Note: Descriptions are shown in the official language in which they were submitted.
3~$7
W091/19745- 1 - . PCT/US91~04187
CHIMERIC TOXINS WITH IMPROVED INTER-DOMAIN GEOMETRY
Backqround of the Invention
This invention relates to the use of
recombinant DNA techniques to construct chimeric toxin
molecules.
The highly selective effects asserted by many
hormones, toxins, and oth~ biologically active proteins
are in part possible because such proteins possess more
than one functionally distinct polypeptide domain. Some
plant and bacterial toxins, e.g., have evolved with
separate domains responsible for cell binding, membrane
translocation, and intoxication. The combination of
properties conferred by the various domains results in
extremely potent bioactive molecules.
The diphtheria toxin (DT) is an example of a
lS naturally occurring multi-domain protein. DT consists
of a number of domain~, each of which confers a
particular function, and all of which, in combination,
result in an extraordinarily active toxin molecule. DT
can be characterized, starting at the amino terminal end
of the molocule, as follows: a hydrophobic leader
~ignal equence ~ ~amino acids Val_2s - Ala_l);
n~ymat~cally-activ- Fragment A tamino acids Glyl -
A~gl93) whlch tnclud-~ a domain which catalyzes the
th- nicotinamide adenine dinuclootide-depondont
ad no-ln dipho~phate (ADP) ribosylation of tho
u~iryotic protein synthe~is factor termed "Elongation
Factor 2"; the ~rotease-sen~itive disulf~de loop 1
~amino acld8 Cysl86 - Cys201), wh~ch contains a
cl-a~age domain; and Fragment B ~amino acids 8erl9~ -
~0 8-r535), which includes a translocation domain and a
g n rali~ed binding domain flanking a ~econd disulfide
. .
~ ~3~7 - 2 - PcT/us91/o4~8?
loop (12, amino acids Cys461 - Cys471).
process by which DT intoxicates sensitive eukaryotic
cells involves at least the following steps: (1) the
binding domain of diphtheria toxin binds to specific
receptors on the surface of a sensitive cell; (ii) while
bound to its receptor, the toxin molecule is
internalized into an endocytotic vesicle; (iii) either
prior to internalization, or within the endocytotic
vesicle, the toxin molecule undergoes a proteolytic
cleavage in the 11 cleavage domain between Fragments A
and B; (iv) as the pH of the endocytotic vesicle
decreases to below 6, the toxin spontaneously inserts
into the endosomal membrane; (v) once embedded in the
membrane, the translocation domain of the toxin
facilitates the delivery of Fragment A into the cytosol;
- (vi) the catalytic activity, which resides in a domain
in Fragment A, causes the death of the intoxicated
cell. The mechanism of cell killing by Pseudomonas:
exotoxin A, and possibly by certain other
naturally-occurring toxins, is very similar.
me therapeutic potential of man-made toxin
molecules containing various functional domains (whether
polypeptide or nonpeptide) has been appreciated for many
years. Paul Ehrlich, Ehrlich (1906) in Collected
2S 8tudl-s on Immunity 2:~2-~7, was the first to suggest
th- oon~truotion of bifunctional molecules that combined
a mol-cul- with an af~inlty for a speoiflc target ~a
targeting or cell-binding domaln) with a molecule that
ct~ a~ a cytotoxic agQnt (a cytotoxic domain).
~ince that time numerous biological and
chemioal moieties have been coupled in a variety of way~
ln attempts to create molecules that exhibit a degree of
~-lecti~e, i.e., targeted, cytotoxicity. Early efforts
w-r- typified by the con~ugation of non-peptide toxins
, . , , .......... ., . , .- . - . .
.. .
' ' . . '. . ~ "' ~ . ''; ' ,` ' " ' , ;'`
. ' ' .: , . . .... ~ . -
Z ~ 37
W O 91/19745 _ 3 _ PC~r/US91/04187
to solubilizing molecules or to antibodies. More
recently, the advent of molecular genetics has allowed
the engineering of chimeric genes that encode novel
multi-domain chimeric toxin proteins. See, e.g., Murphy
S U.S. Patent No. 4,675,382, hereby incorporated by
refeeence, which teaches the construction of hybrid
proteins that include the enzymatically active (toxin)
domain of DT, the cleavage domain of DT, the
tran~location domain of DT, and a cell-specific binding
domain derived from a second protein. The hybrid
proteins described in Murphy suPra combine DT toxin
domain~, which confer toxicity, with a domain from a
different protein, e.g., interleukin 2 (IL-2), which
confers extremely selective cell binding properties.
lS Improvements in the intrinsic properties of the
constituent components, i.e., the u~e of more
highly-spec~fic cell-binding agents, e.g., monoclonal
antibodies, and the use of toxins of increased potency,
e.g., plant or bacterial toxins, have been the primary
route~ to improved toxin conjugates. The way in which
the cell binding and cell-killing entities of
blfunc:io~al molecule are coupled has also received
attention in attempts to improve the performance of
the-e molecules. These efforts ha~e been directed to
2~ pr-~-rving the act~vitie~ of the primary component~,
incr-a~ing ~olubllity, lncre~ing the ratio of cytotoxic
~g nt bound to o~ eclflc bindlng agent, or
l~cr~ g the ea~e with which a reguired step in
intoxloation, e.g., the cleavage of a toxin domain from
~0 th- r-~t of the molecule, is carried out.
Various spacer or l~n~er mQmbers have been
plaoed ~etween the domains ~or nonp~ptide functional
e~tlti-r) of con~ugate-toxins in efforts to realize the
advantag ~ discu~sed above. Rowland et al. (1975)
. ~ . . . ~ .
, ............. , - . ;, . .,..... ., : .
,, , . , . . : ~ ~.. . .
.. : , . . . . . .
- ` ' - ~:, :. ' ' ' - ,
Wo91/19745 2 ~ S~ '7 PCT/US91/W187
Nature 25s:487 reports that the use of polyglutamic acid
(molecular weight=35,000) as a linker between chemical
toxins and antibodies was preferable to direct linkage
of the toxin to the antibody.
"By minimizing interference with the
chemical structure of the Ig in this linkage
step, a conjugate is produced with both a
high concentration of drug and little loss
of antibody activity . . . The concept of
linking cytotoxic drugs to antibody through
an inert intermediate carrier offers a wide
scope for improved cancer chemotherapy in
the future, with the possibility of using a
variety of drugs, different carriers and
antibody preparations of greater purity."
Monsigny et al. (1980) FE~S Letters ll9:l8l reports
miaximizing the activity of a drug, e.g., daunorubicin
con~ugated to a carrier, e.g., an antibody, by the
insertion of a peptide containing spacer between the
drug and the carrier. The spacer arm,
2-(l-thio-B-D-glycopyranosyl)-ethanoyl-L-arg-L-leu, ~a~
be cleaved by lysosomal but not by serum proteases.
~For technical reasons, and
because the activity of a drug is
partially or totallY lost when it is
substituted or chem~cally modified, we
devised a spacer arm such that the
drug carrier con~ugate is stable in
serum and can be specifically split by
2S lysosomal proteases leading to the free
drug in~ide the target cells."
~rnon t al. (~9~2) ~mmunol Rev. 6Z:S re~orts the use of
d-xtr n to llnk daunomycln to an antibody.
