Note: Descriptions are shown in the official language in which they were submitted.
:.
WO 92/01474 2 0 8 716 3 PCr/SE91/00497
HE12ROBIFI~NCIIONAL REAGENTS AND CONJUGATES WITH OXAALKYLENE
UNITS FOR AMPHIPHILIC BRIDOE STRUCl'tlRES
.
The term conjugate is a common designation for
substances prepared by covalently linking different or
identical organic compounds together so that properties
from the compounds will be conferred to the conjugate.
The conjugate substance, the reagent and the novel -
polyethers of the present invention have as the common
structural feature sequentially linked ethoxy groups, i.e.
the structure -(OCH2CH2)n-, where n is an integer = or > 1.
The conjugate of the invention complies with the general
formula A-B-A' wherein A and A' comprise residues from
organic compounds F and F', respectively. At least one of
the compounds is a polymer, the polymeric structure of
which being present also in the residue. B represents an
organic bridge lir;ln~ A and A' together and comprising the
structure -(OCH2CH2)n-. A and A' may comprise several
identical residues originating from the compounds to be
conjugated (F and F'). ~ -
For the preparation o~ conjugates one often utilizes
hetero- or homobi~unctional reagents of the type Z-B'-Z',
where Z and Z' are reactive functional groups that are
inert with respect to reaction with each other (in pairs
they are nucleophllic or electrophilic) and B' is an inert
bridge. By the term lnert is contemplated that the bridge
is stabile and has no groups that are able to neutralize
the reactivity of Z and/or Z'. -~
In order to prepare conjugates in which one of the
compounds (F or F') is a polymer, it is difficult to -
30 prepare uniform conjugate substances. The substances -
obtained will often consist of a mixture of more or less
similar conjugate molecules. Common varieties of the
individual molecules of a given conjugate substance are: ~-
different numbers of A bound to one and the same A' and
3S vice versa, different binding positions, B exists as inter-
and/or intramolecular cross-linkings etc.
., . .. . .. . . ~. ... , . . . - ,., .. . . - .. . ... . - . -- . . . . , - . : . . .
WO92/01474 PCT/SE91/0049?
2087~3 2
The length and the structure of the bridge -B- are of
great importance in order to confer properties of the
compounds to the conjugate. For compounds that are poorly
soluble in water, a hydrophobic bridge structure may result
in conjugates that are insoluble and/or poorly active. If
the bridge is too short and biological active compounds are -~
conjugated, the activity will often be impaired. In the
extreme case an erroneous or too short bridge may
completely deteriorate the activity.
The structure -(OCH2CH2-)n is present in polyethylene
glycol (PEG). The molecular weights of PEGs are given as a
mean values, i.e. PEG is normally a mixture of various
molecules in which the integer n varies. PEGs have appeared -
to have unique amphiphilic properties which can be utilized
when they are linked to biopolymers.
The structure -(OCH2C~2)n- is also present in certain
known amino-PEG-carboxylic acids. For instance
NH2CH2CH2(0CH2CH2)nO~CH2)2COOH with n - 1-10 (Houghton and
Southby, Synth. Commun. 19(18)(1989)3199-3209) that have
been suggested as starting material for the preparation of
cyclic ~olyether amides. The authors have also suggested
that homologues with m > 2 may find the same application.
NH2CH2CH2(0CH2CH2)20CH2COOH has been suggested as starting
material for macrocycle-based ethers (Jullien et al,
25 Tetrahedron Letters 29(1988)3803-06.
Recently (20.1.91) amino-PEG-carboxylic acids of the
formula NH2cH2cH2(ocH2cH2)nocH2cooH (n )~
their reactive derivatives, and protein conjugates prepared
from them have been disclosed (EP-A-410,280).
Conjugates exhibiting the structure -(OCH2CH2)n~ in the
bridge have been synthesized earlier than our priority
; date. Slama and Rando have linked cholesterol to a
monosaccharide through both hydrophilic and hydrophobic
bridges (Biochemistry 19(1980)4595-4600 and Carbohydrate
; 35 Research 88(1981)213-221; a heterobifunctional coupling
reagent). For their purposes the best conjugates had the
; bridge:
W O 92/01474 2 0 8 716 3 P(~r/SE91/00497
3 . :
.,
(-NHCH2CH2(OcH2cH2)nocH2co-)n~
where n = 1 and n~ = 1 or 2. Slama and Rando did not
manifold the -OCH2CH2- structure by increasing the integer
n. For unknown reason they instead duplicated the complete
structure -NHCH2CH2(OCH2CH2)nOCH2CO- by making n' equal 2.
Enzyme-antibody conjugates containing the bridge
-(OCH2CH2)n~ in which n may be various integers are known
(EP-A-254,172 and FR-A-2,626,373). In the latter
application commercially available polyethylene glycol
(PEG) was used as one of the starting materials for the
coupling reagent. This has rendered it difficult to obtain
uniform conjugate substances with respect to the length of
the bridge. Oxaalkylene structures, e.g. -OCH2CH2-, have
been suggested in heterobifunctional reagents for the
selective coupling at aldehyde groups and thiol groups,
respectively (EP-A-240,200 and WO-A-89/12624).
In addition to the publications given above the Swedish
Patent Office has cited in an International Type Search -
Report: EP-A2-345,789, WO-Al-88/03412, Biochem. Biophys.
Res. Comm. 164~1989~ 3, as publications of particular
r~levance with regard to the claims of the priority
applications.
The present i~ventlon provides con~ugate substances in
which the spectrum of individual molecules have an improved
uniformity and a good bioavailability, particularly in
hydrophilic aqueous media. This goal is achieved by
inserting an amphiphilic bridge structure of uniform and
defined length. The conjugates of the invention is ~ -
particularly adapted for in vivo and $n vitro diagnostic
uses as well as for therapeutic uses (drugs).
The conjugate of the invention is characterized in that
the bridge -B- comprises the structure
~srRcoNHcH2cH2(ocH2cH2)n(cH2)mcoy- (I)
The free valencies in formula (I) link to A and A', -
respectively. This takes place either directly or through
further divalent inert structures that are comprised within
the bridge -B-. The length of -B- is usually shorter than
:
., ,: ; ' ^ ' ' ', ', . . ' . ' ' ., ` '~ ' ' ," i ', '' ,, ' , '" , ' ',` ' `- ~.
2 0 8 ~ ~ 6 4 PCT/SE91/00497
180 atoms, such as < loo atoms, but longer than 13,
preferably longer than 16 atoms.
n is an integer > O, e.g. 1-20, and such that n is :
uniform for bridges linking identical positions together in
individual molecules of the conjugate (conjugate
substance). m is 1 or 2. From the synthetic point of view n
is preferably 10 or < 10. In order for the conjugate to
express the uni~ue amphiphilic properties of PEG, the
integer n should be higher than 2, preferably higher than 3
or higher than 4. Thus based on different combinations of
criteria the intervals for the integer n may be 1-20, 1-10,
1-9, 2-20, 2-10, 2-9, 3-20, 3-lo, 3-9, 4-20, 4-lo, 4-9, 5-
20, 5-10, and 5-9.
Sr binds directly to a saturated carbon atom at each of
its valencies. r = 1 or 2, that is Sr represents a
disulfide group or a thioether group. If Sr is binding
directly to A, one of the sulphur atoms may originate from
F.
Y is -NH-, -NHNH- or -NHN-CH- that at their left ends
bind to the CO group shown in the right terminal in formula
I and at their right ends to a saturated carbon atom or to
a carbonyl group ~only when Y eguals -NHNH-). The atoms
binding to the right ~nd o~ Y ar~ not shown ~n ~ormula I.
R is preferably alkylene ~having 1-4 carbon atoms, often
1 or 2 carbon atoms), that possibly is substituted with one
or more (1-3, in the preferred case < 2) hydroxy (OH)
groups. At most one oxygen atom is bound to one and the
same carbon atom in R. With respect to availability in
hydrophilic media R may in many cases be equivalently
substituted for a higher alkylene selected from the group
comprising straight, branched and cyclic alkylene, with the
provision that the higher alkylene shall exhibit
hydrophilic substituents compensating for an enhanced
hydrophobicity.
A further condition is that for r = 1, B may contain a
l-aza-2,5-dioxo-cyclopentan-1,3-diyl group that at its 3-
position binds to the sulphur atom and at its l-position to
.1 .
' '
.. : . .. : :, ~: .. : ...... : - .... - :. - ., - . . . ... . - . - . , . -
:: . :: , : , .: - , , - - , - -
W O 92/01474 2 0 8 716 3 PC~r/SE91/00497
R, or the analogous 1,4-diyl group that at its 4-position
binds to the sulphur atom.
According to a preferred embodiment the bridge -B- must
not contain any aromatic ring.
The polymer may be a synthetic one or a biopolymer. The ~ -
term polymer stands for a compound in which three or more,
preferably more than 10, repeating monomeric units bind
sequentially to each other. The term polymer also
encompasses inorganic polymers, such as glass and other
polymeric silicates.
Examples of synthetic polymers are poly(hydroxyalkyl
(meth)acrylate), poly((meth)acrylamide), poly(vinyl
alcohol), etc and derivatized forms of these polymers.
The term biopolymer means a polymer in which the basic
skeleton is of biological origin. The expression
encompasses also biopolymers that have been derivatized.
Biopolymers exhibit as a rule nucleic acid or
polysaccharide and/or polypeptide structure. Proteins
including polypeptides (e.g. albumin and immunoglobulins)
and polysaccharides, e.g. soluble and insoluble ones
(dextran, starch, heparin cellulose etc), are important.
Polymers of immediate interest ~.g. those of
polypeptide and/or polysaccharide structure) usually have ~-
functional groups such ag hydroxy (-OH), carboxy (-COO~),
am~no-(primary or secondary) and/or mercapto (-SH). To the
extent that a given polymer does not have the appropriate
functional group, chemical modification can as a rule be
carried out to the effect that the group will be inserted.
The compounds F and F' are selected according to the
properties that they shall confer to the conjugate. The
compounds may thus be: carrier compounds, bioaffinity
compounds, analytically d~ectable compounds, compounds
that are insoluble or soluble in agueous media,
therapeutically active compounds (drugs), enzymes, immune - -
`/ ~5 stimulators, toxins etc. Particularly important toxins are
the peptide cytotoxins that exert their effect
intracellularly and thus comprise one peptide segment
'
,.- : -.
WO92/01474 8 7 1~ 3 PCT/SE91/00497
responsible for penetration of the cell wall and another
segment for the intracellularly toxicity (Diphteria toxin,
ricin, Pseudomonas exotoxin etc). Another type of peptide
toxins are those that exert their effect through immune
stimulation (e.g. by activating cytotoxic T-cells via
simultaneous binding to T-cells and cells carrying Class II
MHC antigens). An example of the latter type is
staphylococcal enterotoxin A.
Bioaffinity compounds, i.e. compounds that exert
biospecific affinity, are particularly interesting, because
they as a part of a conjugate may be utilized as a targeter
for their bioaffinity counterparts. Examples are antibodies
and their antibody active fragments and corresponding
antigens/haptens, Fc-fragments/Fc-parts of immunoglobulins
(Ig) and corresponding receptors (for instance IgG binds to
Protein A and G ), avidin/strepavidin and biotin, lectins
and corresponding carbohydrate structures, enzymes and
respective substrate, coenzyme, cofactor, and cosubstrate,
etc. Antibodies o~ various classes (in particular IgG) and
subclasses and various specificlties may be one part of the
con~ugate according to the invention. The specificities of
the antibodies (and fragment thereof) may e.g. be for
tumour cells and/or tumour antigens/haptQns, hor~ones,
hormone receptors etc.
2S Particularly interesting insoluble polymers are those
ones that are used as adsorbents in connection with
chromatography, immunoassays, blood fractionation etc.
Within the field of in vitro and in vivo diagnostics,
conjugates- between an analytically detectable compound and
a compound (targeter) showing biospecific affinity are of
great importance for detecting and localizing the
counterpart to the targeter. The analytically detectable
compounds may be radioactive, enzymatically active
(including enzyme substrates, cosubstrates, coenzymes etc),
fluorogenic, chemiluminogenic, biochemiluminogenic,
particulate (e.g. latex) etc.
' . ' ' ' :! .:' : ' . ' . ,
' ' .. ' .'. . ~ ' ' ~ . ~ .', . . . ' ' ' . , .'. . . ' ' '
.', ~ ~ ` ' . ': .,, :. ' . ' ;' ' ' , ' '. ,
,~ ': . '` .- . . . ' ' ' . . ' '' , '. : - :
:. ' '.... .. . '.. : .' ,., ' . . , ',, - . , ', ~ . . , ' , :. ' :
' . ' : ', ' ., ' ,i'.. . . .. ' ' " . :' ' ' , '' ': . ,. ' ., ' . ,
W O 92/01474 2 0 8 716 3 PC~r/SE91/00497 ~-
For therapeutic purposes bioaffinity compounds may be
conjugated to drugs and other substances that exert a -
therapeutic effect.
For the synthesis of the conjugate of the invention we
have developed a novel heterobifunctional reagent of the
type Z-B'-Z'. This reagent complies with the general
formula (II):
zlRcoNHcH2cH2(ocH2cH2)no(cH2)mzl (II)
m and n have the same meaning as above for formula (I).
Preferably m and n are uniform, i.e. the reagent substance
is not a mixture of compounds having different m and
different n. R is an alkylene group of the same meaning as
above. Zl is an HS-reactive electrophilic group, thiol (-
SH) or protected thiol (e.g. AcS-), with the provision that
a thiol group and a hydroxy group must not be bound to one
and the same carbon atom in R. Examples of HS~reactive
electrophilic groups are:
(i) halogen that is bound to a saturated carbon atom,
preferably in the form of an alfa-halo-alkylcarbonyl
(e.g. ZlCH2C0-, where Zl preferably is bromo or iodo);
(ii) activated thiol, preferably a so called reactive
disulfide (-SSRl) that is bound to a saturated carbon
atom;
tiii) 3,5-dioxo-1-aza-cyclopent-3-en-1-yl. With
respect to reactivity against thiol groups one can
equivalently use other carbon-carbon double bonds that
form conjugated pi-electron systems with a carbonyl
group, a nitro group or a cyano group instead of 3,5-
dioxo-l-aza-cyclopent-3-en-1-yl;
3~ Reactive disulfides are well Xnown in the context of
synthetic chemistry (See EP-A-063,109, 064,040, and
128,885). Rl is defined by the chemical reaction between
-S-S-R1 and HS- releasing Rl-SH that is thermodynamically
stabilized to be withdrawn from further thiol-disulfide -
exchange reactions. Many thiol compounds (Rl'SH) comply
with this criterion by spontaneously tautomerizing to a
thione form in aqueous solutions, i.e. the thione form is
': '
, :
... . . : .: . -...... . . ~ :: ~ . . ;. - .. . - , . .. . .
