..3~
(D) OTHER l~ORMATION: /label= Primer_6
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
AA~... ~.AG ATCAGCAGCT GTGGAGACTG GGCTGG 36
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 33 ba~e pairs
B TYPE: nucleic acid
,C, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/XEY: mi~c_feature
(B) LOCATION: l..33
(D) OTHER INFORMATION: /label= Primer 7
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GCAGTAATAA ACTCCGACGT CCTCAGCCTC CAC 33
~2) INFORMATION FOR SEQ _D NO:30:
(i) SEQUENCE CHARACTERISTICS:
~Al LENGTH: 45 ba~e pair~
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
~D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (primer)
(ix) FEATURE:
(A) NAME/KEY: mi~c feature
(B) LOCATION: 1..45
(D) OTHER INrORMATION: /label= Primer 8
(xi) s~ur;r_r DESCRIPTION: SEQ ID NO:30:
GGAAGCTTTA ATTTCGACCT TGGTACCCTG Gccr-AArGTG AATGG 45
(2) lN~K~ATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 base pairE
(B) TYPE: nucleic acid
(C) STR~ND~nNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/REY: mi~c_feature
(B) LOCATION: 1 741
(D) OTHER INFORMATION: /note= "Sequence encoding I ~ni ~ed
B3(Fv)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
ATGGATGTGA AGCTGGTGGA G~GGGGGA GGCG,CG,GC AGCCCGGGCG ~,CCC,GAAA 60
~ G CAACCTCTGG ATTCACTTTC AGTGACTATT ACATGTATTG GGTTCGCCAG 120
GCCCCGGGCA AGGGCCTGGA GTGGGTCGCA TACATTAGTA ATGATGATAG TTCCGCCGCT 180
TATTCAGACA CTGTAAAGGG CCGG.,~ACC ATCTCTAGAG ArA~ATAr-cAA ~AA~cpcccTc 240
TACCTGCAAA TGAACCGTCT GCGCGCCGAG GA~CAGCCA TATATTCCTG TGCA~r-Ar~-~ 300
CTGGCCTGGG GAGCCTGGTT TGCTTACTGG GGCCAAGGGA CTCTGGTCAC ~ C~.CA 360
GGCGGAGGCG GA-CCG~GG TGGCGGATCT GGAGGTGGCG GAAGCGATGT GCTGATGACC 420
CAG~ ~AT TGAGTTTACC TGTCACCCCG GGAGAGCCGG CCTCCATCTC TTGCAGATCT 480
AGTCAGATCA TTGTArATPr- TAATGGAAAC ACCTATTTAG AATGGTACCT GCAr,AAPrrA 540
GGCCAGTCTC CACAGCTGCT GATCTACAAA ~ CCAACC GA ~-- GG GG~CC;Ar-A~- 600
AGGTTCAGTG GCAGTGGATC AGG~A~Ar,AT TTCACACTCA AGATCAGCAG AGTGGAGGCT 660
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GAGGACGTCG GAGTTTATTA ~.G~... AA GGTTCACATG TTCCATTCAC GTTCGGCCAG 720
GGTArr~Ar7G TCGAAATTAA A 741
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 15 amino acids
B TYPE: amino acid
C STRPNnFn~FSS: ~ingle
,D, TOPOLOGY: linear
( ii ) ~r~T~T''CuT~F TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Peptide
(B) LOCATION: 1..15
~D) OTHER INFORMATION: /label= LINKER
(xi) ~hyu~._h DESCRIPTION: SEQ ID NO:32:
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
(2) lNrORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 772 ba~e pairs
B TYPE: nucleic acid
C STRANnT~'n~SS ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 27..767
(xi) Shyu~h DFCrRTpTIoN: SEQ ID NO:33:
TTTAACTTTA ~G~Ar-r-~r-~T ATACAT ATG GAT GTG AAG CTG GTG GAG TCT GGG 53
Met Asp Val Lys Leu Val Glu Ser Gly
GGA GGC TTA GTG CAG CCT GGA GGG TCC CTG AAA CTC TCC TGT GCA ACC 101
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr
10 15 20 25
TCT GGA TTC ACT TTC AGT GAC TAT TAC ATG TAT TGG GTT CGC CAG ACT 149
Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr
30 35 40
CCA GAG AAG AGG CTG GAG TGG GTC GCA TAC ATT AGT AAT GAT GAT AGT 197
Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile Ser Asn A~p Asp Ser
45 50 55
TCC GCC GCT TAT TCA GAC ACT GTA AAG GGC CGG TTC ACC ATC TCC AGA 245
Ser Ala Ala Tyr Ser Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg
60 65 70
GAC AAT GCC AGG AAC ACC CTC TAC CTG CAA ATG AGC CGT CTG AAG TCT 293