"Th- rea~on for employing this
procedure was two-fold. Fir~t, it i8
exp4cted to re~ult in higher extent of
_ drug bint1ng per antibody molecule,
wh~ch ~hould lead to higher cytotoxic
actlvity; econd, the use of a
macromolecule as a spacer arm between
th- drug and the carrier could prove
advantageous in permitting higher
...
',, , . , . . - . ' ' , .: . ' -
. ; .; . ~ :
, ~.. ..
:,. . . .. , ~ .: : .
. ,~ , ~ - .. , ...
~r . . . - .
~ 7
Wogl/19745 _ 5 _ PCT/US91/04187
exposure of the drug moiety on the
conjugate surface with less steric
hindrance and hence higher efficacy "
Truet et al (1982) Proc Natl Acad Sci USA,
79 626-629 reports coupling daunorubicin to succinylated
bov~ne serum albumin (BSA) by a spacer arm one to four
am~no acid residues in length The purpose of the
spacer arm was to allow lysosomal cleavage between the
drug and the ~SA molecule
Neville et al (1989) JBC 264 14653-14661
reports that a cleavable cross-linker enhances potency
of a DT-antigen conjugate three to ten fold The
crosslinkers are cleavable at acid pH, and are thus
cleaved in an acidic compartment The increased potency
i8 believed to be due to an enhanced intracellular
toxln-toxin receptor interaction which leads to
increased translocation the con~ugate is thought to be
~terically hindered prior to, but not after, cleavage
A nonpeptide crosslinker, bis-maleimidoethyoxy propane,
was used
Greenfield et al PCT/US85/00197 describes a
toxin-antibody con~ugate wherein the tox~n and antibody
are coupled by means of a ~pacer peptide
"the ~nvent~on i~ designed to
provide a toxin con~ate which has the
2~ a~pro~ri-te geometry ~or tran810cating
th- cytotox~c fr~gment into the t~rget
c-ll, th- c-~aclty to ret-in its
blndlng ~r-gm nt ~r~or to ~uch
trunrloc~tlon, and/or the ability to
olublli~- the cytotoxic portion In-
on a~poct of the invention, the spacer
1~ de~lgned ~o a~ to permit the
cytotoxlc ~ortion of the molecule ready
acc-~ to the c-ll membrane A~ the
~ of a typical antibody ~binding)
fragm nt 1~ v ry much greater than that
of mo~t cytotoxlc fragments, there i8
con~id rable teric hinterance of the
. . . . . .
. . -, . , . . , , :
: . ~ . . - . . -.
:. . . . - . , - . -.
:..... ; - , . . . . .
,. . , ` ::: :. , `............ . -
~ . . . ,::, .
- - .. . .
. . .
: . . ~ . .. . :- ... . .
.. . . . - - . ~ , . . ..... -
.~ . . .. . . . . . . . .
WO9l/19745 - 6 - PCT/US91/WI87
2~ 7 '~~
access to the cell membrane by the
cytotoxic portion imposed by the sheer -
bulk of the antibody or antibody
fragments In order to effect
this translocation, the spacer needs to
be sufficiently flexible to allow the A
S portion to reach the cell membrane, and
sufficiently extended to permit it to
have sufficient reach "
Summarv of the Invention
In general, the invention features a chimeric
toxin including protein fragments joined together by
peptide bonds, including, in sequential order, beginning
at the amino termi~al end of the chimeric toxin (a)
the enzymatically active Fragment A of diphtheria
toxin; (b) a fragment including the cleavage domain
11 adjacent Fragment A of diphtheria toxin; (c) a
fragment includinq (i) at least a portion of the
hydrophobic transmembrane region of Fragment B of
diphtheria toxin, the fragment having a deletion of at
least 50, preferably of at least 80, diphtheria toxin
amino acid residues, the deletion being C-terminal to
the portion of the transmembrane region, and the
fragment not including domain 12~ or, (ii) a fragment
including at least a portion of the hydrophobic
transmembrane region of Fragment B of diphtheria toxin
2~ wher-in said Fragment B o diphtheria toxin does not
lnclud- any diphther~ toxln 6eguences C-terminal to
amlno acld r-rldue 386 of native diphtheria toxin; (d)
a ~paa-r ~d-flned lnfra); and ~e) a portion of a
c-ll-rp~cl~ic polypeptide ligand, the cell-specific
poly~ ptlde ligand being a cell growth factor preferably
a lym~ho~ine, g , interleukin 2 (IL-2), or interleukin
) The portlon of the cell-specific polypeptide
ligand lnclud-~ at lea~t a portion of the binding domain
of th polypeptide l~g~nd, the portion of the binding
...., .,, ~; ..
-: , : . ~ . . .
2~3~
WO91/19745 _ 7 _ PCT/US91/04187
domain being effective to cause the chimeric toxin to
bind selectively to the target cell e.g., lymphocytes,
e.g., T-cells or ~-cells bearing receptors for the
ligand. Cell growth factor, as used herein, means a
S protein that binds to a cell surface receptor found on a
mammalian cell and causes proliferation of the cell.
Preferred cell growth factors are lymphokines, i.e.,
cell growth factors that bind to and stimulate the
proliferation of lymphocytes.
In preferred embodiments DAB389 is the
cytotoxic portion, i.e., (a), (b), and (c) above.
(DA~389 consists of me~hionine followed by residues
1-386 of native DT followed by residues 484 and 485 of
native DT.) The construction of DAB389 is discussed
IS in US8N 488,603, filed March 2, l990, hereby
~ncorporated by reference.
Preferred embodiments include those in which
the spacer: is at least 5 amino acids in length,
preferably 10-30 amino acids in length; when placed
between the seguence of DT fragment DAB~85 (DAB485
consists of methionine followed by the first 484 amino
acld residues of native DT) and amino acid residues
2-133 of IL-2, ha~ a Bnorm value of l.000 or greater,
more preferably of 1.12~ or greater, and most preferably
2~ o~ 1.135 or greater; is ¢ompo-ed of at least 60~, and
~r-f-r~bly of ~t l-a~t 80~ of amino acids from the group
o~ ly~lne, ~-rlne, glyclne, proline, aspartic acid,
glutumlo acld, glutamin-, threonine, aspara~ine, or
~rglnlne; i~ at least 60%, and preferably at least 80~,
~0 ho~ologous to any of
Ala-Pro-Thr-8er-8er-8er-Thr-~ys-Lys-Thr, hereinafter
r-f-rred to a~ ~l-lO), Pro-Ly8-8er-Gly-mr-Gln-Gly,
h reinafter referr-d to a~ ~l-7 Gly),
P~ro-Thr-8er-8er-8er-Thr-Lys, (hereinafter referred to as
, - . ~
. - , . . . ............................... .. .
.. .. . . . . .. .. . ....... . .