W092tO1474 2 0 8 7 ~ 6 3 PCT/SE91/0049?
more stabile than the corresponding thiol form. One
prerequisite for this type of tautomerization may be that
the sulfur atom of the thiol group is bound to a carbon
atom that constitutes a part of an aromatic ring that (a)
is heterocyclic having the thiol sulfur atom located at a
distance of an odd number of atoms from a heteroatom in the
ring, or (b) is non-heterocyclic and substituted with
electron-withdrawing groups.
Examples of R1 are 5-nitro-2-pyridyl, 5-carboxy-2-
1~ pyridyl, 2-pyridyl, 4-pyridyl, 2-benzthiazolyl, 4-nitro-3-
carboxyphenyl and the N-oxides of the pyridyl groups just
mentioned.
Zl' is activated carboxy, i.e. an electrophilic group.
Examples are carboxylic acid halides (-COCl, -COBr, and
-COI), mixed carboxylic acid anhydrides (-COOOCRl),
reactive esters, such as N-succinimidyloxycarbonyl, -
-C(=NH)-OR2, 4-nitrophenylcarboxylate (-CO-OC6H4NO2) etc.
R1 and R2 may be lower alkyl (C1-C6) and R2 also benzyl or
C2_C3 alkylene with one of its valencies substituting H in
NH (giving cyclic structures such as in oxazolin-2-yl that
possibly may be substituted with lower alkyl (CI-C6) or
benzyl in its 3- and/or 4-position).
The Zl' terminal may b~ reacted selectively with a
compound F' showing a nucleophilic group selected among:
- 25 (i) -NHR1, such as in primary and secondary a~ines
(Rl is selected from hydrogen and lower alkyl (C1-C6))
and in hydrazine/hydrazide, i.e. NH2NH2 and compounds
in which NH2NH- and NH2NH-CO- are bound to an
aliphatic carbon atom, preferably saturated.
(ii) -OH, such as in an alcohol.
The chemical reaction at the Zl' terminal of the reagent
(II) with a compound F' means that F' will become -~
covalently attached to -(CH2)m in formula (II) via an amide
group or a hydrazide group (-CONHNH- and -CONHN=C-,
respectively) for groups according to (i) above and via an
ester group for groups according to (ii) above. When
appropriate, Zl may then be transformed (reduced) to a
~ .
'~ ` ,' ,' ' - ' '' i ' ." '. ' ' ' . ~ ' " , ' ~ " ' " . '. ' ' . ' ", - ' , . . . ' , ' ', ' ,
~ '.: ' ' . ' " ' . ' " ' . ' " ' . ' '' . , ' ' ' . ' ' " ' '' ', ' ' ' ' ' ' ' ' ,
WO92/01474 2 0 8 716 3 PCT/SE91/00497
9 . ,.
thiol group in a thiol-disulfide exchange reaction. In the ~ -
latter case F' will become thiolated.
The Z1 terminal may selectively be reacted with a
compound F having a thiol group (SH) or a HS-reactive
electrophilic group. If F has a thiol group reaction can
take place directly. In the final product compound F will -
be bound to R in formula (II) via a thioether (-S-) or
disulfide (-S-S-) group.
The use of the reagent (II) i9 carried out in a manner
known per se. The reaction medium is selected so that side
reactions f Zl and Zl' are avoided. When F and/or F' are
biopolymers it is preferred to run the reaction in aqueous
media, and in order to achieve selectivity pH shall be 8-
9.~? for reaction at Zl' and < 8 for reaction at Zl Aprotic
media are often i.ert against Zl' which means that they
generally speaking are the preferred ones. The result will
be a conjugate in which F and F' are linked together
through a bridge complying with formula I.
Extended bridges can be introduced by reacting the
reagent of formula (II) at either lts Zl or Zl' terminal by
suitable bifunctional compounds. For instance, if Zl' is
reacted with an alkylene diamine, alkylene dihydrazine,
alkylene dihydrazide etc and only one of their H2N- groups
is consumed, the remaining f?ree NH2-group can be used for
reaction with other~compounds, e.g. F or F' (exhibiting
karboxy, optionally after activation).
Elongation of the bridge may also be accomplished by ,
reacting the compounds F and/or F' with appropriate
bifunctional reagents of the type A-B'-Z' (see page i)
prior to linking them together by the use of the reagent of
the invention. B' may be selected from the same group as R
above. If each of the compounds F and F' are initially -
reacted with the same reagent Z-B'-Z' at the Z' group and
then linked together through the so introduced Z group the -
group B' will appear twice in the conjugate (head to head
linking).
.
':
' ' . ' " ' ::. ' ' .. '. : ' " , , ~, ' .. ' ' . :'. ` ' , .'; : ' ., ' .
' ' ' . ' . ' . . .. : " . , ' ,`: ' , ' . ` . - ,
W092/01474 2087 ~ ~3 PCT/SE91/0049?
Known techniques encompass a large number of
bifunctional reagents that are useful for chain elongation,
either starting from a reagent of formula (II) or from one
of the compounds F and F'. Specific examples are N-
succinimidyl 4-(N-malein-imidyl)-butyrate, N-succinimidyl
iodoodacetate, N-succinimidyl S-acetyl-2-mercaptoacetate,
and N-succinimidyl 3-(pyridyl-2-dithio)propionate, alkylene
diamine, alkylene diacylhydrazide etc. Alkylene has the
same meaning as previously.
In a manner known per se for the synthesis of
conjugates, a vicinal diol, e.g in a carbohydrate
structure, may be oxidized to two aldehyde groups and
subsequently reacted with a -CONHNH2 group to give a -
CONHN=C- group. Important compounds F and F' having
carbohydrate structures are the glycoproteins. The group
-CON8NH2 may be present in a bifunctional reagent Z-B'-Z',
optionally after reaction with a polymer.
Chain elongation by starting from a reagent of the
present invention may result in con~ugates in which -B- is: -
(1) -COR'~Sr~RCONHcH2cH2(0cH2cH2)no(cH2)mcoy ;
CO binds to NH,
(2) -coR~-sr-RcoNHcH2cH2(ocH2cH2)no(cH2)mcoNHNR NHN ;
N- is usually bound t~ a sp2-hybridized
carbon atom in A which means that the structure
25 - -Ns is part of the structure -CzN-. This
structure may have been formed by reaction
between an aldehyde group and a NH2-NHCO-
', group,
(3) CO(CH2~mO(CH2CH20)nCH2CH2NHCOR'-Sr-(cont.)
0 -RcoNHcH2cH2(ocH2cH2)no(cH2)~coy-;
CO- is bound NH,
~- (4) CO(CH2)mO(CH2CH20)nCH2CH2NHCOR'-Sr-(cont.
RcoNHcH2cH2(ocH2cH2)no(cH2)mcoNHNR~'NHN=;
N= is bound as above in (2),
3~ More variations are possible. r has the same meaning as
above.
~' -,.
.~ ''. .
.: . : . . : : :. . . . :: .
WO92/01474 2 0 8 71 6 ~ PCT/SE9l/00497
11 ~ . ,
By varying the reagents that are employed, different
chain elongations may be constructed starting from the Y-
terminal.
R and R~ are alkylene selected in the same way as R in
formula (I).
The reagent (Formula II) can be prepared starting from
compounds complying with formula III: -
NH2CH2CH2(OcH2cH2)no(cH2)mcooH (III)
m equals an integer 1 or 2. n equals an integer 1-20, such
as 2-20.
The synthesis of certain compounds complying with
formula III with m = 1 and 2, and n = 1-10 have been
described before (Jullien et al, Tetrahedron Letters
29(1988)3803-06; Houghton and Southby, Synth.Commun.
15 19(18)(1989)3199-3209; EP-A-410,280 (publ. 20.1.91); and -
Slama and Rando, Biochemistry ls(ls8o)459s-46oo and
Carbohydrate Research 88~1981)213-221).
The reagents complying with formula II can be
synth~sized by reacting a compound of formula (III) with a
bifunctional reagent of formula Z-B'-Z' and known per se
(see page 1), where Z Z Zl~ B' = R that is as previously
defined, and Z' - activated carboxy. See above for suitable
reagents. After the reaction the -COOH function is
transSormed to an activated carboxy group, e.g. Zl' ' -
activated ester, such as N-succinimidyloxycarbonyl, 4-
nitrophenyloxycarbonyl, 2,4-dinitrophenyloxycarbonyl etc.
Compounds complying with formula III (m = 1 and n =
2-20) and derivatives having the NH2- and/or -COOH groups
replaced with groups that easily can be transformed to NH2- ~ ;-
and -COOH groups, respectively, are novel and relate to a
separate aspect of the present invention. This aspect
provides a number of discrete bifunctional substances
having pronounced amphiphilic properties, i.e. the property
of being simultaneously soluble in water and organic
solvents and lipids. This is a desirable property for
bridge forming reagents that are to be used for the
preparation of conjugates involving biomolecules.
. : . . ... :: ' . . ' : . ' ' '' ' ' ; ' -
'. '' : '. : . ' : . . ' ~ :: `. :: ~ ' '' .' . : ' '
WO92/01474 3 12 PCT~SE91/00497
The novel compounds of formula (III) and their novel
derivatives comply with polyethers having the general
formula:
XCH2CH2(OcH2cH2-)nocH2y (IV)
n is an integer 2-20, preferably 3-20. X is H2N- including
the protonated form thereof (+H3N-) or substituted H2N-
that is transformable to H2N-, preferably by hydrolysis or
reduction. Examples are unsubstituted amino (H2N-); nitro;
amido (- carbamido), such as lower acylamido (formylamido,
l0 acetylamido ........ hexanoylamido) including acylamido
groups that have electron-withdrawing substituents on the
alpha carbon atom of the acyl moiety and then particularly
CF3CONH-, CH3COCH2CONH- etc; phtalimidyl which possibly is
ring substituted; carbamato (particularly Rl'OCONH- and
(Rl'OCO)(R2'0CO)N-, such as N-(t-butyloxycarbonyl)amino
(Boc), N-(benzyloxycarbonyl)amino and di(N-
(benzyloxycarbonyl))amino (Z and diZ, respectively) which
possibly are ring substituted: alkyl amino in which the
carbon atom binding to the nitrogen atom is alpha to an
aromatic system, such as N-mono~enzylamino and
dibenzylamino, N-tritylamino (triphenylmethylamino) etc
including analogous groups where the methyl carbon atom
(including benzylic carbon atom) atom is replaced with a
silicon atom (Si), such as N,N-di(tert-butylsilyl)amino;
and 4-oxo-l,3,5-triazin-l-yl including such ones that are
substituted with lower alXyl in their 3- and/or 5-
positions.
Above and henceforth Rl' and R2' stand for lower alkyl, -
particularly secondary and tertiary alkyl groups, and a
methyl group that is substituted with 1-3 phenyl groups
that possibly are ring substituted. Lower alkyl and lower
acyl groups have 1-6 carbon atoms.
Y is carboxy (-COOH including -COO~) or a group that is -
transformable to carboxy, preferably by hydrolysis or
oxidation. The most important groups are the ester groups
in which the carbonyl carbon atom or the corresponding atom
in orto esters binds to the methylene group in the right
W092/01474 2 0 8 716 3 PCT/SE91/~97
13
terminal of formula (I). Examples are alkyl ester groups
(-COOR1'); orto ester groups (-C(OR3')3) and reactive ester
groups, such as N-succinimidyloxycarbonyl, 4-
nitrophenyloxycarbonyl, alkyl imidate groups (-C(=NH)O-R1')
including 5-membered cyclic forms (oxazolin-2-yl) with or
without lower alkyl in their 4- and/or 5-positions). R3'
has the meaning as previously given for R1
Other groups Y are -CHO, -CN, -CONR1'R2', where Rl' and
R2' have the same meaning as previously, and -CONH2.
The compound of the invention may be synthesized from
known starting materials by combining methods that are
known per se. Appropriate synthetic routes are:
A. Formation of the chain.
B. Transformation of terminal functional groups.
C. Transformation of a symmetric polyether to an
unsymmetric ether.
D. Splitting of a bisymmetric chain into two identical
fragments. ,~
` Convenient starting materials that have the repeating
unit -OCH2CH2- ~re commercially available. Examples are
oligoethylene glycols having 2 to 6 repeating units. Other
suitable compounds with identical terminal groups are
corresponding dicarboxylic acids and diamines.
Convenient starting materials that have di~fQrent
25 terminal groups are omega-hydroxy monocarboxylic acids in ~i
which the terminal groups are spaced apart by a pure
~olyethyleneoxide bridge. Such compounds having up to 5
repeating units have been described in the prior art
(Nakatsuji, Kawamura and Okahara, Synthesis (1981) p.42).
A. Formation of the chain.
Williamson's ether synthesis can be applied to the
synthesis of chains having the repeating unit -OCH2CH2- and
identical or different terminal groups. The method means
i 35 alkylation of an alcohol (QZ'' + HOQ' --> Q-O-Q' or the
other way round QOH + Z''Q' --> Q-O-Q'). By selecting
.
.~ .
~' - .
.. :. , ~ .. ... , . ... . , ., - :. ~ - :, . : , - : . .
W092/01474 PCT/SE91/0~97
2 0 ~ 3 14
(i) Q = X'CH2~0CH2CH2)p- in which X' is XCH2- or a group
that in one or more steps is transformable to XCH2-
and stabile under the conditions for Wil}iamson~s
ether synthesis, and
(ii) Q' = -(CH2CH20)rCH2Y' in which Y~ is Y or a group that
in one or more steps is transformable to Y and stabile
under the conditions applied.
p and r are O or positive integers such that p + r = n.