A~p A~n Ala Arg A~n ~hr L~u Tyr Leu Gln Met Ser Arg L~u Ly~ Ser
75 80 85
GAG GAC ACA GCC ATA TAT TCC TGT GCA AGA GGA CTG GCC TGG GGA GCC 341
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Glu Asp Thr Ala Ile Tyr Ser Cy~ Ala Arg Gly Leu Ala Trp Gly Ala
100 105
TGG TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCC TCA GGC 389
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly
110 115 120
GGA GGC GGA TCC GGT GGT GGC GGA TCT GGA GGT GGC GGA AGC GAT GTG 437
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val
125 130 135
CTG ATG ACC CAG TCT CCA TTG AGT TTA CCT GTC AGT CTT GGA GAT CAA 485
Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Va Ser Leu Gly Asp Gln
140 145 150
GCC TCC ATC TCT TGC AGA TCT AGT CAG ATC ATT GTA CAT AGT AAT GGA 533
Ala Ser Ile Ser Cy8 Arg Ser Ser Gln Ile Ile Val Hi~ Ser Asn Gly
155 160 165
AAC ACC TAT TTA GAA TGG TAC CTG CAG A~A CCA GGC CAG TCT CCA AAG 581
A~n Thr Tyr Leu Glu Trp Tyr Leu Gln Ly~ Pro Gly Gln Ser Pro Ly~
170 175 180 185
CTC CTG ATC TAC AAA GTT TCC AAC CGA TTT TCT GGG GTC CCA GAC AGG 629
Leu Leu Ile Tyr Ly~ Val Ser A~n Arg Phe Ser Gly Val Pro A~p Arg
190 195 200
TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC AGC AGA 677
Phe Ser Gly Ser Gly Ser Gly Thr A~p Phe Thr Leu Lys Ile Ser Arg
205 210 215
GTG GAG GCT GAG GAT CTG GGA GTT TAT TAC TGC TTT CAA GGT TCA CAT 725
Val Glu Ala Glu AQP Leu Gly Val Tyr Tyr Cy8 Phe Gln Gly Ser Hi~
220 225 230
GTT CCA TTC ACG TTC GGC TCG GGG ACA AAG CTG GAA ATT AAA 767
Val Pro Phe Thr Phe Gly Ser Gly Thr Ly~ Leu Glu Ile Lys
235 240 245
GCTTT 772
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 247 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Met Asp Val Ly~ Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Lys Leu Ser CYB Ala Thr Ser Gly Phe Thr Phe Ser A~p
Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Ly6 Arg Leu Glu Trp
Val Ala Tyr Ile Ser Asn ADP A~p Ser Ser Ala Ala Tyr Ser A~p Thr
Val Ly~ Gly Arg Phe Thr Ile Ser Arg Aqp Asn Ala Arg Asn Thr Leu
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Tyr Leu Gln Met Ser Arg Leu Ly~ Ser Glu A~p Thr Ala Ile Tyr Ser
~ys Ala Arg Gly Leu Ala Trp Gly Ala Trp Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser A~p Val Leu Met Thr Gln Ser Pro Leu
130 135 140
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala S~r Ile Ser Cy~ Arg Ser
145 150 155 160
~er Gln Ile Ile Val His Ser A~n Gly Asn Thr Tyr Leu Glu Trp Tyr
165 170 175
~eu Gln Lys Pro Gly Gln Ser Pro Ly~ Leu Leu Ile Tyr Ly~ Val Ser
180 185 190
A~n Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
195 200 205
Thr A~p Phe Thr Leu Ly~ Ile Ser Arg Val Glu Ala Glu ABP Leu Gly
210 215 220
Val Tyr Tyr Cyc Phe Gln Gly Ser Hi~ Val Pro Phe Thr Phe Gly Ser
225 230 235 240
~ly Thr Lys Leu Glu Ile Ly~
245
~2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
~A LENGTH: 58 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
~Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (gen ic)
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 3..47
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
CA CAT GTT CCA TTC ACG TTC GGC TCG GGG ACA AAG CTG GAA ATT AAA 47
His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Ly~
1 5 10 15
TAATGAATTC C 58
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acidc
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) ~LyuL.._L DESCRIPTION: SEQ ID NO:36:
Hi~ Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Ly~