WO 91/19745 ~ 7 - 8 - PCI`/US91/04187_
( 1-7 Lys), or multiples thereof (the number of multiples
hereinafter referred to with a superscript, e g ,
~ o)2, which indicates 2 tandem copies of the (1-10)
subunit; results in an affinity of the chimeric toxin
for the tarqet cells that is greater than the affinity
of a second chimeric toxin for the target cells, the
second chimeric toxin being identical to the chimeric
toxin except that the second chimeric toxin lacks the
spacer; or, results in the chimeric toxin exhibiting
cytotoxicity for the target cells that is a least 2
times greater than the cytotoxicity exhibited by a -
second chimeric toxin for the target cells, the second
chimeric toxin being identical to the chimeric toxin
except that the second chimeric toxin lacks the spacer
Diphtheria toxin, or native diptheria toxin, as
used herein, means the 535 amino acid residue mature
form of diphtheria toxin protein secreted by
CorYnebacterium diDhtheriae The seguence of an allele
of the gene wh~ch encodes native diphtheria toxin can be
found in Greenfield et al (1983) Proc Natl Acad Sci
U8A 80 6853-6857, hereby incorporated by reference
Enzymatlcally active Fragment A, as used herein, means
umlno acid residues Gly 1 through ~rg 193 of native D~,
or an enzymatically active derivative or analog of the
2S natural seguence Cleavage domain 11, as used herein,
m an~ th- protea-e en~ltive domain within the region
~annlng Cy~ 186 and Cy- 201 of nitive DT Fragment B,
~ ur~d h-r-ln, m n~ th- region from 8er 19~ through
8-r 535 of natlve DT The hYdrophobic transmembrane
~0 r-glon, or hydrophobic domain, of Fragment B, as u~ed
h r-ln, means the amino acid ~eguence bearing a
~tructural l~ilarity to the bilayer-sp~nning hellces of
lnt-gral membrane proteins and located approximately at
or d rlv d from amlno acid residue 3~6 through amino
. ;.: .. . . ,,: . .. -.. . . : :
. : . , ,, : ,.. , . . .::
- - , . ~
, , ., -
- . .. ~ . .- .:
.. . .
~S~ 7
WO91J19745 _ ~ _ PCT/~S91/04187
acid residue 371 of native diphtheria toxin Domain
12, as used herein, means the region spanning Cys 461
and Cys 471 of native DT The generalized eukaryotic
binding site of Fragment B, as used herein, means a
region with~n the C-terminal 50 amino acid residues of
native DT responsible for binding DT to its native
receptor on the surface of eukaryotic cells The
generalized eukaryotic binding site of Fragment B is not
included in the chimeric toxins of the invention
A spacer, as used herein, is a polypeptide
which possesses one or more of the following
characteristics (1) when placed between the sequence
of the diphtheria toxin fragment DAB485 and amino acid
re~idue~ 2-133 of IL-2, it possesses an amino acid
lS residue with a normalized B value (Bnorm) (as defined
in Karplu~ et al (1985) Naturwissenschaften 72 212,
hereby incorporated by reference) of 1 000 or ~reater,
preferably of 1 125 or greater, and most preferably of
1 135 or greater; (2) at least 60% and preferably at
least 80% of its ~mino acid residues are from the group
of ly~ine, serine, glycine, proline, aspartic acid,
glutamic acid, glutamine, threonine, a~paragine, or
arglnlne; (3) it possesses the seguence, or multiples of
th- 8eguence Ala-Pro-Thr-8er-~er-8er-Thr-Lys-Lys-Thr;
25 ~) lt 1~ ~t lea~t 60% and preferably at least 80%
homologou~ to the ~-guence, or to multiples of the
~ gu-nc~ t-d ln ~3) (S) lt possesse~ the sequence,
or multlple8 of the sequence
Pro-Ly~-8er-~ly-Thr-41n-Gly; ~6) it is at least 60% and
~0 ~r-f-rably at least 80% homologous to the seguence, or
multl~ of the seguence, li8ted in ~S) ~7) lt
~0-~ 8 the seguence, or multiples of the seguence of
~ro-Thr-8-r-8er-8er-Thr-Lys; or (8) it 18 at least 60%
~d ~r-f-rably at least 80% homologou~ to the seguence,
.. ...
.. .,.. .. . . . ~.
,;, ~ . ~ . , .. . . . . . -
. . ~ .... . . . . . .. . .
: - : .' : .. ,. .. :
3~7
Wog1/19745 - 1o - PCT/US91/0418
or multiples of the sequence, listed in (7).
The Bnorm value of residues in a polypeptide
sequence can be determined with computer programs, e.g.,
with FLEXPRO (Intelligenetics, Mountain View (CA)) which
predicts the flex~bility between alpha carbon atoms at
each point of a selected protein sequence, using the
method (the Bnorm method) of Karplus et al., suPra.
FLEXPRO calculates the chain flexibitiy at a selected
amino acid residue from the average values of the atomic
temperature factors (also called B values or
Debye-Waller factors) of the alpha carbon atoms in
ad~acent amino acids. The B value is the mean sguare
displacement of the atom from its average position in
the protein. Flexible locations have high ~ values
because their displacement can be large.
The Bnorm value for an amino acid in FLEXPRO
is affected by the Bnorm values of its neighboring
amino acids. The predicted flexibility at an amino acid
calculated by FLEXPRO is the weighted sum of the Bnorm
value~ (taking account of neighbors) of the seven amino
acids closest to that point in the sequence. The weight
for the two outermo~t amino ac~ds is 0.25; for the two
n-xt to them, 0.5; for the two ad~acent to the central
amino acid, 0.75; and for central amino acit it~elf, l.
For aoh amino wld, the welght is multiplied by the
Bnorm v-lU- ~t~klng account of neighbor~). When an
uml~o acid -qu-nce i~ input to FLEXPRO, the program
calculat-~ the ~norm values and displays 7-amino ac~d
r-~ldue ~ections and the pea~ Bnorm value for each of
~0 th- 7-amino acid residue sections.
A normalizod B value of less than l indicates a
rigid umino acid; ~ value greater than l indicate~ a
fl-~ible amino acid. Observations in a number of
prot-in~ ~uggest that the amino acids alanine, valine,
.. . . . . .. .
' ' ' - ': '' . '
, - , , . ~. . .. .
.
.
. , .
, , ., :.
: - - . .. .. . . . .. .
3~ 3'7
Wo91/19745 - 11 - pcT/uss
leucine, isoleucine, tyrosine, phenylalanine,
tryptophan, cysteine, methionine, and histidine tend to
be rigid and that the amino acids lysine, serine,
glycine, proline, aspartic acid, glutamic acid
glutamine, threonine, asparagine, and arginine tend to
be flexible (Karplus et al suPra)
The sequence used as a spacer may be derived
from any source e g , from one of the polypeptides used
to construct the chimeric toxin, from other naturally
occuring sequences, or from synthetic sequences
regardless of whether they are naturally occuring
The invention also features a chimeric toxin
encoded by a fused gene including regions coding for the
protein ragments, a DNA sequence encoding the chimeric
toxin, an expression vector containing the DNA sequence
encoding the chimeric toxin, a cell transformed with the
expre~ion vector, and a method of producing the
chimeric toxin including culturing the transformed cell
~nd isolating said chlmeric toxin from the cultured cell
or supernatant
The invention also features spacer peptides
having the seguence
Ala-Pro-Thr-8er-Ser-Ser-~hr-Lys-Lys-Thr, or tandem
repeats of that 10 residue subunit ~with or without one
or two additional re-~due- at each end (or between the
indi~idual ubunit~ of the tand~m repQat) that ari-e
~ro~ th- inolu~ion o~ function-l or disfunctional 1/2
r-~trlction n~yme r-cognltion site linker sequences in
th- DNA that encodes the spacer), DNA segments encoding
th- ~pac-r ~eptides ~with or without functional or
dl-~unctional 1/2 re-triction enzyme recognition site
lln~-r ~-quence~), vector~ containing these DNA
~-gm-nt~, c-lls trunsformed with the~e vectors, and
m thod~ of ma~ing the ~pacor peptide including culturing
:. -. . ......... . : . .