Wi.lliamson's ether synthesis may also be applied to the
synthesis of the type o~ bisymmetric chains that is
described in Part D.
For the case that Y' is not Y, one select Y' preferably
among groups that are stabile against strong bases. For
instance Y' may be -CH20R4', -CH=CR5'R6', where R4' are
selected from lower alkyl groups (Cl-C6), preferably
secondary or tertiary al~yl groups of at most 5 carbon
atoms and possibly substituted in their alpha positions
with at most three phenyl groups that possibly are
substituted, and R5' and R6' are hydrogen, lower alkyl (Cl-
C6) or phenyl that possibly is substituted.
In the formulas given above Z'' is a leaving group of
moderate to high reactivity and may be halo, alXane-
6ul~0nate, arenesul~onate, pre~erably toluenesulfonate
(tosylate), and per~luoroal~anesul~onate, pre~erably
tri~luoromet~anesulfonate etc. Williamson's ether synthesis
may be carried out in inert solvents and normally in the
presence of a base - often strong bases, such as sodium
hydride etc. By combining components of appropriate lengths
one can in principle develop any chain -(OCH2CH2-)n by a
sequence of elongation steps.
The chain can also be elongated with one OCH2CH2 unit
through Michael addition of HOQ or HOQ~ to a compound
X''-C(X''')=CH2, where Q and Q', respectively~, have the
same meaning as previously. The ~roups X'' and X''',
respectively, have to be selected such that -OQ and OQ' are
guided to the 2-carbon atom (=CH2) in the compound X''-
C(X''')=CH2 and such that the terminal groups in Q and Q',
.: . -
: : :
W O 92/01474 2 0 8 71 6 3 PC~rlSE9l/00497
.
respectively, are stabile during.the conditions applied. :
X''' may be hydrogen and X'' may be a group that is easily
transformable to amino or substituted amino, e.g. nitro.
Alternatively X'' and X''', respectively, may be groups
that after the transformation enable the group ,
X''-C(X''')CH2- to be converted to the group HOOCCH2- or a
derivative thereof. Preferably X'' is methylsulfinyl and
X''' methylthio, and this requires hydrolysis of the
product obtained in the addition step, said hydrolysis
giving an aldehyde that subseguently may be oxidized to the
corresponding carboxylic acid. The conditions for Michael
addition are similar to those ones for Williamson's ether
synthesis.
A third alternative for chain elongation is reduction of
thione ester by the use of Raney Nickel or corresponding
reagents having a high affinity for sulfur. Suitable thiono
esters comply with the formula:
X~cH2(ocH2cH2)qocH2cs-(ocH2cH2)s-cH2y
or
X~cH2(ocH2cH2)po-cscH2(ocH2cH2)g-cH2y~ ``;
where X' and Y ' have the same meaning as previously and q
and s are 0 or positivQ integers such that q + g - n - 1,
where n has the same meaning as previously.
Thione esters can be synthesized through rearrangement
of the corresponding thiol esters by the action of an
alkylating agent, e.g. methyl iodide, dimethyl sulfate or
diazo methane. Thiol esters may be synthesized by reaction
of a carboxylic acid halide with a thiol compound. Another
alternative is reaction of a carboxylic acid ester with a
sulfur transferring reagent that is selective for double
bonded oxygen when present in a carboxylic acid ester.
Examples of such reagents are phosphorous pentasulfide or
preferably Lawesson's reagent (2,4-bis(4-methoxyphenyl)-
1,2,3,4-dithiaphosphetane-2,4-bis-sulfide). In this
rearangement the functional terminal groups must not
contain a carboxy oxygen.
~.
.
WO92/01474 PCT/SE91/00497
208~ ~3 16
_. Transformation of terminal groups.
A compound complying with the formula:
X~-CH2~(OcH2cH2-)nocH2y
where X'- is XCH2- or a group that in one or more steps is
transformable to XCH2-, and -Y' is Y or a group that, in
one or more steps is transformable to Y, may be subjected
to reaction conditions that give the desired transformation
and end product or where appropriate give an intermediary
product that can be used in a coupling step.
Exemples of X' (besides XCH2-) are hydroxymethyl,
R4'OCH2-, CR5'R6'=CH-, where R4', R5' and R6' have the
same meaning as previously. In the case that X' is
hydroxymethyl the transformation can take place, e.g. by
reaction with a sulfonyl halogenide in the presence of a
base and followed by reaction with ammonia and, if
necessary, further reagents to give the appropriately
substituted amino group, such as phtalimido. In the case
that X'- is R4'OCH2- the group R4' is removed, e.g. by acid
catalyzed hydrogenolysis. The exposed hydroxymethyl group
is then trans~ormed to a group containing a nitrogen atom
as previously given. In the case that X' is CR5R6=CH-, the
=CH- of the alkenylene group is oxidized to an aldehyde
group, e.g. by ozonisation. The aldehyde group is then
transformed to an aminomethyl group by reductive amination.
Examples of -Y'-(in addition to -CH2Y) are
hydroxymethyl, -CH2OR4', -CH=CR5'R6', where R4', R5' and -
R6' have the same meaning as previously. In the case that
Y' is hydroxymethyl the reaction may take place by -
oxidation to a carboxy group (-COOH). In the case that -Y'
is -CH2OR4', one removes R4', for instance by acid
catalyzed hydrolysis, and, if R4' contains at least one
alpha positioned phenyl, the removal can be performed by - ~ ~
catalytic hydrogenolysis. The exposed hydroxymethyl group ~ -
may then be transformed according to what has been said
above. Alternatively, one oxidizes the group directly to
the corresponding carboxy group (-COOH). In the case that -
Y' is -CH=CR5'R6', the -CH= of the alkenylene group may be
.
.::
WO92/01474 2 0 8 71 6 3 PCT/SE91/~497
17
' .
oxidized to a carboxy group (-COOH), possibly via an
intermediary aldehyde (e.g. by ozonisation) that may be
further oxidized. The oxidations may be run in the presence
of an appropriate tertiary alcohol, for instance t-butanol,
in such a way that the corresponding ester will be formed
directly.
:: .
C. Transformation of a symmetric polyether
XlCH2~CH2~2LnQÇ~2~l to a~ U~syn~Letrical ether.
Symmetrical ethers can be synthesized from shorter
polyethylenoxide ethers by chain elongation, for instance
by the use of Williamson's ether synthesis. Transformation
of one of the two identical groups Xl, where Xl is X' or Y' -
as given previously, to another group can be performed by
partial reaction of the desired kind and subsequent
separation of the unsymmetrical product from the unreacted
starting material and the double-reacted side product.
Examples of such transformations are:
a) When Xl is a carboxy group the dicarboxylic acid is
trans~ormed to the corresponding di(acyl halide) that in
turn can be reacted with a deficient amount af ammonia
~ollowed by hydrolysis of the remaining acid halide groups.
The formed amide carboxylic acid is separated ~rom the
reaction mixture wherea~ter its ami~e ~unction is
selectively reduced to an aminometh l group. Alternatively,
the separation is carried out after the reduction step by a
sequence of ion exchange separation steps.
b) When X} is aminomethyl the diamino compound is reacted
with a deficient amount of an acylating carboxylic acid
30 derivative, such as carboxylic acid chloride or -
corresponding anhydride. The monoacylated product is then
separated from the starting material and the diacylated
product, whereafter its aminomethyl group is oxidized, for
instance through the corresponding aldehyde, to a carboxy
group, and its acyl group removed by hydrolysis. A
particularly useful variation of this method is the
reaction with cyclic anhydride. In this latter case ion
, ., . .. . .. , . . ~- ; , . : ,
, .. : .. . . , - ~ - -.,', , - '-~ :. .' , ' - i , .
, . . . , , . :. : : . . . ~, . . . .
: .. . .
.. . .. . . .
-: : . . , , : : .
WO92/01474 PCT~SE91/0049?
20~7 ~3 - 18
exchange separation will become facilitated because the
intermediary product is an amino acid, the starting
material a diamine, and the side product a dicarboxylic
acid.
c) If Xl is a group of moderate to high lipophilicity, for
instance trityloxymethyl or allyloxymethyl, partial removal
or transformation of one of the groups will facilitate
separation by liquid partition due to a significant
difference between the partition coefficients for the
product, the starting material and the side product.
Analogous critera are valid if the appropriately lipophilic
group has been introduced onto to a symmetric polyether
having hydrophilic groups Xl, for instance oligoethylene
glycol that is monosubstituted with trityl.
d) When Xl is hydroxymethyl the reaction can be performed
by Williamson's ether synthesis, for instance by adding an
excess of haloacetic acid. After the reaction the mixture
is esterified with a suitable lower alcohol (Cl-C6), for
instance isopropanol. By selecting the proper alcohol, the
diSforences between the partition coefficients (water and
organic solvents) of the components in the reaction mixture
will become more pronounced. This will facilitate the
separation o~ the product from the starting material and
~rom the disubstituted side product. For instance a diester
will be transSerred Srom an aqueous phase to an organic
solvent in neutral solution, while the monoester will be
transferred at an acid pH. For compounds that are
relatively volatile, the major part of the starting
material may be removed by destillation. Exemples are
di-thylen- glycol or triethyleno glycol.
,~ " '
'-, .
.,
.
~:
WO92/01474 2 0 8 71 6 3 PCT/SE91/~97
19
D. Splitting of a bisymmetric chain into two identical
fragments.
A compound having the formula: -
A) X~cH2-(ocH2cH2)n-ocH2cH=cHcH2o-(cH2cH2o)n CH2
5 B) Y~cH2-(ocH2cH2)n-ocH2cH=cHc~I2o-(cH2cH2o~n 2
where X' and Y', respectively, have the same meaning as
previously, provides a special case. The splitting is
carried out by oxidation of the double bond; in case A
directly to two identic~l carboxy groups and in case B to
two identical aldehyde groups that in turn is transformed
to aminomethyl groups by reductive amination. -
The starting ;
H2 (CH2CH2)n 0cH2cH=cHcH2o-(cH2cH2o)n-cH2x~ and
Y~CX2-(OCH2CH2)n-ocH2cH=cHcH20 (CH2cH2)n C 2
_j respectively, are preferably synthesized through
Williamson's ether synthesis by reacting HOCH2C~=CHCH2OH
with two equivalent~ of X'CH2-(OCH2CH2)n-Z'' and
Y'CH2-~OCH2CH2)n-Z'', respectively, where X', Y' Z'' and n
have the same meaning as previously. An alternative route
is to react Z''-CH2CH~CHCH2-Z'' with two eguivalents of
X'CH2-(0CH2CH2)n-OH and Y'CH2-tOCH2CH2)n~OH, respectively.
Analogous methods are also available. For instance one may
employ compounds in which the group -C~2C~-CHC~2- i~
replaced by the group
-H2CCH-HCc~2
`A~
where A is a possibly substituted lower alkylidene
(preferably Cl-C6), preferably dimethylmethylene. In this
case the group A is removed by acid hydrolysis, whereafter
the intermediary l,2-diol formed is oxidized, for instance
by periodate and subsequent transformation of the aldehyde
group formed to an aminomethyl group or to a carboxy group
in a manner known per se.
The invention is defined by the appending claims that
are a part of the specification.
.~.
,.......... . . . " , . .. , , , ,, .~: . .. .. . .. . . . .. .. .
. - - . . . . . . . . . ` ~ - . .
;--:. - :: . ::. , . . : :: : - : . ,, . ~ :
WO92/01 ~ 8 7 ~ ~ 3 PCT/SE91/00497
The exemplification below is divided into three parts:
Part 1 illustrates the synthesis of amino-PEG-carboxylic
acids complying with formula IV ( m = 1),
Part II illustrates the synthesis of heterobifunctional
reagents complying with formula II and of conjugates having
a bridge structure according to formula I, and
Part III sums up the results obtained for conjugates
between the T-cell immune stimulator (superantigen)
staphylococcal enterotoxin A (SEA) and antibodies directed
against tumour antigens.
Examples 5 and 6 ~Part 2) illustrate the synthesis of a
conjugate between an immunoglobulin and a peptide.
Comparative experiments have shown that, by having a bridge
according to the invention, the solubility of the conjugate
will be increased and therefore also the availability of
the peptide in aqueous media. The comparison has been made
against a conjugate comprising the same peptide and
antibody, but having a short hydrophobic bridge that is not
according to the invention.
W092/01474 2 0 8 71 6 3 PCT/SE91/0~97
21
EXPERINENq!AL PORT~:ON . PART 1.
Iso~ropyl 8-hvdroxv-3,6-dioxa-octanoate (l~. -
Sodium (23 g, l.O mole) in form of chips was added in :
5 portions to diethylene glycol (500 ml) under nitrogen ~ i
atmosphere. When the sodium had reacted completely, the
mixture was cooled to room temperature and bromoacetic acid
I was added (76 g, 0.5 mole) under stirring. After 18 hours
at 100C the excess of diethylene glycol was distilled off
at about 4 mm Hg. Thereafter isopropyl alcohol (400 ml) and
in portions acetyl chloride (51 g, 0.65 mole) were added.
~ After stirring for 18 hours at 65C the mixture was cooled
; to room temperature and neutralized with sodium acetate
(3.5 g, 0.15 mole). The mixture was filtered and the
filtrate evaporated nearly to dryness, whereupon it was
dissolved in water (200 ml). The water phase was extracted
with l,l,l-trichloro-ethane (3xSOml). The pooled organic
phases were washed with water (20 ml). The product was
extracted from the pooled water phases with dichloromethane
~ 20 ~50 ml) that aft-r evaporation gave an oil (55 g). ~ ;
Isopropyl ll-hydroxv-3.6.9-trioxa-undecanoate t2). ~ -
~ Sodium (23 g, l.O mole) in form Or chips was added in
`~ portions to triethylene glycol (700 ml) under nitrogen
4 25 atmosphere. When thé sodium had reacted completely, the
mixture was cooled to room temperature and bromoacetic acid
was added (76 g, O.S mole) under stirring. After 18 hours
, at 100C the excess of diethylene glycol was distilled off
', at about 4 mm Hg. Thereafter isopropyl alcohol (400 ml) and
in portions acetyl chloride (51 g, 0.65 mole) were added.