l 5 lO 15
(2) INFORMATION FOR SEQ ;D NO:37:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 9 ba~e pairs
B TYPE: nucleic acid
C STRANnEnNEss: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) ~LyU~ DESCRIPTION: SEQ ID NO:37:
CC~, 9
(2) INFORMATION FOR SEQ ID NO:38:
( i ) S~YUL.._~: CHARACTERISTICS:
'A' LENGTH: 9 base pair~
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID No:38:
TTGAGTTTA 9
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: l6 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) ~LyuLhCE DESCRIPTION: SEQ ID NO:39:
CCAG~ ~ . C~A CTCTCC l6
(2) INFORMATION FOR SEQ ID NO:40:
yD~:~ CHARACTERISTICS:
'A) LENGTH: lO amino acid~
B) TYPE: amino acid
C) STR~NDEDNESS: ~ingle
,D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(xi) sr;yu~N~ DESCRIPTION: SEQ ID NO:40:
Met Clu Val Gln Leu Val Glu Ser Gly Gly
l 5 lO
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: lO amino acids
B TYPE: amino acid
C ST~ANnFnNFSS: single
~D~ TOPOLOGY: linear
(ii) M~T-T~'CUT-Ti' TYPE: peptide
(xi) ~yuL..~T~' DESCRIPTION: SEQ ID NO:41:
Met Glu Val Ly~ Leu Val Glu Ser Gly Gly
l 5 lO
(2) Il.rv~ATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
rA LENGTH: lO amino acid~
BI TYPE: amino acid
C ST~PNnFn~FSS: ~ingle
,D, TOPOLOGY: linear
(ii) MOTFC~TTF TYPE: peptide
(xi) SEQUENCE DT~'SCRTPTION: SEQ ID NO:42:
Gly Gly Gly Ser A~p Val Val Met Thr Gln
l 5 l0
(2) IN~OR~ATION FOR SEQ ID NO:43:
(i) sr;yur..._~ CHARACTERISTICS:
'A' LENGTH: l0 amino acids
B TYPE: amino acid
C STRANDEDNESS: ~ingle
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) sr;yur,~_~ DESCRIPTION: SEQ ID NO:43:
Gly Gly Gly Ser Asp Val Leu Leu Thr Gln
- l 5 lO
(2) INFORMATION FOR SEQ ID NO:44:
(i) SLyUL~_L CHARACTERISTICS:
~A' LENGTH: 8 amino acids
B TYPE: amino acid
C STRANnT~.n~T'~S ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(xi) S~YU~N~: DESCRIPTION: SEQ ID No:44:
Ser Gly Gly Pro Glu Gly Gly Ser
1 5
(2) INFORMATION FOR SEQ ID NO:45:
(i) ~yu~ ~ CHARACTERISTICS:
'A LENGTH: 119 amino acid~
Bl TYPE: amino acid
C STRANDEn~SS: ~ingle
D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Protein
(B) LOCATION: 1..119
(D) OTHER INFORMATION: /note= "B3 VH region"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
A~p Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Ly~ Leu Ser Cy~ Ala Thr Ser Gly Phe Thr Phe Ser A~p Tyr
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
Ala Tyr Ile Ser Asn A~p A~p Ser Ser Ala Ala Tyr Ser Aap Thr Val
Ly~ Gly Arg Phe Thr Ile Ser Arg A~p A~n Ala Arg Asn Thr Leu Tyr
Leu Gln Met Ser Arg Leu Ly~ Ser Glu A~p Thr Ala Ile Tyr Ser Cy~
Ala Arg Gly Leu Ala Trp Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID No:46:
(i) ~yu~N~ CHARACTERISTICS:
~AI LENGTH: 119 amino acids
B TYPE: amino acid
C STRANDFnNESS: ~Lngle
~Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..119
(D) OTHER INFORMATION: /note= "Human fetal i noglobulin
56Pl'CL VH region"
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Se~ Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
Ala Met Hi~ Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
Ala Val Ile Ser Tyr A~p Gly Ser A~n Ly~ Tyr Tyr Ala A~p Ser Val
Ly~ Gly Arg Phe Thr Ile Ser Arg Asp A~n Ser Lys Asn Thr Leu Tyr
ao
Leu Gln Met A~n Ser Leu Arg Ala Glu ABP Thr Ala Val Tyr Tyr Cy~
Ala Arg Arg Ser Ala Arg Thr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
'A LENGTH: 119 amino acid~
B TYPE: amino acid