. : ,. ............... . - ... ..
.. . . , ., . - :: : : - .. . -- - .
W091/19745 2 C~ '7 PCTlUS91/0418
the transformed cell, and isolating the spacer from the
cultured cell or supernatant
Preferred embodiments include a DNA segment
encoding the spacer wherein the spacer is a tandem
repeat of the subunit
Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr, and the DNA
~eguence encoding each occurence of the subunit in the
spacer is nonhomologous with the DNA sequence encoding
every other occurence of the subunit in the spacer or
the chimeric toxin, the nonhomology being sufficient to
prevent recombination between seguences encoding
tandemly repeated subunits
The invention also features a method of
preventing recombination between the tandemly repeated
lS subunits of spacer-peptide-encoding DNA by choosing the
codons of each subunit-encoding sequence such that the
DNA encoding each subunit is nonhomologous with the DNA
encoding e~ery other ~ubunit, the nonhomology being
sufficient to prevent recombination between tandemly
repeated subunits
~ he invention also features DNA encoding a
spacer (with or without functional or disfunctional 1/2
rertriction enzyme recognition site linker sequences),
an expression vector containing that DNA, a cell
tran~formed with that expre~sion vector, and a method of
~roducing the ~ac-r includlnj culturing the transformed
c-ll nd lrol~tinq ~id ~ac-r from the cu~tured cell or
cU~ rn~t~nt
Molecules of the invention exhibit improved
~0 blnding affinity and improv d cytotoxicity for cells
b-arlAg th rece~tor to which the ligand portion of the
ohim rlc toxin bind- In the management of autoimmune
dlr-ar-, allograft re~ection, and other
lym~hocyt~-dependent conditions, a chimeric toxin that
. . .
. . . .' ,., ' . ,: ' . ' ' " .
: - ., - ,
.: :; , - - , . .: .
: ' ' . :. : , ,
- . .. : . - :
, . : . .
, . ., - .. ~ .
. - . . :-. ' :. ' - . , . - ' . :
. . ., : . . - ~ - . - -,, ~:
. ~. . . - - . . .
WO91/1974~ - 13 - ~ ~ S3~ ~7. - PCT/US91/~187
employs a cell bindinq portion that recognizes an
interleukin (or other growth factor~ receptor must
compete with indigenous interleukin (or other growth
factor) for sites on the target cell. Thus,
optimizat~on of the early, cell-binding, step is
partlcularly cr~tical in chimeric toxins in which the
cell binding portion recognizes a ligand such as an
interleukin receptor.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments and from the claims.
Description of the Preferred Embodiments
The drawings will first be briefly described.
Drawinq~
lS Fig. 1 i5 a diagram of the DT molecule and
various fu~ion proteins:
Fig. 2 i~ a depiction of the construction of
the plasmids of a preferred embodiment;
Structure and 8Ynthe~is of chimeric toxins with imProved
inter-domain qeometrY
DA~86-(1-10)-IL-2 i8 a chimeric toxin
~olypeptide con~ifiting of, in the following order: Met;
umino acid residues 1 through His~84 of mature native
DT: ~ly; the amino acid residues
Al~-Pro-Thr-8-r-8er-8er-Thr-Lys-~y~-Thr (indicated a8
~1-10)); ~i~; Ala~85 of mature natlve DT; and ~mino
aold r~ldu-~ 2 through 133 of S~-2. The DT portion of
th ohim ric toxin DAB~8C-(1-10)- IL-2 includes all of
DT fragm nt ~ and the portion of DT fragmont B extending
- ~0 to r-~ldu- ~85 of mature nat~ve DT. See Fig. la for the
~t~uotur- of DT. Fig. lb ~hows the structure of
DAB~6-1~--2. ln Fig. lb a wide bar indicate8 the
fu~lon ~rotein, narrow connecting lines repre~ent
d~l-tion~, numbers above the bars ~re amino acid residue
. .
. ; : - : - , . -
W091/19745 ~ 7 - 14 - PCT/USs1/04182
num~ers in the DAB nomenclature (described below),
numbers below the bars correspond to the amino acid
residu numbering of native DT, cross hatching indicated
amphiF -hic regions, darkened areas correspond to the
transr ~brane region, IL-2-2-133 indicates amino acid
re81d 3 2-133 of IL-2, Ala ~ alanine, Asn = asparagine,
A~p ~ ;partic acid, Cy8 - cysteine, Gly = glycine, His
~ hist;dine, Ile - isoleucine, Met = methionine, Thr =
threonine, Tyr = tyrosine, and Val = valine. (The
nomenclature adopted for DT-IL-2 toxins is illustrated
by DAB486-(1-10)-IL-2, where D indicates diphtheria
toxin, A and B indicate wild type sequences for these DT
fragments, the number in the parenthesis represents a
spacer polypeptide, and IL-2 indicates mature human
lS interleukin-2 seguences. The numerical subscript
indicates the number of DT-related amino acids in the
fusion protein, the last of which is at the C-terminal
end of the spacer where a spacer is inserted. Note that
the last two codons of DT also function as a l/2 S~hI
site. 8ince the deletion of the tox signal sequence and
expres~ion from the trc promoter re6ults in the addition
of a methionine residue to the N-terminus, the numbering
o DAB-IL-2 fu~ion toxins is ~l out of phase with that
of native diphtheria toxin.)
DAB~86-~1-lO~-rL-2 was ¢onstructed from
DAJ~J6-IL-2. pDW2~, which c~rries DAB~86-IL-2 wa~
¢on~truct-d a~ follow~. pUCl8 (New England 8ioLabs) was
dige-ted with P~tI and B9lI and the PstI-~glI fragment
¢arrying th~ E.coli origin of replication, the
~olylln~-r region, and the 3' portion of the
B-lacatama~e gene ~ampr) was recovered. Plasmid
pR~-233-2 ~Pharmacia) was digested w~th PstI and L~lI
and th- P8tI-B~lI fragment carrying, two transcr~ption
t~r~nator~ and the 5' portion of the ~-lactamase gene
'
, - ~ : :
- . ,., - .
W091/19745 - 15 - 2~ S~ 7 PCT/US91/~187
was recovered. pDW22 was constructed by ligating these
two recovered fragments together.
pDW23 was constructed by isolating a BamHI-SaII
fragment encoding human IL-2 from plasmid pDW15
~Williams et al. (1988) Nucleic Acids Res. 16:10453-
10467) and ligating it to BamHI/SalI digested pDW22
~described above).
pDW24 was constructed as follows. A BamHI-NcoI
fragment carrying the trc promoter and translational
initiation codon (ATG) was isolated from plasmid
pKX233-2 (Pharmacia). The DNA seguence encoding amino
acid residues 1 through 485 of DT was obtained by
digesting pABC508 (Williams et al. (1987) Protein
Eng~neering 1:493_498) with SDhI and HaeII and
recovering the HaeII-SPhI fragment containing the
~equence encoding amino acid residues 1 through 485 of
DT. A NcoI/HaeII linker (5'CCATGGBCGC 3') was ligated
to the HaeII-8~hI fragment and that construction wa6
then ligated to the previously isolated 8amHI-NcoI
fragment carrying the trc promoter. This results in a
BamHI-80hI fragment bearing, in the following order, the
trc promoter, the NcoI site (which supplies the ATG
lnltiator codon for Met), and the seguence encoding
re~idue~ 1 through ~85 of native DT. This fragment was
ln--rt d lnto DDW23 that had been dlge~ted wlth ~amHI
and a~l. Th- r-rulting pla-mid wa~ de-ignated pDW24.