, After stirring for 18 hours at 65C the mixture was cooled -
, to room temperature and neutralized with sodium acetate
i~ (3.5 g, O.l5 mole). The mixture was filtered and the
filtrate evaporated nearly to dryness, whereupon it was
dissolved in water (200 ml). The water phase was extracted
with l,l,l-trichloro-ethane (3x50ml). The pooled organic
phases were washed with water (20 ml). The product was
;' '
.,~ . ~ .
- , . . , ~ : , ; . - - ;: , , - . . . .
, - . ~ : . . . ~ . :
: ., -.. , , , . ... . . :
WO92/01474 2087 ~3 22 PCT/SE91/0049?
extracted from the pooled water phases with dichloromethane
(50 ml) that after evaporation gave an oil.
H-n.m.r.(CDCl3); 1.26(d,6H);3.07(s,2H);3.6-3.8(m,12H);
4.11(s,2H);5.09(m,1 H)
8-rN-~htalimidovl~-3 6-dioxa-octanol (3~. -
8-Chloro-3,6-dioxa-octanol (365 g, 2.2 mole, prepared from
from triethylene glycol and SOC12) was dissolved in
dimethyl formamide (400 ml) and potassium phtalimide (370
g, 2.0 mole) was added under stirring. After stirring for
18 hours at 110C dimethyl formamide was distilled off at
reduced pressure. The residue was suspended in toluene (1.5
1) at 40-50C and potassium chloride was filtrated off. The
product crystallizes at cooling (-10C). A second fraction
is available from the mother liquor by concentrating it and
repeating the crystallization procedure.
H-n.m.r.(CDC13); ~2.90(s,1H);3.51-3.58(m,2H);3.60-
3.68(m,6H);3.73-3.78(t,2H);3.89-3.94(t,2H);7.70-7.89(m,4H).
Isopropyl 17-(N-phtalimidoyl~-3.6.9.12.15-pentaoxa-
heptadecanoate ~4~.
A solution of pyridine (2.8 ml, 35 mmole) in
dichloromethane (30 ml) was added dropwise under stirring
at about -5C to a solution o~ 8-~N-phtalimidoyl)-3,6-
dloxa-octanol ~3) (8.5 g, 36 mmole)-and
trifluoromethanesulfonic acid anhydride (10.2 g, 36 mmole)
in dichloromethane. After about 30 minutes the organic
phase was washed with 0.5 M hydrochloric acid and water.
After drying (Na2S04) and filtration isopropyl 8-hydroxy-
3,6-dioxa-octanoate (l) (12 g, 48 mmole) and Na2PO4 (6.5,
46 mmole) were added, and the mixture was vigorously
stirred for 20 hours at room temperature. The reaction
mixture was filtrated and the filtrate evaporated. The
residue was partitioned between l,l,l-trichloroethane and
water. Evaporation of the organic phase resulted in an oil
(13 g). -
... . ; :~ . . : . , ,.. , . -
W O 92/01474 2 0 8 71 6 3 PC~r/SE91/00497
23
. '.' '.". '
1H-n.m.r.(CDC13); ~1.26(d,6H);3.58-3.76(m,18H);3.90(t,2H); '
4.11(s,2H);5.09(m,1H);7.70-7.89(m,4H).
17-(N-DhtalimidoYl)-3.6.9 12.15 ~entaoxa-he~tadecanoic acid
ts).
Isopropyl 17-(N-phtalimidoyl)-3,6,9,12,15-pentaoxa-
heptadecanoate (4) (13 g) was dissolved in tetrahydrofuran
(50 ml) and hydrochloric acid (conc., 50 ml)). After 16
hours at room temperature the solution was diluted with
water (200 ml) and tetrahydrofuran was removed at reduced
pressure. The water phase was washed with toluene (lx) and
extracted with dichloromethane (2x). Drying (Na2SO4) and
evaporation of the organic phase resulted in the product in
Sorm of an oil (8.5 g) -
1H-n.m.r.(CDC13): ~3.57-3.76 (m,18H);3.91(t,2H);4.11(s,2H);
4.8(br,2H);7.65-7.90(m,4H) ~-
.
Isopropyl 17-amino-3 6.9.12.15-pentaoxa-heptadecanoate (6)
17-(N-phtalimidoyl)-3,6,9,12,15-pentaoxa-heptadecanoic acid
(5) (8.5 g) was dissolved in 150 ml ethanol and 3 ml
hydrazine hydrate. The solution was stirred at room
temperature ~or 16 hours, whereupon HCl ~100 ml, 3M) was
added and the solution was then refluxed ~or 3 hour~ ter
cooling to room temperature and ~iltration, pH was adjusted
~pH 9, NaOH) and the filtrate was evaporated almost to
dryness. Water was added and re-evaporation almost to -
dryness was carried out, whereupon the pH of the solution
was adjusted (pH 4, HCl) followed by evaporation to
dryness. ~he product was treated with isopropanol (100 ml)
and acetyl chloride (2 ml) at room temperature during the
night and evaporated. The residue was collected in water
and extracted into dichloromethane at an alkaline pH (7-
11). Evaporation resulted in the product (3.3 g).
, lH-n.m.r.(CH30D): ~1.26(d,6H);3.17(t,2H);3.65-3.80(m,18H);
- 35 4.16(s,2H);5.07(m,1H)
~,.
.
. - , . . . . ,: . . . . .. . . . .
W O 92/01474 PC~r/SE91/00497
2087~ 24
7,7.7-Triphenyl-3,6-dioxa-heptanol (7).
Triphenylmethyl chloride (28 g, 0.1 mole) was added to a
solution of pyridine (8 ml, 0.1 mole) in diethylene glycol
(100 ml, 0.94 mole) and the mixture was stirred at 50C for
20 hours. The crystals formed were filtrated off and washed
with water. Yield 31 g. M.p. 103-105C.
lH-n.m.r.(CH30D): ~2.42(~r,1H);3.25(t,2H),3.55-
3.72(m,6H),7.20-7.32(m,9H);7.44-7.53(m,6H)
10,10.10-Triphenvl-3.6.9-trioxa-decanol ~8~.
Triphenylmethyl chloride (187 g, 0.67 mole) was added to a
solution of pyridine (54 ml, 0.67 mole~ in triethylene
glycol (1,000 ml, 7.3 mole) and the mixture was stirred at
60C for 16 hours, The product mixture was divided into 4
portions and each portion (=250 ml) was shaken with water
tl,000 ml) and dichloromethane (250 ml), The organic phases
were pooled and evaporated. Yield about 259 g.
. .
13.13,13-TriDhenvl-3.6.9.12-tetraoxa-tetradecanol (9).
Triphenylmethyl chloride ~129 g, 0.46 mole) was added to a
solution o~ pyridine (38 ml, 0.48 mole) in tetraethylene
glycol (800 ml, 4.2 mole) and the mixture was stirred at
80C ~or 20 hours. Water (800 ml) was added and the mixture
j Was extracted With dichloromethane (3X200 ml). The pooled
organic phases were washed with water (150 ml),-whereafter
the product was obtained as a syrup upon evaporation, Small
amounts of the disubstituted and the unreacted materials -
were present in the product. Yield 200 g.
- ;.
16,16,16-Triphenyl-3r6,9,12,15-pentaoxa-hexadecanol (10).
'~ Triphenylmethyl chloride (2.8 g, 10 mmole) was added to a
' solution of pyridine (1 ml, 12 mmole) in pentaethylene
glycol (25 g, 0.1 mole) and the mixture was stirred at 80C
, for 2 hours. Water (100 ml) was added and the mixture was
extracted with dichloromethane (40 ml). The water phase was
evaporated by use of azeotropic distilllation in the
presence of toluene and ethanol, The residue was treated -
.. '
-.
WO92/01474 2 0 8 71 6 3 PCT/SE91/00497
with pyridine (1 ml) and triphenylmethyl chloride ~2.5 g)
in the same manner as above. The same procedure was applied
once more. The three organic phases were pooled and
evaporated. Small amounts of the disubstituted and of the
unreacted materials were present in the product. Yield
10 g. -
19.19 19-Tri~henyl-3.6 9.12 15.18-hexaoxa-nonadecanol ~
Triphenylmethyl chloride (9.7 g, 34 mmole) was added to a
solution of pyridine (2.8 ml, 12 mmole) in hexaethylene
glycol (100 g, 36 mole) and the mixture was stirred at 80C
for 3 hours. Water (400 ml) was added and the mixture was
extracted with dichloromethane (150 ml). The water phase
was treated once more as described in the example above,
but the amounts and reaction conditions were as described
in this example. The pooled organic phases were evaporated
and washed with water (100 ml) and then extracted with
dichloromethane (50 ml). Small amounts of the disubstituted
and of the unreacted materials were present in the product.
Yield 41.6 g.
7.7.7-Triphenyl-3.6-dioxa-he~tY1 4-methylbenzenesulfonate
. '
p-Toluenesul~onyl chloride ~5 g, 26 mmol~) was added to a
solution o~ 7,7,7-triphenyl-3,6-dioxa-heptanol (7) (3.5, 10
mmole) in pyridine (10 ml) . The mixture was stirred at
room temperature for 30 minutes, whereupon water (1 ml) was
added and the stirring continued for 10 minutes more.
Dichloromethane (100 ml) was added and the mixture was
extracted with hydrochloric acid ~l M) until the pyridine
had been removed completely . Thereafter the organic phase
was washed with NaHCO3 (saturated). After drying (Na2SO4)
and evaporation, the product crystallized from
diethyl ether/hexane. Yield 2g.
. . . , . ... . - -, - . ;.......... : .. ,. i.................... .
: ~ - :, : : . .: . . :: - ;- . . - ,
W O 92/01474 Pl~r/SE91/00497
2087~ ~3 26
10.10.10-Triphenyl-3.6 9-trioxa-decvl 4-methvl-
benzenesulfonate (131-
p-Toluenesulfonyl chloride (5 g, 26 mmole) was added to a
solution of 10,10,10-triphenyl-3,6,9-trioxa-decanol (8)
(4.8 g, 12 mmole) in pyridine (lo ml). ~he mixture was
stirred at room temperature for 1 hour, whereupon water (1
ml) was added and the stirring continued for 10 minutes
more. The mixture was diluted with dichloromethane (50 ml)
and washed with 1 M hydrochloric acid (2xloO ml), water (50
ml) and NaHC03 (saturated, 50 ml). The organic phase was
evaporated and the product was purified on silica gel
~toluene:ethyl acetate, 19:1), whereupon the product
crystallized from dichloromethane/ether. Yield 4.2 g.
1H-n.m.r.(CDCl3): ~2.20(s,3H);3.24(m,2H);3-60-3-80(m8H);
4.20(m,2H);7.20-7.90(m,19H).
8-(N-phtalimidovl)-3.6-dioxa-octvl 4-methylbenzenesulfonate
8-~N-phtalimidoyl)-3,6-dioxa-octanol (3) (82.2 g, 0.3 mole)
was dissolved in pyridine 30 ml, 0.37 mole) and p-
toluenesulfonyl chloride t56.9 g, 0.3 mole) was added
during 1 hour and stirring at 0C. The mixture was then
stirred at 10C for 16 hours. A solid cake had been formed
and ice (0.5 kg) mixed with HCl ~conc., 200 ml) was added
and the mixture stirred at 50C for 4 hours, whereafter
ethyl acetate ~200 ml) was added. Filtration of the
complete reaction mixture resulted in the product in solid
form. Yield 97 g.
1H-n.m.r.(CDC13): ~2.44(s,3H);3.52-3.73(m,8~);3.87(t,2H);
4.09(q,2H):7.31-7.86(m,8H)
17-(N-phtalimidoyl~-3.6.s.12.15-Dentaoxa-heptadecanol (15~.
lO,lO,lo-Triphenyl-3,6,s-trioxa-decanol (8) (0.78 g, 2
mmole) dissolved in dimethyl formamide (15 ml) was added
dropwise to sodium hydride (80%, 0.24 g, 8 mmole, washed
-` with 2x hexane). The mixture was warmed to 30C for 30
minutes whereafter 8-(N-phtalimidoyl)-3,6-dioxa-octyl 4-
, . . .' : , -
W O 92/01474 2 0 8 71 6 3 P(~r/SE91/00497
27
::
methylbenzenesulfonate (14) (0.87 g, 2 mmole) was added in
solid form. After stirring during the night at room
temperature, acetic acid anhydride was added (5 ml), and
the mixture was allowed to stand for three hours more at
room temperature. Water (2 ml) was added and after 30
minutes the product was partitioned between toluene (25 ml)
and a solution of NaHC03 (50 ml). The toluene phase was
evaporated and contained about 1 g substance that was
collected in dichloromethane and treated with 0.2 ml
tri~luoroacetic acid and about 0.2 ml water at room
temperature for 10 minutes. The reaction mixture was then
evaporated. The product was purified on a silica column
(chloroform:methanol, 19:1). Yield about 0.7 g.
lH-n.m.r.(CDC13):~3.59-3.67(m,20H);3.71-3.75(m,4H);
3.90(t,2H);7.71-7.86(m,4H)
tert-Butyl 17-~N-Dhtalimidoyl~-3.6.9.12.15-pentaoxa-
heptadecanoate (lC~.
Pyridinium dichromate (0.64 g, -4 eg.), acetic acid
anhydride (1 ml - 20 eq.) and t-butyl alcohol (1 ml = 30
e~.) were added in the order given to a solution of 17-(N-
phtalimidoyl)-3,6,9,12,15-pentaoxa-heptadecanol ~15) (190
mg) in dichloromethane t5 ml). The r~aaction mixture was
stirred at room temperature ~or 3 hours, whereafter ethyl
acetate ~25 ml) was added. A~ter 10 minutes the liq~id was
allowed to pass through a column containing silica gel
(= 5 cm x 5 cm 0) and the column was eluted with more ethyl
acetate. After evaporation the residue was purified on a
silica gel colum~n (chloroform:methanol, 19:1) Yield about
0.1 g.
lH-n.m.r.(CDC13): Sl.47(s,9H);3.58-3.76(m,18H);3.90(t,2H);
4.02(s,2H);7.70-7.87(m,4H)
14.14.14-Tri~henyl-4.7.10.13-tetraoxa-tetradecene ~17).