C STR~Fn~F~cs: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Protein
(B) LOCATION: 1..119
(D) OTHER INFORMATION: /note= "F -ni 7e~ B3 VH region"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
A~p Val Ly~ Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Ly~ Leu Ser Cy~ Ala Thr Ser Gly Phe Thr Phe Ser A~p Tyr
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Ly~ Gly Leu Glu Trp Val
Ala Tyr Ile Ser Asn Asp Asp Ser Ser Ala Ala Tyr Ser Asp Thr Val
Ly~ Gly Arg Phe Thr Ile Ser Arg A~p Asn Ser Ly~ A~n Thr Leu Tyr
Leu Gln Met A~n Arg Leu Arg Ala Glu Asp Thr Ala Ile Tyr Ser Cys
Ala Arg Gly Leu Ala Trp Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
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(2) IN~ORhATION FOR SEQ ID NO:48:
(i) S~U~:N~ CHARACTERISTICS:
A' LENGTH: 112 amino acids
B TYPE: amino acid
~C STRANDEDNE~S: ~ingle
~D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Protein
(B) LOCATION: 1..112
(D) OTHER INFORMATION: /note= B3 VL region"
( Xi ) S~U~N~: DESCRIPTION: SEQ ID NO:48:
A~p Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Aep Gln Ala Ser Ile Ser Cy~ Arg Ser Ser Gln Ile Ile Val His Ser
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
Ser Hi~ Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
(2) INrORMATION FOR SEQ ID NO:49:
( i ) S~yU~N~ CH M ACTERISTICS:
~A'I LENGTH: 112 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Protein
(B) LOCATION: 1..112
(D) OTHER INFORMATION: /note= Human IgM GM607 VL region"
(Xi) ~Q~N~ DESCRIPTION: SEQ ID NO:49:
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gln Gln Ser
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Pro Gln Leu Leu Ile Tyr Leu Gly Ser A~n Arg Ala Ser Gly Val Pro
A~p Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr ~yr Cys Met Gln Gly
Leu Gln Thr Pro Gln Thr Phe Gly Gln Gly Thr Ly~ Val Glu Ile Ly~
100 105 110
(2) INFORMATION FOR SEQ ID NO:50:
~i) SEQUENCE CHARACTERISTICS:
,A'I LENGTH: 112 amino acids
B TYPE: amino acid
C, STRANDEDNESS: single
,D TOPOLOGY: linear
( ii ) ~nr-FCuT-F TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Protein
(B) LOCATION: l..112
(D) OTHER INFORMATION: /note= "F -ni ze~ B3 VL region"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
A~p Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
l 5 l0 15
Glu Pro Ala Ser Ile Ser Cy~ Arg Ser Ser Gln Ile Ile Val Hi~ Ser
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro Gln Leu Leu Ile Tyr Lys Val Ser A~n Arg Phe Ser Gly Val Pro
A~p Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cy~ Phe Gln Gly
Ser His Val Pro Phe Thr Phe Gly Gln Gly Thr LYB Val Glu Ile Lys
l00 105 ll0
(2) INFORMATION FOR SEQ ID NO:5l:
(i) SEQUENCE CHARACTERISTICS:
'A) T~ n: 4 amino acids
B) TYPE: amino acid
,C) STRANnF-nNESS: ~ingle
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) S~Q~.CE DESCRIPTION: SEQ ID NO:51:
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Ly~ Aap Glu Leu
(2) I~rOR~ATION FOR SEQ ID NO:52:
(i) SrQuL.J~ CHARACT~RlSTICS:
~A'l LENGTH: 4 amino acid~
B TYPE: amino acid
,C STRANDEDNESS: single
D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~ uL.._r; DESCRIPTION: SEQ ID NO:52:
Arg Glu ABP Leu
(2) INFORMATION FOR SEQ ID NO:53:
( i ) ~LQULN~ CHARAC~ERISTICS:
A' LENGTH: 5 amino acids
B TYPE: amino acid
C STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MnT-T~-CUTT~' TYPE: peptide
(xi) S~ur;l._r; DESCRIPTION: SEQ ID NO:53:
Arg Glu Asp Leu Leu
l 5