~D~2~ 1J ~OWn ln Fig. 2. The inr-rt correspondlng to
DAB~86-IL-2 18 ~hown a8 a heavy llne. In Fig. 2
flll d clrcl-~ indi¢ate NcoI sites, open circlos
i~dicat- N~lI slt-~, open dlamond~ ~ndicate ClaI sltes,
~lll d gu~res lndioate HDalS sltes, open ~guare-
lndi¢at- 8~hI slt-s, and fllled trlangles indicate 8alI
it--. The fusion protein ~DAB~86~ 2) encoded by
~DN2~ i~ expres~ed from the trc promoter and consists of
,
.
' ' '~ ~ ',, ' '. ' ' '~. , ' , ' ' .' , ' ' .
' ,' . ~,'. ,., ' ,`"'.,' .',' "'~' ', ,,"'.' '" " '' '' ' .
. ' : . ' ' . .: : . ' '
. . : ' . ' : ' ' . . . ' .
W091/19~45 ~ ~ 3 ~ S 7 - 16 - PCT/US91/W187,_
Met followed by amino acids l through 485 of mature DT
fused to amino acids 2 through 133 of human IL-2.
DNA encoding the polypeptide spacer, (l-lO),
was synthesized and inserted into pDW24. pDW24 was cut
S at the SphI site at the 3' end of the DT sequence and a
synthetlc seqyence encoding a spacer (or multiples
thereof) inserted. The sequence of the synthetic
seguences encoding 1, 2, and 3 copies of the (l-lO)
spacer are shown in Table l.
.~ `
. ' : ; . ' ''
' ' ~ ', : ,: . ,' :
~ 91/1~45 - 17 - 2~ pcT/usgl/o4l~ ~
TABLE 1: SPACER SEQUENCES
1 2 3 4 5 6 7 8 9 10
1.Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr His
CT CCG ACC AGC TCT AGC ACT AAA AAG ACT CAT G
GTA CGA GGC TGG TCG AGA TCG TGA TTT. TTC TGA
S Functional SphI site Disfunctional SphI site
1 2 3 4 5 6 7 8 9 10
2. Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr-
GT GCA CCG ACT AGC AGC TCT ACT AAG AAA ACA-
GTA CCA CGT GGC TGA TCG TCG AGA TGA TTC TTT TGT-
Di~functional SphI ~ite
11 12 13 1~ 15 16 17 18 19 20 . .
Ala Pro Thr 8er Ser Ser Thr Lys Lys Thr His
lS GCT CCT ACC TCT TCT AGC ACG AAG AAG ACG CAT G
CGA GGA TGG AGA AGA TCG TGC TTC TTC TGC
Functional SphI site
1 2 3 4 5 6 7 8 9 10
20 3. Gly Ala Pro Thr Ser 8er Ser Thr Lys Ly~ Thr-
~T GCA CCG ACT AGC AGC TCT ACT AAG AAA ACA-
OTA CCA CGT GGC TGA TCG TCG AGA TGA TTC TTT TGT-
Dl~u~ctlo~l 8~h1 lte .
2~ 11 12 13 1~ lS 16 17 18 19 20 21 22
Al~ Pro Thr 8er 8er 8er Thr Lys rys Thr H1~ Ala-
oCT CCT ACC TCT TCT AGC ACG AAG AAG ACG CAT GCT-
CCA G4A TG4 AGA AaA TCG TGC TTC TTC TGC GTA CGA-
Functional 8phI site
~O
23 2~ 25 26 27 28 29 30 31 32
Pro Thr 8er 8~r 8er Thr ~ys Lys Thr ~i~
CCC ACC AGC TCT AGC ACT AAA AAG ACT CAT G
~GC TG~ TCG AGA TCG TGA TTT TTC TGA .
Disfunctional SphI site
;~f ~ 7
WO91/19745 - 18 - PCT/US91/04187~
A 4-base extension capable of annealing to a l/2 SPhI site
is present at either end of the DNA fragment encoding each spacer
sequence In each spacer, codon substitutions destroy one of these
SPhI recognition sites such that only one SPhI site is regenerated
Refering to Table l, sequence l, the use of T in place of G in the
third position of the 10th codon prevents the creation of an SPhI
~lte ~t the 3' end of the spacer In sequence 2, the use of G in
place of C in the 2nd position of the Gly codon prevents the
creation of an S~hI site at the spacer's S' end Sequence 3 is made
by maintaining the functional SPhI site at the 5' end of sequence 1
and ligating the spacer of sequence 2 at that site, leaving a
regenerated SPhI site 3' of the spacer of sequence 2, and
disfunctional SPhI sites at either end of the spacer of sequence 3
Thus, insertion of sequence l from Table l into the SphI
15 site of DAB486-IL-2 or DAB389-IL-2 results in a fusion protein
with splice/~unctions as follows the His48 residue of native
DT; Ala-Pro-Thr-8er-8er-Ser-Thr-Lys-Lys-Thr; a His residue encoded
by the 3' extension; Ala485 of native DT; and the second amino
acid of the IL-2 sequence
The insertion of sequence 2 from Table l into the SphI site
of DAB~86-IL-2 or DA~389-IL-2 results in a fusion protein with
rplice/~unctions as follows the His48~ residue of native DT; a
Gly residue derived from the 3' extension at the 5' end of the
oligonucleotide; Ala-Pro-Thr-8er-8er-8er-Thr-Lys-Lys-Thr-
2~ Ala-Pro-Thr-8er-8er-8er-Thr-Ly8-Lys-Thr; a His re~idue encoded by
th- 3' xt-n~ion; A1~85 of n~tive DT; and the second amino acit
o~ th- IL-2 ~-~u-nc-
~ h in~-rtion of seqUence 3 from Table l into the 8PhI site
o~ D~8~86-IL-2 or DAB389-IL-2 results ln a fusion protein with
~0 rplic-/~unctlon~ as follow8 the His48~ re~idue of nttive DT; a
Oly r--~due der~ved from the 3' extension at the 5' end of the
oligonucl-otid-;
Al~-Pro-Thr-8-r-8er-8er-Thr-Lys-Lys-Thr-Ala-Pro-Thr-8er-8er-8er-Thr-
Ly~-~y~-Thr-His-Ala-Pro-Thr-8er-8er-8er-Thr-Lys-Lys-Thr; a His
.~ , . . . . -... ,
- .: . . . .
~ . . . .
.. . . . - :. .. . .
.. . . . . . . . .
.
... ... . . ..
.. .. . . . ..
WO 91/19745 -- 19 _ 2~?~37 PCI`/US9l/04187
residue encoded by the 3' extensioni Ala 85 of native DT; and the
second amino acid of the IL-2 sequence. Three subunits may be added
by ligating sequence 1 and 2 in vitro to achieve sequence 3, which
i8 then inserted into the chimeric toxin, or, seguence 1 may be
S inserted into the SphI site of a chimeric toxin that harbors
6equence 2.