Allyl bromide (2 ml, 1.1 eq.) and 10,10,10-triphenyl-3,6,9-
trioxa-decanol (8) (8.5 g, 22 mmole) were dissolved in
dimethyl formamide (25 ml) and added dropwise into sodium
~-- -: : . : ' :
' ` ' ~ ` : :
'
- . . .. . . . .. . .
WO92/01474 PCT/SE91/0049?
2087~3 28
hydride (1 g) at 0c during 30 minutes. After stirring for
three hours at room temperature, methanol was added until
the solution became transparent. Toluene (loo ml) and water
(lO0 ml) were added and the reaction mixture was extracted.
The toluene phase was washed with a saturated salt solution
and was then dried (Na2SO4). The product was obtained as a -
syrup (10 g) after evaporation of the solvent.
3.6.9-Trioxa-ll-dodecen-l-ol (18).
14,14,14-Triphenyl-4,7,10,13-tetraoxa-tetradecene (17)
~5.5 g, 13 mmole) was dissolved in dichloromethane (50 ml)
whereafter trifluoroacetic acid (1.2 ml, 1~.6 mmole) and
water ~1 ml, 55 mmole) were added and the reaction mixture
stirred vigorously for 10 minutes. The product (1.5 g) was
obtained after purification on a silica gel column
( CHC13: MeOH, 9 ~
3.6.9.12.15.18-Hexaoxa-20-heneicosen-1-ol (19~.
3,6,9-Trioxa-ll-dodecen-l-ol (18) (2.07 g, lO.9~mmole) and
10,10,10-triphenyl-3,6,9-trioxa-decyl 4-methyl-
benzenesulfonate (13) ~5.95 g, 10.9 mmole) were dissolved
in dimethyl formamide and added dropwise under stirring to
sodium hydride for lO minutes at room temperature. After 2
hours dichloromethane and water were added. The phases were
separated, and trifluoroacetic acid ~1 ml, 13 mmole) and
water (1 ml, 55 mmole) were added to the organic phase. The
mixture was evaporated to dryness after vigorous stirring
for one hour. Methanol (70~) was added whereupon triphenyl ~-
methanol (2 g) was filtrated off and the solvent
evaporated. The product (2.5 g) was obtained after
purification on a silica gel column (CHCl3:MeOH, 97:3).
H-n.m.r.(CDC13): ~3.56-3.74(m,24H);4.00(m,2H);5.20tm,2H);
5.89(m,lH)
3.6.9.12.15.18.21-Heptaoxa-23-tetracosenoic acid t20).
Bromoacetic acid (0.15 g, 1.1 mmole) and sodium hydride
(0.15 g, 5 mmole) were added to a solution of
:
.
. .' ' . . ' . ' . . . , ' ' . . .'
W O 92/01474 2 0 8 71 6 3 P~r/SE~1/00497
29
.
3,6,9,12,15,18-Hexaoxa-20-heneicosen-1-ol (19) (0.33 g,
1 mmole) in tetrahydrofuran. The mixture was stirred at
room temperature for three hours, whereupon water (50 ml)
was added in order to degrade the excess of reagent and pH
was adjusted to pH 1 (HCl). The mixture was washed with
diethyl ether (50 ml) and was then extracted with
dichloromethane (2x50 ml). Drying (Na2S04) and evaporation
of the solvent gave the product (0.26 g).
23-(N-tert-butoxvcar~onvlamino)-3.6 9.12.15.18.21-he~taoxa-
tricosanoic acid (21).
Ozone was bubbled slowly through a solution of
3,6,9,12,15,18,21-heptaoxa-23-tetracosenoic acid (20)
(0.21 g, 0.55 mmole) in methanol (30 ml) for 30 minutes at
approximately -60C. The mixture was allowed to stand for
30 minutes, and then air was bubbled through the solution
for 30 minutes, whereupon dimethyl sulfide (50 /ul, 0.65
mmole) dissolved in methanol (5 ml) was added dropwise. The
solution was tempered to room temperature during one hour,
1 20 whereupon a solution of ammonium chloride (O.2 g, 3.7
i ~mole) in water (5 ml) and sodium cyano borohydride (0.2 g,
3.1 mmole) was added. After 16 hours at room temperature
the pH of the solution was adjusted to about 1 (HCl),
wher~upon the solvent was remov~d by distillation. The dry
solid phase was ex~racted with dichloromethane that then
was evaporated. Dissolution in sodium hydroxide (1 M),
extraction with dichloromethane and evaporation resulted
in an oil (0.25 g). A pure product was obtained by
preparing the tert-butoxycarbonyl derivative of the amino
function; 0.17 g of the oil and sodium hydroxide (0.4 g)
were dissolved in water (3 ml) and di-tert-butyl
dicarbonate (0.12 g) dissolved in dioxane (5 ml) were
added. After stirring for 16 hours the dioxane was
evaporated and the water phase was washed with n-hexane
(2xlO ml) and pH was adjusted to about 4 (HCl), whereupon
the mixture was extracted with dichloromethane (5x50 ml). --
Drying (Na2S04) and evaporation of the solvent gave an oil
: ~ . : - -
-.,
., ,. , : .
~. : . - -
. ~ .
WO92/01474 PCT/SE91/00497
2087163 30
that was purified on a silica gel column (CHC13:MeOH:AcOH,
45:4:1). The yield was 0.15 g.
1H-n.m.r.(D2O, 500 MHz): 61.31(s,9H);3.14(t,2H);3.48(t,2H);
3.59(m,24H);3.84(s,2H)
11-(N-phtalimidoyl)-3 6.9-trioxa-undecanoic acid ~22).
Sodium hydride (3.8 g, 80%, 130 mmole) and bromoacetic acid
(7.6 g, 55 mmole) dissolved in tetrahydrofuran (5 ml) were
added in the order given to a solution of 8-(N-
phtalimidoyl)-3,6-dioxa-octanol (3) ~10.1 g, 36 mmole) in
tetrahydrofuran tl50 ml). After stirring for four hours at
room temperature acetic acid anhydride (40 ml) was added
and the mixture was stirred for 18 hours more, whereafter
the mixture was filtrated and evaporated in the presence of
some water. Purification of the product on silica gel
(CHC13:MeOH, 3:2) resulted in the product. Yield 1.2 g.
Methyl ll-amino-3.6.9-trioxa-undecanoate ~231.
Hydrazine hydrate (0.7 ml, 14 mmole) was added to a
solution o~ 11-tN-phtalimidoyl)-3,6,9-trioxa-undecanoic
acid ~22) (1.2 g, 3.5 mmole) in ethanol (25 ml) and the
mixture was stirred at room temperature for 18 hours,
whereupon hydrochloric acid (3.7 ml, conc.) and water were
added. The mixture was re~luxed ~or two hours and was then
evaporated almost to dryness whereafter water (25 ml) was
added and pH adjusted to about 9 (NaOH). After evaporation
to dryness methanol (200 ml) and acetyl chloride (2 ml)
were added and the mixture was stirred at room temperature
for 66 hours. The methyl ester formed was purified on a
silica gel column.
H-n.m.r.(D2O):~ 3.17(t,2H);3.65-3.80(m,13H);4.20(s,2H);
3.6.9.12.15.18.21.~4.27-nonaoxa-2s-tricontenoic acid (24).
Sodium hydride (50 mg, 1.6 mmole) was added to a solution -
3S of 3,6,9,12,15,18-hexaoxa-20-heneicosen-1-ol (19) (0.6 g,
1.9 mmole) and 7,7,7-triphenyl-3,6-dioxa-heptyl 4-
methylbenzenesulfonate (12) (0.9 g, 1.8 mmole) in dimethyl-
~ - .
WO92/01474 2 0 8 71 6 3 PCT/SE91/00497
31
formamide (10 ml). The mixture was stirred at room
temperature for 56 hour, whereupon water (10 ml) and
dichloromethane (15 ml) were added. The mixture was shaken
and the organic p~ase was evaporated and purified on a
silica gel column (CHC13:MeOH, 9:1). The product was
dissolved in dichloromethane (20 ml) and trifluoroacetic
acid (3 drops) and water (5 drops) were added, whereafter
the mixture was stirred for 4 hours and evaporated.
Extraction of the residue with methanol:water (70:30) and
filtration gave upon evaporation an oil that was dissolved
in tetrahydrofuran (10 ml) to which sodium hydride (about 5
eq.) and bromoacetic acid (about 2 eq.) were added,
whereafter the mixture was stirred at room temperature for
16 hours. Water was added in order to destroy excess of
sodium hydride, and the mixture was evaporated and then
purified on a silica gel column (CHC13:MeOH, 3:1). Yield
0.14 g.
lH-n.m.r.(CDC13): ~2.89(s,2H);2.97(s,2H);3.50-3.78(m,30H); -
4.02(d,2H);5.20(m,2H);5.91(m,1H)
29-(N-tert-butoxycarbonvlamino)-3.6.9.12.15.18.21.24 27-
nonaoxa-29-nonacosanoic acid (25~.
Ozon was bubbled smoothly through a solution of
3,6,9,12,15,18,21,24,27-nonaoxa-29-tricontenoic acid (2~)
(0.11 g, 0.23 mmole) in methanol (25 ml) for 30 minutes at
about 70C. 30 minutes later, air was bubbled for 30
minutes through the solution, whereafter dimethyl sulfide
(50 /ul, 0.65 mmole) was add d. The solution was tempered
i to room temperature during a period of one hour, whereafter
~ 30 a solution of ammonium hydrochloride (0.1 g, 1.9 mmole) in
i water (5 ml) and sodium cyano borohydride (0.1 g, 1.6
mmole) were added. After 16 hours at room temperature pH
was adjusted to about 1 (HCl), and the solvent was
evaporated. The dry material was extracted with
` 35 dichloromethane which was then evaporated. The residue was
an oil (0.1 g). A pure product was obtained by preparing a
tert-butoxycarbonyl derivative of the amino function; the
.. .
-; .. ;~ ,,, , ~, , ., ,. . -, . . , . ~ -,- ,. . .
- .. ~, . . ,, .. , . . .. . . .... . , ,:-, .. :: : : . . .
W O 92/01474 P ~ /SE91/00497
2 0 8 7 ~ 6 3 32 ~ :
oil and sodium hydroxide (0.4 g) were dissolved in water (3
ml) and di-tert-butyl dicarbonate (0.16 g) dissolved in
dioxane (5 ml) was added. After stirring for 16 hours the :
dioxane was evaporated and the water phase was washed with - -
5 n-hexane (2xlO ml) and the pH was adjusted to about 4 :
(HCl), whereupon the product was extracted with
dichloromethane.
': ' - i :- ' -' . . . . ..... .....
W O 92/01474 2 0 8 71 6 3 P(~r/SE91~00497
33 :~.
FORMULAE OF SYNTHESIZED AMINO-PEG-CAR~OXYLIC ACIDS
O
H--(OCH2CH2)n OCH2 J~CH(CH3)2 PhtN-cH2cH2-(ocH2cH2)4 OCH2 J<
n=2 16
2 n=3
Phh~--CH2CH2--(OCH2CH2)2 OH CH2=CHCH2--(OCH2CH2)3 OTr
O 17 -:
PhtN-CH2CH2-(OCH2CH2~4 OCH~OCHlCH3)2CH2=CHcH2 (OCH2CH2)n OH
4 ~ 18 n=3 .
O 19 n=6 O
J~ J~ ' .
PhtN--CH2CH2--(OCH2cH2)4 OCH2 OH CH2=CHCH2 (OCH2CH2)6 OCH2 OH
O 20
I Jl ,11~
H2N--CH2CH2--(OCH2CH2)4 OCH2 OH >Lo NHCH2CH2(OCH2CH2~6 OCH2 OH
6 21 O
OCH2CH2ln OH PhtN-CH2CH2--(ocH2cH2)2 OCH2 OH
7 n=2 O .
8 n-3 l
10 n-5 H2N-CH2CH2--10CH2CH2)2 OCH2/ J~O /
lS--~OCH2CH2)n OTs 23 O
13 n=3CH2=CHCH2--(OCH2CH2)8 OCH2 OH
24
PhtN--CH2CH2--(OCH2CHz~2 arS O O
14 ~
>Lo NHCH2CHz~OCH2CH2)8 OCH2 OH
PhtN--CH2CHz--(ocH2cH2)4 OCH2CH2H . ~
:~ ~ 15
~ ~ '
."~ -
.
-: .
W O 92/01474 PC~r/SE91/00497
2087~3 34
,, ~.. - :
PREPARATION OF BIFUNCTIONAL REAGENTS AND COUPLING
PRODUCTS
Structural formulae are set forth on a separate page.
5 Example 1. Preparation of N-hydroxysuccinimide ester
of 17-iodoacetylamino-3,6,9,12,15 --
pentaoxaheptadecanoic acid
.
A. Preparatlon of 17-iodoacetYlamino -3,6,9,12,15-
pentaoxaheptadecanoic acld (A)
Isopropyl 17-amino-3,6,9,12,15-pentaoxahepta-
~ decanoate (see part I of the experimental part)
i (1.1 g, 3.2 mmole) was dissolved in 3 ml of 1 M
~; 15 sodium hydroxide solution and left at room tempera-
1 ture for 30 min. 1.5 ml of 6 M hydrochloric acid was
added and the mixture was evaporated to dryness. The -~
residue was ta~en up in dichloromethane and filtered
to give 545 mg of 17-amino-3,6,9,12,15-pentaoxa-
heptadecanoic acid after evaporation of the solvent.
460 mg (1,39 mmoles) of this compound were dissolved
, in 10 ml of borate bufer pH 8.4. ~he solutlon was
deaerated with nitrogen gas. A solution of 432 mg
~ (1.52 mmoles) of N-succinimidyl 2-iodoacetate in
;~ 25 5 ml of dioxane was added dropwise during 1 min pH
~; was kept at 8.4 by addition of 5 M NaOH. The reac-
tion solution was stirred for 15 min during inlet of
nltrogen gas. Accordlng to thln layer chromatography
(eluent: CH2C12-MeOH 60:35) the rèaction was com-
pleted in some few minutes. After 15 min the pH of -
the reaction solutlon was adjusted to 3 and the
solution was frozen and lyophilized. ~he reaction
mixture was fractionated on a reversed phase column
PEP-RPC HR 30/26 (Pharmacia Biosystems AB) using a
gradient of 0-13 ~ acetonitrile with 0.1 ~ tri- - ;
fluoroacetic acid followed by isocratic separation
at 13 % acetonitrile, 0.1 ~ TFA. Fractions from the ~-;
: .