Insertion of sequences 2 or 3 from Table 1, which encode 2
~nd 3 copies o (1-10) respectively, result in analagous
structures. When repeats of the (1-10) se~uence are present codons
are chosen such that DNA homology between sequential (1-10) encoding
8eqUenCe8 i8 minimized. This prevents rearrangements that would
arise from homolo~ous recombination between sequential copies of
(1-10) encoding DNA .
The presence of the Gly and His residues (encoded by the
lS linkers at each end of the spacer seguence) do not affect the
Bnorm value of the linker (data not shown).
The (1-10) spacer geguence was identified by applying the
FLEXPRO program (described above) to the seguence of DAB486-IL-2.
Table 2 shows the 10 most flexible 7-amino acid segments in
DAB486-IL-2 as determined by FLEXPRO.
TABLE 2: FLEXPRO Analysis of DA84g6-IL-2
Rank From To Bnorm Sequence
1 ~87 ~93 1.13S Pro-Thr-Ser-8er-Ser-Thr-Lys
2 39 ~S 1.12S Pro-Lys-8er-Gly-Thr-Gln-Gly
3 1~2 1~8 1.107 Ala-Glu-Gly-8er-8er-Ser-Val
S~l 587 1.107 Leu-~ys-Gly-8er-Glu-Thr-Thr
S 172 178 1.102 Gly-Lys-Arg-Gly-Gln-Asp-Ala
6 7 13 1.098 Val-A~p-8er-8er-Lys-8er-Phe
7 232 238 1.097 8er-Glu-8er-Fro-Agn-Lys-Thr
8 63 7~ 1.036 Val-Asp-Asn-Glu-Asn-Pro-Leu
~ 9 ~11 417 1.08S Phe-Gln-Gly-Glu-Ser-Gly-His
266 272 1.08 Thr-Val-Thr-Gly-Thr-Asn-Pro
The ~o~t flexible seven amlno acid stretch of the fusion
~rot-in wa~ found to be am~no acids, residues ~87
.- - . . . ~ . ~ . . . . . .
"'; ' ,. ' ' ,` ",` ' "', ., ,' ' '', . '' ' ' ' . "~ ' . ' " ~ . . --, , ' " ~ ' ,. .
, , . . ~ - :
,- - - : : - , .. ,-
WO91/19745 ~ f S~ 7 - 20 - PCT/USsl/04187
through 493 of DA~486-IL-2, with a Bnorm
1.135. These seven amino acids correspond to amino acid
residues 2-8 of IL-2. These seven amino acids were also
the most flexible found in DAB389-IL-2 and
DAB295-IL-2 (a molecule entirely devoid of the toxic
ch~racteri~tics of the other DT chimeric toxins
te~ted). 9ecause the first 10 amino acid residues of
IL-2 are known not to orm an orderly array in crystals
~Brandhuber (1987) Science 238:1707-1709), and are thus
flexible, the entire first 10 amino acid residues of
IL-2 were used as the (1-10) spacer. When inserted into
DA~486-IL-2, the (1-10) spacer is the most flexible
seqyence of the new construction, as shown in Table 3.
2~
~O
WO 91/19745 - 21 - ~ ~ 3~ ~r ~7 PCr/US91/04187
TABLE 3:
A: FLEXPRO Analysis of DAB486-(1-1o)-IL-2
Rank From To B~Norm] Sequence
S 1 487 493 1.135 Pro-Thr-Ser-Ser-Ser-Thr-Lys
2 498 504 1.135 Pro-Thr-Ser-Ser-Ser-Thr-Lys
3 39 4S 1.125 Pro-Lys-Ser-Gly-Thr-Gln-Gly
~ 142 148 1.107 Ala-Glu-Gly-Ser-Ser-Ser-Val
592 598 1.107 Leu-Lys-Gly-Ser-Glu-Thr-~hr
6 172 178 1.102 Gly-Lys-Arg-Gly-Gln-Asp-Ala
7 7 13 1.098 Val-Asp-Ser-Ser-Lys-Ser-Phe
1O 8 232 238 1.097 Ser-Glu-Ser-Pro-Asn-Lys-Thr
9 68 74 1.086 Val-Asp-Asn-Glu-Asn-Pro-Leu
411 417 1.085 Phe-Gln-Gly-Glu-Ser-Gly-His
B: FLEXPRO Analysis of DAB486-(1-10)2-IL-2
RANK FROM TO BtNorm] SEQUEUOE
15 1 488 4941.135 Pro-Thr-Ser-Ser-Ser-Thr-Lys
2 ~98 50~1.135 Pro-Thr-Ser-Ser-Ser-Thr-Lys
3 509 51S1.135 Pro-Thr-Ser-Ser-Ser-Thr-Lys
39 ~5 1.125 Pro-Lys-Ser-Gly-Thr-Gln-Gly
S 1~2 1481.107 Ala-Glu-Gly-Ser-Glu-Thr-Thr
6 603 6091.107 Leu-Lys-Gly-Ser-Glu-Thr-Thr
7 172 1781.102 Gly-Lys-Arg-Gly-Gln-Asp-Ala
20 8 7 13 1.098 Val-Asp-Ser-Ser-Lys-8er-Phe
9 232 2381.097 Ser-Glu-Ser-Pro-Asn-Lys-Thr
68 74 1.086 Val-Asp-A~n-Glu-Asn-Pro-Leu
~ =.
2~
` ' ,`',, ` "' . ~, ." '. ".'' '. ',' ..'. .',' . ;' `' ,'' ., .. - .: ':
wO9l/l9745 ~ 7
- 22 - PCT/US91~W187
DA~389-(1-10)-IL2 was constructed from
DAB389-IL-2 DAB389-IL-2 was constructed by
removing a 309 bp HpaII - SPhI restriction fragment from
pDW24 and replacing it with oligonucleotide linker
261/274 (Table 4) to generate plasmid pDW27 (Fig 1)
TABLE 4 Oligonucleotide Linkers
con~truct oligonucleotide linker
DAB389-IL-2 5~-CG-GGT-CAC-AAA-ACG-CAT-S' 1/2 HpaII-1/2SphI
CCA-GTG-TTT-TGC
S5 Thi~ linker restores fragment B sequerces from Pro383 to
Thr387, and allows for in-frame fusion to IL-2 seguences
at this position Thus, in DA9389-IL-2 the 97 amino
acid~ between Thr387 and His~85 have been deleted
DA9389-~1-10)-IL-2 was constructed by
inserting DNA encoding the spacer (see Table 1) into
pDW27 at the 8phI site, as de~cribed above
DA~389-~1-10)-IL-2 may al~o be generated directly from
~DW2~ ~DA9~86-IL-2) by remo~al of the 309 bp
H~aII-8phI fragment and replacing it with a linker that
2~ r-~tor-- fragm nt ~ ~eguences from Pro383 to Thr387,
ncod - th ~oly~ ~tid- linker, and allows for the
ln-~rum ~u~on to th IL-2 ~equence~
The -gue~ae of DT i8 given in Greenfield et
ul ~1983) Proc Natl Acad 8ci ~8A 80 6853-68S7 The
~0 ~ qu-~ce enaoding IL-2 wa~ synthesized on an A~plied
~o-y~t-m- DNA-8ynthe-izer, a8 de8aribed in Williams et
~1 ~1988) Nucleic Acids Res 16 10~S3-10~67, hereby
incorporat d by r-feronce The sequence of I~-2 i8
~ound in Williams et al ~1988) Nucleic Acid~ Res
- . . . ~ :
. . , , . ,, - :
~, . .
WO91/19745 - 23 ~ 35~7 PCT/US9~/04187
16:10453-10467. Fusion of the seguence encoding mature
DT to ATG using an oligonucleotide linker is described
in Bishai et al ( 1987) J. Bact 169 5140-5151 hereby
incorporated by reference
Chimeric toxins in which DT fragments were
fused to murine interleukin 4 (IL-4) with or without
the presence of a spacer were also constructed
486 2 3~9
DAB389-(1-10) -IL-4 were constructed by methods
known to those skilled in the art Briefly
DAB389-(1-10)2-IL4 was constructed by first
digesting a plasmid containing the
DAB389-(1-10)2-IL-2 fusion (derived from pDW27) with
SphI and HindIII to remove the IL-2 coding sequence and
lS then inserting a ~egment of DNA that encodes IL-4 The
IL-4 encoding fragment includes linkers to allow
ln~ertion and in-frame fusion to the 3 end of the
spacer encoding DNA DAB486-IL-4 and DA~389-IL-4
were made by analogous treatment of pDW24 and pDW27
respectively The æequence of murine IL-4 may be found
in Lee et al (1986) Proc Natl Acad Sci U8A
83 2061-2065 hereby incorporated by reference Thie
-guenae u~ed in these construction~ was obtained from
DN~X ~Californi~)
Ollgonuoleotite8 and nuoleic acids were
~ynth-~lr-d and mani~ulat-d ~ follow~
Ollgonucl-otid-~ w r- ynthe-i~ed u~ing cyanoethyl
~iho~phor~midlt- chemi-try on an Applied Biosystems 380A
D~A ~ynth~ r (Ap~lied Biosystems Inc Foster City
CA) Following ynthe~is oligonucleotides were
purlfl-d by chromatography on Oligonucleotide
Purification Cartridg~s ~Applied Bio~ystem8 ~nc Fo8ter
City CA) a- dir-cted by the munufacturer Purified
~ligonualeotide~ were re~u~pended in IE buffer (10 mM
r ' ,.
. ' '` ,'', - . :" ," '............... ' ' ' - ' ~ ' ':
.~ . . . , ~: . , , , , ,:
: ., '; . . '
WO9l/19745 2 ~ ~ 7 - 24 - PCT/US91/0418
Tris base, 1 mM EDTA, pH 8.0). To anneal complementary
strands, equimolar concentrations of each strand were
mixed in the presence of 100 mM NaCl, heated to 90C for
10 min, and allowed to cool slowly to room temperature.
S Plasmid DNA was purified by the alkaline
lysi6/cesium chloride gradient method of Ausubel et al.
~1989) Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. D~A was digested with restriction
endonucleases as recommended by the manufacturer (New
England Biolabs, Beverly, MA and ~ethesda Research
Laboratories, Gaithersburg, MD). Restriction fragments
for plasmid construction were extracted from agarose-TBE
gels, ligated together (with or without oligonucleotide
linkers) and used to transform E. coli using standard
1S methods. 8ee Ausubel et al (1989), su~ra, and Maniatis
et al. (1982), Molecular Cloning Laboratory Manual, Cold
8pring Harbor Laboratory, Cold Spring Harbor, N.Y.
Plasmid DNA seguencing was performed according to the
dideoxy chain termination method of Sanger et al. (1987)
Proc. Natl. Acad. 8ci. U&A 74:5463-5467, as modified by
Kraft et al. ~1988) Bio Technigues 6:544-5~7, using
8eguenase (United 8tates Biochemicals, Cleveland, OH).
ExDression ~nd Purification of Chimeric Toxins
Expression and purification of chimeric toxins
2S wa- a~ follow~. All DT-related I8-2 fusion proteins
u~-d h r-ln w r- expre--ed in the cytoplasm of E. coli
~tr~ln JM~Ol ~rom th- trc promoter, Amann et al. (l9~S),
~-n- ~0:183-190, hereby incorporated by reference.
R-comblnant E. coli were grown in M9 minlmal medium
~MaAiatis et al. (1982) supra) supplemented with
10 m~ml ca~amino acids ~Difco, Detroit, MI), 50 ~g/ml
ampiclllin, and O.S ng/ml thym~ne in 10 liter volumes in
a Microgen Fermentor tNew 8runswick 8clenctific, Edl~on,
N.J.). Bacterial cultures were grown at 30-C, and
,, , . . , . . - , .:: . ,' '
~ . .. .
, ' ' ~ , ' ' ' ~ .: `: .
. .j .
WO91/1~45 - 2s - 2~$~7 PCT/US91/~187
sparged with air at 5 L/min. When the absorbance
(A590nm) of the culture reached o.3, expression of
chimeric tox gene was induced by the addition of
isopropyl-B-D- thiogalactopyranoside. Two hours after
induction, bacteria were harvested by centrifugation,
re~uspended in buffer ~lOl (50 mM ~H2P04, lO mM
EDTA, 750 mM NaCl, 0.1% Tween 20, pH 8.0), and lysed by
sonication (~ranson Sonifier). Whole cells and debris
were removed by centrifugation at 27,000 x g, and the
clarified extract was then filter sterilized and applied
to an anti-diphtheria toxin immunoaffinity column.
~ound proteins were eluted with 4M guanidine
hydrochloride, reduced by the addition of
~-mercaptoethanol to 1% and then sized by high pressure
lS liguid chromatography on a 7.5 x 600 mm G4000PW column
~To~oHass). Prior to uce, fusion toxins were
exhauetively dialysed against HEPES buffered Hank's
balancet salt solution (Gibco), pH 7.4. Purified
diphtheria toxin was purchased from List Biological
Laboratories (Campbell, CA). The concentration of all
purified proteins was determined by using Pierce Protein
As~ay reagent ~Pierce Chemical Co., Rockford, IL).
CYtotoxicltv
The do~e re~ponse capacity of various chimeric
2~ toxln- to block ~l~C~-leuclne lncorporation by HUT
~0~/6I~ c-llJ ~whlch bQar the high afinity IL-2
r-c-ptor) and YT2C2 cells (whi¢h bear only the
interm-diate affinity ~p7S) receptor) was determined.
Table ~ shows the concentration in mole~ of
toxln r-gulred to lnhibit ~l~C~-leucine incorporation
by ~0% ~IC50).
. ~ ~ ... . . .. . . . .
.` ' . ' -. ' ' .
' . '; I , - . ' '. , ' .
,
,.,' ~, ,, ' '' :
.
2f 3~ 7
wosl/1s74s - 26 - PCT/US91/~18
TABLE s CYTOTOXICITY
TOXIN ICso ~M)
IL-2 -_ __
DAB486-IL-2 120 x 1o-12 70 x lo-9
DA~389-IL-2 40 x lo-12 11 x lo-9
DAB389-~1-1o)-IL-2 8 x 1o-12 2 x lo-9
DAB389-(1-10)2-IL-2 8 x 1o-12 2 x lo-9
DAB389-(1-10)3-IL-2 11 x 1o-12 2.2 x lo-9
As seen in Table S, the toxicity of the DAB389 - IL-2
chi~orie toxin is increased approximately 5-fold by the
addition of ~ sp~eer peptide. The effect of two copies
of the (1-10) spacer (~AB389-(1-10)2-IL-2) or
three eopies of the (1-10) spaeer (DAB389-(1-10)3-IL-2)
ha~e essentially the same effeet as does one copy of the
8pacer (DA8389-(1-10)-IL-2),
Chimeric toxins in which DA~6 or DAB389
wa~ fu-ed to I~-~ (with or without the same spacer used
on th- SL-2 con-truetion-, se- Table 1) were also
an~ly~-d. ~h-n t-~t-d for eytotoxicity to
rL-~-r-a~tor-b-aring o~ DAJ389-(1-10)2-IL-4 was
8 - -A to b- ~-10 tim 8 more cytotoxic than DAB389-IL-
~whieh wa~ n to be about 10 timos more cytoto,xic thun,
DA~ 86-S~
The addition of a spaeer to fusions o~ DT
fra~ nt~ to melanoeyte ~timulating hormone had no
ff-ct on aytotoxicity.