'~ .
WO92/01474 2 0 8 71 6 3 PCT/SE91/00497
desired peak were pooled and lyophilized giving 351
mg of 17-iodoacetylamino-3,6,9,12,15-pentaoxa-hepta- -
decanoic acid (A). Yield: 76 ~.
The structure of the product was established by the
aid of its NMR spectrum. lH NMR spectrum (D20) ex-
pressed as ~-values:
ICH2C ~.23 s, OCH2COH 3.76 9
0 0
-OCH2CH20- 3.71-3.76, -NHCH2CH20- 3.65 t,
-NHCH2CH20- 3.41
-- . :
B. Preparation of ~-hYdroxysuccinimide ester of 17-iodo-
acetvlamlno-3,6,9,12,15-pentaoxaheptadecanoic acid
(B)
Hydroxysuccinimide (4.5 mg, 39 ~mole) was weighed in
the reaction vial. 17-Iodoacetylamino-3,6,9,12,15-
pentaoxaheptadecanoic acid (A) (18.3 mg, 39 ~mole)
was dissolved ln 0.55 ml drled dloxane and added to
the reaction vlal. The v~al wa~ deareated with
nltrogen gas and then a solutlon of 8.0 mg
(39 ~mole) dlcyclohexylcarbodilmide in 0.15 ml of
dried dioxane was added dropwise to the reaction
vial. The vial was filled with nitrogen gas, ciosed
and placed in the dark. The reaction solution was
stirred for 3.5 h. The prec~pitate formed was re- ~-
moved by filtration. The percentage formed product B
in the filtrate was determined by NMR-analysis to be
89 %.
,'
.:
~ ' ' ' : , . , .' - ,' ~: ., ' . : ' . . ' -, ' ' : ' ' ',. ' ' ` ' . ' " : : '
': ' . ' ~ '' . ' , ' ' . ', ' . ` ,' ' ., . ,', ' -, ' ' " ' ~ : ', '
.' ~ '~ .': , , ' , ' ' .' ' ' ' ' .'. '': ., .. ' ' :' ' ' ':
W O 92/01474 PC~r/SE91/00497
2 o 87 ~ ~ 3 36 ;~
. . .
.~ ,: . '
Example 2, Preparation of (17-iodoacetylamino-
3,6,9,12,15-pentaoxaheptadecanoylamino)- -
immunoglobulin (C)
A. Monoclonal antibody Mab C215
''. . ,.:
A monoclonal antibody of immunoglobulin class IgG2a
(Mab C215) (34 mg, 0.218 ,umole) dlssolved in 17.7 ml
of 0.1 M borate buffer pH 8.1 containing 0.9 % sodium
chlorlde was added to a reaction vlal. 146 ~1 of a
dioxane solution containing 3.6 mg (6.4 ,umole) of N-
hydroxysuccinimide ester of 17-iodoacetylamino- ~
3,6,9,12,15-pentaoxaheptadecanoic acid (B) was in- -E
jected into the buffer solution and the reaction was
completed during stirring for 25 min. at room temper-
ature. The reactlon vlal was covered with folie to `
exclude llght. Excess of reagent B was removed by
fractionation on a Sephadex G 25 K 26/40 column using
0.1 M phosphate buffer pH 7.5 contalnlng 0.9 % sodlum
chlorlde as eluent. Fractlons containing the desired
product C were pooled. The solutlon (22 ml) was con-
centrated in an Amlcon cell through a YM 30 filter to
8 ml. The concentration and degree of substitutlon
were determlned with amlno acld analysls to be
4.7 mg/ml and 18 spacer per Mab C215 respectively.
B. Monoclonal antibody Mab C242 ,
. :....
A monoclonal antibody (Mab C242) of the immunoglobu-
lin class IgG 1 was reacted with 15, 20 and 22 times
molar excess of N-hydroxysucclnimlde ester of 17- '
iodoacetyl-amino-3,6,9,12,15-pentaoxaheptadecanoic
acid (B) respectively accordlng to the procedure
described in example 2.A giving nona, dodeca and
tetradeca(l7-iodoacetylamino)-3,6,9,12,15-
pentaoxaheptadecanoylamino)-Mab C242. (C)
.
' . '
'~"
~ . , .. . ., . . . .. : . . . . . . . - . . .. :
~092/01474 2 0 8 71 6 3 PCT/SE91/0~97 ~
37
C. Monoclonal antibody Mab C
A monoclonal antibody tMab C) of the immunoglobulin
class IgG 2a was reacted with 14 and 18 times molar
excess of N-hydroxysuccinimide ester of 17-
iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic
acid (B) respectively according to the procedure de-
scribed in example 2A giving tetra and hepta(17-iodo-
acetylamlno-3,6,9,12,15-pentaoxa-heptadecanoylamino)-
Mab C. (C)
Exam~le 3. Preparation of 2-mercaptopropionylamino-
Eu3-labelled-staphylococcal enterotoxin A
(SEA)
A. PreDaration of Eu3l labelled SEA (D)
SEA (freezed drled product from Toxln Technology
I Inc.) (2 mg, 72 nmole) was dlssolved in 722 ~l
,~ 20 mllll-Q water and added to a 15 ml polypropylene
i tube. 100 ~l of 0.1 M borate buffer pH 8.6 was added
and then 2160 nmoles of Eu3+-chelate reagents
~Pharmacia Wall~c Oy) ln 178 ~1 of mllli-0. The
reaction was completed at room temperature over
night. Excess reagent was removed by fracti~nation
of the reaction solution on a Sephadex G Z5 PD 10
column (Pharmacla Biosystems AB) using 0.1 M -~
phosphate buffer pH 8.0 as eluent. Fractions with
the desired product D were pooled. The solution
(3 ml) was concentrated in an Amicon cell through an ~-
~; YM5 filter to a volume of 0.8 ml. The concentration
'` ~ - was determined with amino acid analysis to be
, ~ ~
1.7 mg/ml. The degree of substitution was determined
by comparing with a EUCl3 standard solution to be
0.8 Eu3+ per SEA.
..: .
WO92/01474 3 3a PCT/SE91/00497
Bl. Preparation of 3-(2-pyridvldithio)propionylamino Eu3+
labelled SEA ( E) and 3-mercaptopropionylamino Eu3+ - -
labelled SEA (F)
Eu3+-SEA (l.24 mg, 44.8 nmoles) in 0.75 ml of O.l M .. .
phosphate buffer pH 8.0 was added to a 15 ~l poly-
propylene tube. 35 ~l (180 nmole) of a solution of -~ -
1.6 mg of N-succinimidyl 3-(2-pyridyldithio)-pro-
pionate in l ml of ethanol was added to the tube and
the reaction solution was stlrred for 30 min at room
temperature. The obtained product E was not isolated ,
before being reduced to product F.
.~. .
To the reaction solution from above were added 20 ~l
of 0.2 M Eu3+-citrate solution and 50 ,ul of 2 M ~ : .
acetic acid to adjust the pH to 5. Thereafter a
solution of 3.l mg of dithiotreltol (Merck) in
O.l ml of O.9 % sodium chloride was added and the
reaction solutlon was stirred for 20 min at room
temperature. Thereafter the total volume was adjust-
ed to l ml by addltion of 50 ~l of O.9 ~ sodium
chloride solution. The reaction solution (l ml) was
placed on a Sephadex G25 NAP-lO column (Pharmacia
Biosystems A3) and desired product F was eluted by
addition of 1.5 ml of O.l M phosphate buffer p~ 7.5
containing O.9 ~ sodium chloride. The eluted product
F was collected in a 15 ml polypropylene tube and ~ -
immediately used in the synthesis of product G to -
avoid reoxidation to a disulfide compound.
-~
B2. Preparation of 2-mercaptopropionYlaminostaphYlococcal
enterotoxin A (SEA) (F2)
~: , .
Native SEA (freeze dried product from Toxin Technol-
ogy Inc) or recombinant prepared SEA (rSEA) was re-
acted with 2 times molar excess of N-succinimidyl 3-
(2-pyridyldithio)-propionate according to the proce-
dure described in example 3Bl.
~;
W092/01474 2 0 8 71 6 3 PCT/SE9l/~97
39
The degree of substitution was determined with W -
analysis according to Carlsson et al (Biochem. J. -
173(1978)723-737) to be 1.9 mercaptopropionyl group
per SEA.
Example 4. Preparat~on of the SEa-monoclonal antibody
con~ugate tGl och G2)
A. Conjugates between ~u3+-SEA and Mab C215 (Gl)
To the solution oi 4-mercaptopropionylamino Eu3+
labelled SEA (F) described in example 3B was added -
1.2 ml of a solution of octadeca(17-iodoactylamino-
, 3,6,9,12,15-pentaoxaheptadecanoylamlno)Mab C215 (C)
(4 mg) in 0.1 M phosphate buffer pH 7.5 containing
0.9 % sodlum chlorlde. The reactlon was completed by
standlng at room temperature over nlght. Unreacted
iodinealkyl groups were then blocked by addition of
5 ,ul (1.2 ~mole) of a solutlon of 20 ~1 mercapto-
~ 20 ethanol ln 1 ml of water. The reaction solution was
¦ left for 4 h at room temperature and then filtrated.
The flltrate was then fractlonated on a Superose 12
HR 16/50 column (Pharmacia Biosystems AB) using as
eluent 0.002 M phosphate buffer pH 7.5 containing
0.9 % sodium chloride. Fractions with the desired
product G were pooled and analysed. The protein con-
' tent was 0.22 mg/ml determined by amino acid analy-
sis. The degree of substitution was one SEA per IgG
determined by Eu3+ d-termination. The product was
also studied for immunostimulating properties and
antibody binding capacity.
~y increasing the amount of compound (F~ in relation - -
to compound (C) higher degree of substitution was ob-
tained.
.~ .
W092/01474 PCTISE9l/OW97
20~7 ~3 ;
B. Conjugates between rSEA and Mab C215 (G2) -
Octadeca ~`.7-iodoacetylamino-3,6,9,12,15-
pentaoxaheytadec~noylamino)Mab C215 (C~ was reacted
with 1.8 times molar excess of 2-mercaptopropionyl-
amino-rSEA (F2) according to the procedure described
in example 4A. The composition of the con;ugate was
analysed by sodium dodecylsulfate polyacrylamide gel
electrophoresis (SDS-PAGE) on Phast-Gel~ gradient 4-
15 and the bonds were scanned with Phast IMAGE
(Pharmacia 3iosystems AB). The con;ugate obtained was
composed of 6% Mab C215 with three SEA, 15% with two
SEA, 28% with one SEA and 51% unsubstituted Mab C215.
In another experiment 2.7 times molar excess of F2
was used givlng a con~ugate wlth the composition 15%
Mab C215 wlth three SEA, 25% wlth two SEA, 34% with
one SEA and 26% of unsubstltuted Mab C215.
:.
! C. ConJugates between rSEA and Mab C242
Cl. Tetradeca(17-iodoacetylamlno-3,6,9,12,15-
pentaoxaheptadecanoylamino)Mab C242 (C) was reacted
with 3.2 times molar excess of 2-mercaptopropionyl-
amino-rSEA (F2) accordlng to the procedure described
in example 4A. The composition of the conjugate was
analysed as descrlbed ln example 4 B and was found to
be 4* Mab C242 wlth four SEA, 12% wlth three SEA, 28~ -;
wlth two SEA, 36% with one SEA and 20% of unsubsti-
tuted Mab C242.
~ 30
i The same reaction was run but the reaction product
was treated 4 h with 0.2 M hydroxylamine before
column fractionation to remove unstable bonds between
Mab C242 and the spacer and between SEA and the
mercaptopropionyl group. The con~ugate formed had the
composition 1% Mab C242 with four SEA, 12% with three
;-; . . . - . , -.: : .,. . - .; ,. .- - ., -: - , .
. , . , , . . . . ., . . . . ~ -
WO92/01474 2 ~ 8 71 6 3 PCT/SE91/00497
41
SEA, 27% with two SEA, 36% with one SEA and 24% of
unsubstituted Mab.
C2. Dodeca(17-iodoacetylamino-3,6,9,12,15-pentaoxahepta-
decanoylamino)Mab C242 (C) was reacted with 3 times
molar excess of 2-mercaptopropionylamino-rSEA (F2)
according to the procedure described in example 4A.
The con~ugate obtained had the composition 6% Mab
C242 with three SEA, 26% with two SEA, 36% with one
SEA and 31% unsubstituted Mab C242.
C3. Nona(17-iodoacetylamino-3,6,9,12,15-pentaoxahepta-
decanoylamino)Mab C242 (C) was reacted with 3 times
molar excess of 2-mercaptopropionylamino-rSEA (F2)
according to the procedure described in example 4A. -~
The con~ugate obtained had the composition 13% Mab
C242 with two SEA, 39~ with one SEA and 46% unsubsti-
tuted Mab C242.
D. Coniugates between rSEA and Mab C
Dl. Hepta(17-iodoacetylamino-3,6,9,12,15-pentaoxahepta-
decanoylamlno)Mab C was reacted with 5.4 times~molar
excess of 2-mercaptopropionylamino-rSEA (F2) accord- -
ing to the procedure described in example 4A. The ~-
composition of the conJugate was analysed as de-
scribed in example 4B and was found to be 15% with
four SEA, 24% wlth three SEA, 29~ with two SEA, 19%
with one SEA, 3% unsubstituted Mab C and 10% in a
dimeric form.
'
The same reaction was run with 0.2 M hydroxylamine
present to remove unstable bonds between Mab C and
spacer and between SEA and the mercaptopropionyl
group. The con~ugate formed had the following
composition 11% Mab C with three SEA, 24% with two
.. - : . : . . ~. - . : -- . , .. : ., . . - .: . - , : . : .. ... - . - .. .... : . .: - .- . :: ..