Cytotoxicity assays were performed a~ follows.
F~or lS-2 eh~m~ric toxins cultured HVT 102/6TG (Tsudo et
WO91/19745 - 27 - ; r' , , ~ PCT~USsl/04
2~ S~
al. (1986) Proc. Natl. Acad. Sci. USA 83:9694) or YT2C2
(Teshigawari et al. (1987) J. Exp. Med 165:223) cells
were maintained in RPMI 1640 medium (Gibco, Grand
Island, N.Y.) supplemented with 10% fetal bovine serum
(Cellect, GIBCO), 2 mM glutamine, and penicillin and
stceptomycin to 50 IU and 50 ~g/ml, respectively.
Cells were seeded in 96-well V-bottomed plates
(Linbro-Flow Laboratories, McLean, VA) at a
eoncentration of S x 104 per well in complete medium.
Toxin~, or toxin-related materials, were added to
varying concentrations (10 12M to 10 6M) and the
eultures were incubated for 18 hrs at 37C in a 5% CO2
atmosphere. Following incubation, the plates were
eentrifuged for 5 min. at 170 x g and the medium removed
and replaced with 200 ~1 leucine-free medium (MEM,
Gibeo) eontaining 1.0 ~Ci/ml ~14C~-leucine (New
England Nuclear, ~o~ton, MA). After an additional 90
min. at 37-C, the plates were centrifuged for 5 min. at
170 x g, the medium was removed and the cells were lysed
by the addition of 4 M KOH. Protein was precipitated by
the addition of 10% trichloroacetie acid and the
insoluble material was then collected on glass fiber
filters uslng a cell harvester (Skatron, Sterling, VA).
Filters were washed, dried, and counted aecording to
standard method~. Cells cultured with medium alone
r-rv-d a~ th- eontrol. IL-~ ehimerie toxin~ were tested
ln a rlmll~r mann~r exc-~t that CT~R eells ~W~lliam E.
~ul, NSH), ~15 e~ ATCC), or CTL~2 ~ATCC) were
a--d d at 1 x 10~ cells per well and incubated for 40
~0 hourr.
Como-tit~ve disDlacement ex~eriments
T~ble 6 ~how8 the competit~ve displacem-nt of
tl25Il-1 bel d IL-2 from the high affinity IL~2
~-e-~tor ~HnT102 eells) and the intermediate affinity
IL-2 ree-ptor ~Y~2C2 eells) by various chimeric toxins.
, , .. ... ,' ,. ' ~
.. ; .. ,.. .. . -
- ; -. .. .
.. . . . .
: , . -. ... ;, . .
': ~ : 1 , . .
WO91/19745 2 ~ 3 3A-~ 7 - 28 - PCT/US91/041~L~
TABLE 6 Competitive Displacement
Toxin 50% displacement
or HUTl02 YT2C2
Control
.
IL-2 l x lO lO 2 x lO 9
DA~486-IL-2 35 x lO lO 120 x lO 9
DA~389-IL-2 29 x lO lO llO x lO 9
DA~389-(l-lO)-IL_2 13 x lO lO 30 x lO 9
DA~389-(1-10)2-IL-2 12 x lO lO 28 x lO 9
DAB389-(1-10)3-IL-2 13 x lO lO 32 x lO 9
A- r-en in Table 6, the bind~ng affinity is increased by
the lnsertion of the (l-lo) spacer peptide into
DAB389-IL-2 Insertion of two copies of the spacer
(DAB389-~1-10)2-IL-2) or 3 copies of the spacer
~DAB389-~1-10)3-IL-2) has eissentially the sa~e
~ ct a~ does insortion of a single copy of the spacer
~DA1~389--~1-10)-IL-2) .
Compotitive displacement of ~125I]-rIL-2 by
r-co~blnaAt IL-2 ~rlL-2) a~d ch~merlc toxins wa~
2~ d~t-r~in d ar tollow~ The ridiolabeled IL-2 blnding
~aaay waa ~ rtorm d ~a-ntlally ar dercribed by Wang et
~1 ~19~7 J ~x~ M d 166 1055-1069 Cells ~ere
har~ ~t d, warh d with cell culture medium, and
r-aua~nd d to 5 x 106 per ml and ~ncubated with
~ tl2~ rlL~ ~0 7 ~Ci/~mol) in the ~rosence or
~br-~- o~ i~ar---ing ~on~entrationr of unlabel d rIL-2
or chlm rlc toxin for 30 mi~ at 37 C under 5~ CO2
r-action wais then overlayed on a mixture o~ 80~ 550
- : ' ' . .`. ` . - ' .`..... ~ `-. ,. :. ' ',.. . . . , ~ ' :
' ' ''' . ' .` '`, .'''' :'- ' '`. ' ~ '' ' '; ' ., . ' ' ~ ' '
'- '' . ' . : . .: . . ' ' . . . ` . '~ :'- : ' . . . ' ,: ,
- `. ., . . . . , -.. . . . . . .
3~37.
WO91/19745 - 29 ~ PCT/US91/~187
fluid (Accumetric Inc., Elizabethtown, KN) : 20~ parafin
oil (d = 1.03 g/ml) and microcentrifuged. The aqueous
phase and the pellet of each sample, representing free
and bound lig,~nd, respectively, was then counted in a
S Nuclear Chicago gamma counter. Apparent dissociation
con6tants, Kd, were determined from the concentrations
of unlabeled ligand required to displace 50% o
radiolabeled rIL-2 ~inding to receptors.
U
The improved chimeric toxins of the invention
are administered to a mammal, e.g., a human, suffering
from a medical disorder, e.g., cancer, or other
conditions characterized by the presence of a class of
unwanted cells to which a polypeptide ligand can
lS selectively bind. The amount of protein administered
will vary with the type of disease, extensiveness of the
disease, and size of species of the mammal suffering
from the disease. Generally, amounts will be in the
range of those used for other cytotoxic agents used in
the treatment of cancer, although in certain instances
lower amounts will be needed because of the specificity
and increased toxicity of the improved chimeric toxins.
The improved chimeric toxins can be admnistered
u~ing any conventional method: e.g., via in~ection, or
~ia ~ tlm d-relea~e implant. The improved chimeric
toxin~ c n b- oomb~n d with any non-toxic,
ph~r~ac-utically-acc-ptable carrier substance.
Other embodiments are within the following
claimJ.
What i8 claimed is:
., ; -, . , ~ ' :
. . .
;. . - : -
:, . ~ ~.. ... - ..
. . .
- ,. . ...
.
,', '
' ' ' , ." ; .. ,' . ,' ~ :.