W092/01474 PCTlSE91/~97
42
2~ 63 '"
SEA, 30% with one SEA, 18% of unsubstituted Mab C and
17~ in a dlmerlc form.
D2. Tetra(17-iodoacetylamino)-3,6,9,12,15-pentaoxahepta- -
decanoylamino)Mab C was reacted with 5.7 times molar
excess of 2-mercaptopropionylamino-rSEA (F2) accord-
i ing to the procedure described in example 4B. The
con~ugate had the following compositon 8~ Mab C2 with
four SEA, 18% wlth three SEA, 30~ with two SEA, 26~ '
with one SEA, 5% of unsubstituted Mab C and 12% in a
dimeric form.
~' . ."' .
Example 5. Coupling of the pept~de sequence 145-165
derived from the human alloantigen HLA-A2.1
to the monoclonal antibody Mab C215 (~) ;
Octadeca(17-iodoacetylamino-3,6,9,12,15-pentaoxa-
heptadecanoylamino)-lmmunoglobylln G2a (C) (5.4 mg,
34.6 nmole) dlssolved 1n 1.6 ml of 0.1 M phosphate buffer - ~;
pH 7.5 contalnlng 0.9 ~ sodlum chlorlde was added to a
5 ml ~eacti vial. The solution was deaerated by nitrogen
gas and then the HLA-A2.1 peptlde sequence 145-165 Hls-
LysTrpGluAlaHl~ValAlaGlu~lnLeuArgAlaTyrLeuGluGlyThrCysVal
(2.5 mg, 0.8 ~mole) was added ln solid state in small
portions during stirrin~ to the solution. The pH of the
reaction solution was chequed to be 7.4. Before closing
, the vial more nltrogen gas was bubbled through the solu-
tion. The vial was covered with folie and the reaction
solution was stirred over night at room temperature. To
30 block unreacted iodinealkyl groups 10.5 ,ul (1.5 ~mole) of
a solution of-10 ,ul mercaptoethanol in 1 ml water was
added and the reaction solution was stirred for 4 h. The
reaction solution was filtered and fractionated on a
Superose 12 HR 10/50 column (Pharmacia Biosystems AB)
35 using 2 mM phosphate buffer pH 7.5 containing 0.9 %
sodium chloride as eluent. Fractions with the desired
product (H) were pooled. The protein concentration and
~ .
.: .
- : - . : ' .' .. ' . ~ : . `, ' , : . . ..
W092/01474 2 0 8 716 3 PCT/SE91/0~97
43 ~ -
degree of modification were determined by amino acid
analysis to be 176 yg protein per ml and ll peptides per
IgG.
.
In a similar synthesis the peptide was added in an amount ~
of 5 mg ~1.7 ,umole) giving a product with 17 peptides per -- -
IgG.
. .
Example 6, Coupling of the peptide sequence 93-113
derived rom the human alloantigen HLA-~2.1
to the monoclonal antibody Mab C215 (1)
Tridecane (17-iodoacetylamino-3,6,9,12,15-
pentaoxaheptadecanoylamino)-immunoglobulin G2a (4 mg,
15 25.6 nmole) dissolved ln 1.6 ml 0.1 M phosphate buffer pH
7.5 contalnlng 0.9 % sodlum chlorlde was added to a 5 ml
Reacti vial, (This compound was prepared slmilar to com-
pound C, Example 2A,uslng less excess of the reagent B).
The solution was deaerated by nitrogen gas. ~he HLA-A2.1
peptide MetTyrGlyCysAspValGlySerAspTrpArgPh~LeuArgGlyTyr
(4.7 mg, 2.1 ~mole) was suspended in 0.2 ml of ,
acetonltrile and dlssolved by addltion of 0.1 ml of 0,1 M
phosphate buffer pH 7.5 contalning 0.9 % sodlum chloride.
Thls solutlon waq added dropwl~e to the Reacti vial. pH
was chequed to be 7.5. Nitrogen gas was bubbled through
the reactlon solutlon before the vlal was closed. The
vlal was covered with folie and the reactlon solution was
stlrred o~er night. To block unreacted lodlnealkyl group
10 ~1 (1.4 ~mole) of a solution of 10 ,ul mercaptoethanol
in 1 ml water were added. The reactlon solutlon was
stirred for another 4 h, filtered and then fractionated
on a Superose 12 HR 10/50 column using 2 mM phosphate
buffer pH 7.5 containing 0.9 ~ sodium chloride as eluent. - ' -
Fractions with the desired product (I) were pooled. The
35 protein concentration and degree of modification were - -
determined by amino acid analysis to be 196 ~g protein
per ml and 7 peptides per IgG respectively.
W092tO1474 PCT/SE91/0~9?
44
a8~ ~3 Example 7. Preparation of N-hydroxysuccinimide ester
of 17-~3-(2-pyridyldithio)propionylamino]-
3,6,9,12,15-pentaoxaheptadecanoic acid (K)
A. Preparation of 17-[3-(2-pYridYldithio)propionvl-
amino] -3,6,9,12,15-pentaoxaheptadecanoic acid (J)
,~.~ . - -
17-Amino-3,6,9,12,15-pentaoxaheptadecanoic acid ~' ;
(66 mg, 0.2 mmole) was dissolved in 3.5 ml of 1 M
borate buer pH 8.4. The pH decreased to 8.1. N-
Succinimidyl 3-(2-pyrldyldithio)-propionate (69 mg,
O.22 mmole) dissolved in O.8 ml of dioxane was added
to the above solution. The pH of the reaction solu-
tion decreased to 7.6. The reaction was completed in
10 min which was shown by thin-layer chromatography
(Eluent: CH2C12-MeOH 60:35). ;
After 30 min the pH of the reaction solution was ad-
~usted to 4.5 wlth 5 M hydrochloric acid and the
solution was frozen and lyophilized. The reaction
mlxture was fractlonated on a reversed phase column
PEP RPC HR 16/10 (Pharmacia Biosystems AB) using a
gradient of 0.17 ~ acetonitrlle wlth 0.1 % TFA
followed by isocratlc separation at 17 ~ aceton-
ltrile with 0.1 % TFA. Fractlons with the desired
compound J were pooled and lyophilized. The frac-
tionation was repeated 13 times. Yield: 39 mg.
.
The structure of the product J was established by
the aid of its NMR spectrum.
B. Preparation of N-hydroxysuccinimide ester of 17-[3-
(2-pyridYldithio)propionylamino]-3,6,9,12,15-penta-
oxaheptadecanoic acid ( K ) ~ .
'~ 35
Hydrosuccinimide (1.87 mg, 16.3 ~mole) was weighed
in a 5 ml Reacti vial. 17-[3-(2-pyridyldithio)-pro-
pionylamino]-3,6,9,12,15-pentaoxaheptadecanoic acid
.. , . . . . - ....... - .
.: ~ . . . .- , ~ . . - . . .
.,. ~ - ... , , . .. - : :
WO92/01474 2 0 8 71 6 3 PCT/SE91/00497
(J) (8.0 mg, 16.4 ,umole) was dissolved in 0.5 ml of
dried dioxane and added to the vial. Nitrogen gas
was bubbled through the solution. A solution of
6.8 mg (32.8 ,umole) of dicyclohexylcarbodiimide in
200 ml of dioxane was added to the Reacti vial and
more nitrogen gas was bubbled through the reaction
solution before closing the vial. The reaction was
allowed to occur during 24 h and the precipitate
formed was removed by flltration. NMR analysis of
the filtrate showed that the reaction was almost
completed to compound K.
Example 8. Preparation of di~17-~3-(2-pyridyl-
dithio)propionylamino]-3,6,9,12,15-penta-
oxaheptadecanoylamino)immunoglobulin Gl (L)
,
A monoclonal antibody of immunoglobulin class IgGl (Mab
C242) (6 mg, 38 mmole) dissolved ln 2.23 ml of 0.1 M
, borate buffer pH 8.0 containlng 0.9 ~ NaCl was added to a
- 20 5 ml Reacti vial. The solution was dilutsd with 0.77 ml
o the above buffer to a final concentration of 2 mg pro-
tein per ml. A solution of N-hydroxysuccinimide ester of
17-[3-(2-pyridyldithio)propionylamino]-3,6,9,12,15-penta-
oxaheptadecanoic acld (K) (0.26 mg, 447 nmole) dissolved
in 100 ~l of dloxane was in~ected into the solution in
the Reacti vial. The reactlon solutlon was stirred for
25 mln at room temperature and then placed in the refrig-
erator over nlght. The reaction solutlon was fractionated
on a Superdex 75 HR 10/30 column (Pharmacia Biosystems
AB) using 0.1 M phosphate buffer pH 7.5 containing 0.9
NaCl as eluent. Fractions with the desired product (L)
: were pooled (7 ml) and concentrated in an Amicon cell
through an YM 30 filter to 1.5 ml. The concentration and
degree of substitution were determined with amino acid
~' 35 analysis to be 3.51 mg protein per ml an~ 2 spacer per --
IgG respectively.
, . . ..
: .
W O 92/01474 PC~r/SE91/00497 '.
208~3 ;~
Example 9. Crosslinking of two monoclonal antibody
molecules to product (N)
A. Preparation of di(l7-[3-thioproPionylamino]-
3,6,9,12,15-pentaoxaheptadecanoylamino)-immuno-
globulin Gl ( M ) -
: ~,
Di( 17-[3-(2-pyridyldithio)propionylamino]-
3,6,9,12,15-pentaoxaheptadecanoylamino)immuno-
globulin Gl (L) (2.5 mg, 16 mmole) dissolved ln
0.7 ml 0.1 M phosphate buffer pH 7.5 contalning
0.9 % sodium chloride was added to an Ellenman tube.
pH was adjusted to 4.7 with 1 M acetic acid. ~here-
after 100 lul (2.3 mg) of a solution of 6.9 mg of
dithiotreitol in 300 ,ul of 0.9 ~ sodium chloride was
added. The reaction solutlon was standing at room
temperature for 25 min and then desalted on a
Sephadex G25 NAP 10 column (Pharmacia Biosystems
AB). 0.1 M phosphate buffer pH 7.5 containing 0~9
sodium chloride and 2 mg EDTA pe~ ml was used as
eluent. The product M was eluted in a ~olume of
1.5 ml and immediately used in the synthesis of pro-
duct (N) to avoid reoxidation of the free mercapto
groups.
B. Preparation of the crosslinked monoclonal antibody
(N)
To a 5 ml Reacti vial were added 0.7 ml (1.25 mg) of
the solution of (17-[3-thiopropionylamino]-
3,6,9,12,15-pentaoxaheptadecanoylamino)immuno-globu-
lin Gl (M) described in example 9A and 0.35 ml of a
solution of tri(17-iodoacetylamino-3,6,9,12,15-
pentaoxaheptadecanoylamino)immunoglobulin Gl
(1.26 mg). (This compound was prepared similar to
compound C using less excess of the reagent B, and
the monoclonal antibody Mab C242).
:: . . . , - : . - ,,. . - . ,
. .
.:. :. . ., , . . . ,~ . . . . . ..
' - ~ ' .: ~, : - - . ,: . :: : . ,,
-
2087i63
WO92/01474 PCT/SE91/00497
47
Nitrogen gas was bubbled through the reaction solu-
tion. Thereafter the vial was closed, covered with
folie to avoid light and left over night at room
temperature. Unreacted iodinealkyl groups were
blocked by addition of 2.5 ul (0.6 ~mole) of a solu-
tion of 20 ,ul of mercaptoethanol in 1 ml water. The
reaction solution was standing at room temperature
for 6 h and then fractionated on Superose 12
HR 10/30 column (Pharmacla Biosystems AB) using 5 mM
phosphate buffer pH 7.5 containing 0.9 % sodium
chloride as eluent. Fractions with the dimeric pro-
duct (N) were pooled.
Example 10. Preparation of [17-(3-mercaptopropionyl-
1; amino)-3,6,9,12,15-pentaoxaheptadecanoyl-
amino]-rSEA (P)
A. Preparation of 17-[3-(2-pyridYldithio)propionYl-
amino]-3~6,9~12~15-Pentaoxaheptadecanoylamino)-rsEA
(O)
..
A solution of N-hydroxysuccinimide ester of 17-[3-(2-
pyridyldithio)proplonylamino]-3,6,9,12,15-pentaoxa-
heptadecanoic acid (K) (0.53 mg (896 nmoles) ln ~3 ,ul
of dioxane) was injected into a solution of 3.67 mg
(128 nmoles) of rSEA in 1 ml of 0.1 M phosphate
buffer pH 7.5 containing 0.9% of sodium chloride. The
reaction was completed in 30 min at room temperature. -
lO0 ,ul was taken for analysation of degree :f substi-
tution. The rest was stored frozen until it was re-
duced to product P.
The degree of substitution was determined by desalt-
ing 100 ul of the reaction solution on a Sephadex G50
NICK column (Pharmacia Biosystems AB) and analysing
the eluate with W -spectroscopy according to Carlsson
et al (Biochem. J. 173(1978)723-737). 2.7 spacers
were coupled to rSEA.
`: :
:
~ . .. : : . .. . , , . . .. ~ :.
WO92/01474 PCT/SE91/00497 ~-
208~ ~3 48 ~ ;
' :'
B. Preparation of ~17-(3-mercaptopropionylamino)-
3,6,9,12,15-Pentaoxaheptadecanoylamino]-rSEA (P)
.
The pH of the reaction solution with product 0
(0,9 ml) was adjusted with 2 HCl to pH 4.4 and 2.9 mg
of dithiotreitol dissolved in 75 ~1 0.9% sodium
chloride was added. The reduction was completed ln 30
min. The reaction solution was added to a Sephadex
G25 NAP 10 column (Pharmacia Biosystems AB) and
eluted with 1.5 ml of 0.1 M phosphate buffer pH 7.5
with 0.9~ NaC1 and immediately used in the synthesis
of product Q in example 11.
.
Exam~le 11. Preparation of SEA-monoclonal antibody
con~ugate Q with double spacer
Dodeca(17-lodoacetylamino-3,6,9,12,15-pentaoxahepta-
decanoylamlno)Mab C242 (C) (4.2 mg, 27 nmoles in 1.0 ml
of 0.1 M phosphate buffer pH 7.5 with 0.9% NaCl) was re-
acted during 43 h with ~17-(3-mercaptopropionylamino)-
3,6,9,12,15-pentaoxaheptadecanoylamino]-rSEA (P) (1.17
mg, 42 nmoles ln 1 ml of the above buffer) in the dar~ in
nitrogen atmosphere. Thereafter 1.14 ~mole of mercapto-
ethanol was added. After another 1 h the reaction solu-
tion was fractionated on a Superdex 200 HR 16/65 column.The product was eluated with 2 mM phosphate buffer pH 7.5
with 0.9 % NaCl. Fractions with the desired product Q
were pooled and analysed as described in example 4B. The
conjugate was composed of 9% Mab C242 with two SEA, 25
with one SEA and 66% of unsubstituted Mab C242.
- ~ '
~, :
., ~
-:
~':: : - ... : :.
W O 92/01474 2 0 8 71 6 3 P ~ /SE91/00497
IcH2cN~cH2cH2(ocH2cH2)4ocH2cooH - A
O O ,'.'
IcH2cNHcH2cH2(ocH2cH2)4ocH2lclON~ ]
IgG-(NHCCH20(CH2CH20)4CH2CH2NHCCH2I)n C
O O
n = 4, 7, 9, 12, 14, 18
SEA-NHCNH ~ -CH2 N N ~ N (EU3~-SEA)
S \=/
COO COO COO COO
~ ' '.
Eu3+Na+
Eu3+-SEA-(NHCCH2CH2SS ~ )3_4 E
O - :
Eu3+-SEA-(NHCCH2CH2SH)3_4 Fl
O :~, ' .
SEA(NHclcH2cH2sH)l.9 F2
(NHCCH2o(CH2CH2o)4CH2CH2NHjCjCH2SCH2CH2CIlNH-Eu3+-SEA)m
O O O
IgG
(NHlclcH2o(cH2cH2o)4cH2cH2NHclcH2scH2cH2oH)18 m Gl
O O ,.
-~NHlclcH2o(cH2cH2o)4cH2cH2NHclcH2scH2cH2cNH-sEA]m G2 ~-~
' / O O O ~,
. IgG
(cH2cH2o)4cH2cH2NHcllcH2scH2cH2oH]n-m
O
: .
.. :', .
. .. ........ ~ . . , . . - .. . . - .. . .. - . -
- : . . - . .. . . . :, : . .
- -. . .. - . . - ~ ,, .-.
W O 92/01474 PC~r/SE91/00497~ ~
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WO 92/01474 2 0 8 71 6 3 PCI'tSE91/00497
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WO92/01474 PCT/SE91/0~97
2087 ~3 52
EXPERIMENTAL PART II
Effects of superantigen-antibody conjugates on cells
The bacterial toxin used in the following experiments
was Staphylococcus enterotoxin A (SEA) obtained from Toxin
Technologies (WI; USA) or produced as a recombinant protein
from E. Coli.
The antibodies were C215, C242 and Thy-1.2 mAbs. C215
i9 an lgG2a mAb raised against human colon carcinoma cell
line and reacts with a 37kD proteln antigen on several human
colon cell lines. References to these mAbs have been given
above. The con;ugates were prepared as described in the
preceding part.
Before the priority date studies had only been per-
formed with Eu3 labelled SEA-C215 mAb conjugates. During
the priority year the results have been verified with
unlabelled SEA-215, SEA-242 and SEA-Thy-1.2 mAb con~ugates.
The results now incorporated refer to unlabelled con~ugates.
To determine the cytotoxicity mediated by the SEA-C215
mAb con~ugate and unconjugated SEA and C215 mAb agalnst
colon carclnoma cells lacking MHC Class II or expressing low
but undetectabl~ amounts of MHC Class II, we employed
various human SEA expanded T cell lines as effector cells
and a panel of colon carCinoma cells and MHC Cl~ss II+ RaJi
cells as target cells. The colon carcinoma cell lines
Colo205, SW620 and WiDr, all lacked expression of MHC
Class II, as determined by staining with mAbs against
HLA-DR, HLA-DP and HLA-DQ and FACS analysis. The SEA ex-
panded T cell lines were establisned from peripheral blood
by weekly restimulations with mitomycin C treated MHC
Class II BSM lymphoma cells precoated with SEA in the
presence of recombinant IL-2 (20 units/ml~. These T cell
lines were strongly cytotoxic towards Raji or BSM cells
coated with SEA but not to uncoated cells or cells coated
with staphylococcal enterotoxin B (SEB). This SEA induced
killing is dependent on interaction of SEA with MHC Class II
., . .. . . . ~ ~
-': ' ' - ~ ' . ,
. ~ . . . -
.
..
. ' , ': '~ . . , ' . . ~
W O 92/01474 2 0 8 71 6 3 P ~ /SE91/00497
53
on the target cell as determined by the use of blocking
HLA-DR antibodies, MHC Class II Raji mutant cells and
HLA-DR transfected L-cells (Dohlsten et al., Immunology 71
(1990) 96-100. These T cell lines could be activated to kill
C215 MHC Class II colon carcinoma cells by the C215-SEA
conjugate. In contrast uncon~ugated SEA and C215 mAb were
unable to induce more than marginal T cell killing against
the C215 MHC Class II colon carcinoma cells. The staphylo-
coccal enterotoxin antibody conjugate dependent cell-mediated
cytotoxicity was dependent on binding of the SEA-C215 mAb
con~ugate to the C215l tumor cells. The specificlty in this
binding was demonstrated by the fact that excess of un-
con~ugated C215 mAb but not the irrelevant C242 and w6/32
mAbs inhibited the lysis of the colon carcinoma cells. CD4
and CD8~ T cells demonstrated killing of SEA-C215 treated
C215+ colon carcinoma cells, but did not lyse SEA treated
cells. The interaction of T cells with SEA-C215 mAb conju-
gate bound to MHC Class II tumor cell seems to involve
interaction with speclfic V-beta TC~ sequenCes in a similar
manner as earlier demonstrated for SEA induced killing of
MHC Class II cells. This was indicated by the interaction
of an SEA specific but not an autologous SEB specific T cell
llne wlth the C215-SEA conJugate. C242 mAb and Thy-1.2 mAb
con~ugates demonstrate actlvlty ln analogy with the C215 mAb
con~ugate.
Chromium labelling and incubation of the target cells with
SEA
0.75x106 target cells and 150 ,uCi 51chromium (Amersham
Corp., Arlington Hights, England) were incubated for 45
minutes a~ 37C in a volume of 100 ,ul. The cells were kept
in complete medium containing RPMI-1640 medium (Gibco, -
~aisley, GBR) supplemented with 2.8 % (v/v) 7.5 % NaHC03,
1 ~ sodium pyrovate, 2 % 200 mM L-glutamine, 1 % lM Hepes,
l % 10 mg/ml gentamicin and 10 % fetal calf serum (FCS,
Gibco, Paisley, GBR). After the incubation the cells were
washed once in complete medium without FCS and incubated 60
'~'
, . . ~ . - ... , , .. .-. - - - .. .. : .
.. - , . . . : .. ... : ~: : . -
' ` ' ' ' ! ' ' ' . ,, , , , , ,, .. . :, .. . .
'' ' ' ' ' ' ' ' ,' . ' '. '~ . . ' ' ', . ' `
W O 92/01474 PC~r/SE91/00497
2,0~7 ~3 54
minutes at 37C and washed and resuspended in complete
medium containing 10 ~ FCS. 5x103 taryet cells were added to
each well of U-bottom 96-well microtiter plates (Costar,
Cambridge, USA).
Cytotoxicity assay
The effector cells were added to the wells at various
effector/target cell ratios. The final volume in each well
was 200 ~l. Each test was done in triplicate. The plates
were incubated 4 hours at 37~C after which the released
chromium was harvested. The amount 51Cr was determined in a
gamma-counter (Cobra Auto-gamma, Packard). The percentage
cytotoxicity was computed by the formula % cytotoxicity =
(X-M)/(T-M) * 100, where X is the chromium release as cpm
obtained in the test sample, M is the spontaneous chromium
release of target cells incubated with medium, and T is the
total chromium release obtained by incubating the target
cells with 1 % sodium dodecyl sulfate.
RESULTS
SEA-C242, SEA-C215 and SEA-anti-Thy-1.2 mAb con~ugates
bind to cells expressing the relevant epitopes of the mAbs,
respectively, and to MHC Class II cells. Uncon~ugated SEA
on the other hand only binds to MHC Class II~ cells. Un-
con~ugated C215, C242 and Thy-1.2 mAbs bind to the relevant
cells but not to ~a~i cells. (Table 1)
Human T cell lines lysed the MHC Class II SW620,
Colo205 and WiDr cells in the presence of SEA-C215 mAb
con~ugate but not in the presence of uncon;ugated SEA and
C215 mAb (Fig. 1). The lysis of colon carcinoma cells was
seen at 10-100 ng/ml of SEA-C215 mAb conjugate. High levels
of lysis at various effector to target ratios were seen with
SEA-215 mAb conjugate against SW620 (Fig. 1). In contrast,
unconjugated SEA or C215 mAb mediated no cytotoxicity
against SW620 cells at all tested effector to target ratios.
.,~ . . : , -: . . - . -- - :
- - .: -; - - . . :. - .
2087~ 63
W O 92/01474 PC~r/SE91/00497
` :
This indicates that the capacity to lyse MHC Class II
Colo205 cells is restricted to the con;ugate and cannot be
induced by unconjugated SEA and C215 mAb. SEA and SEA-C215
mAb conjugate but not C215 mAb mediated T cell killing of
MHC Class II Raji cells and of interferon treated MHC
Class II Colo205 cells ( Fig. 1).
In order to demonstrate that the SEA-C215 mAb conjugate
mediated lysis involved specific binding of the conjugate to
the C215 mAb molecule on the target cells, we performed
block~ng studies with excess of unconjugated C215 mAb and
mAb C242, which bind to an irrelevant antigen on the colon
carcinoma cells (in regard to C215 mAb binding). Addition of
mAb C215 strongly blocked cytotoxicity, whereas the C242 mAb
had no influence (Fig. 2). Similarly lysis by a SEA-C242 mAb
conjugate was specifically blocked by excess of unconjugated -
C242 mAb but not C215 mAb.
The capacity of SEA-C215 mAb conjugate to induce T cell
dependent lysis of M~C Class II SW620 colon carcinoma cells
was seen in both CD4+ and CD8+ T cell populations (Table 2).
SEA did not activate any of these T cell subsets to mediate
killing of SW620 cells but induced lysis of MHC Class II
RaJi cells (Table 2).
The SEA-C215 mAb conJugate lnduced lysls of SW620 and
Raji cells by a SEA expanded T cell line, but not by a SEB
expanded T cell line (Fig. 3). The specificity of the SEA ~ -
and SEB lines is indicated by their selective response to
SEA and SEB, respectively, when exposed to Raji cells
(Fig. 4). This indicates that the SEA-C215 mAb conjugate
retains similar V-beta TC2 specificity as for unconjugated
SEA.
Legend to figures
Fig. 1. The SEA-C215 mAb conjugate directs CTLs against
MHC class II colon carcinoma cells. Upper left panel
demonstrates the effect of SEA responsive CTLs against SW620
cells at various effector to target ratios in the absence
(-) or presence of SEA-C215 mAb conjugate, SEA, C215 and a
'~, ,'' ~
-
- .
WO92/01474 PCT/SE91/00497
208~3 56
mixture of C215 and SEA (C215+SEA) at a concentration of
1 ,ug/ml of each additive. The other panels demonstrates the
capacity of SEA-C215 mAb conjugate, and SEA to target SEA
responsive CTLs against the C215+M~C class II colon carci-
noma cell lines SW620, Colo205 and WiDr, MHC class II C215
interferon treated Colo205 cells and C215- MHC class II
Raji cells. Effector to target ratio was 30:1. Addition of
unconjugated C215 mAb, at several concentrations, did not
induce any CTL targeting against these cell lines. FACS
analysis on SW620 cells, Colo205 and WiDr ceLls using mAbs
against HLA-DR, -DP, -DQ failed to detect any surface M~C
class II expression, whereas abundant expression of ~LA-DR,
-DP and -DQ was detected on Raji cells and HLA-DR and -DP on
interferon treated Colo205 cells. Colo205 cells were treated
with 1000 units/ml of recombinant interferon-gamma for 48
hours prior to use in the CTL assay.
Fig. 2. SEA-C215 mAb conjugate and SEA-C242 mAb conju-
gate induced CTL targeting against colon carcinoma cells
~, depends on the antigen selectivity of the mAb. Lysis of
Colo205 cells by a SEA responsive CTL line in the presence
; of SEA-C215 mAb and SEA-C242 mAb con~ugate (3 ,ug/ml) is
blocked by addition of unconjugated C215 and C242 mAbs
t30 ~g/ml), respectlvely. The uncon~ugated mAbs or control
- medium ~-) were added to the target cells 10 minutes prior
to the aonJugates.
Fig. 3. Lysis of SEA-C215 mAb conjugate coated colon
carcinoma cells is mediated by SEA but not SEB responding
CTLs. Autologous SEA and SEB selective T cell lines were
used at an effector to target ratio of 10:1 against SW620
and Ra~i target cells in the absence (control) or presence
- of SEA-C215 mAb conjugate, a mixture of unconjugated C215
mAb and SEA (C215+SEA) and unconjugated C215 mAb and SEB
(C215+SEB) at a concentration of 1 ,ug/ml of each additive.
- Fig. 4. Cytotoxicity induced by the SEA-C242 mAb
conjugate and SEA-Anti-Thy-1.2 mAb conjugate against their
- ta~get cells (Colo205 tumour cells and EL-4 tumour cells,
~ r~spectively).
. . - , - . . : . .: . :
~: - ': ` ` ' - - -, ~ . : - ; , . ~ , -
- .~ . - ~ :, - , :
. . . -
.. - - . - : - . . ..
- . . , :
. .
2o~7163
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57
Table 1
.
SEA-C215 mAb conjugate bind to C215 colon carcinoma
cells and MHC Class II Raji cells
Reagent Cell Facs analYsis
. .
SEA-C215 mAb Colo205 Pos
Raji Pos
C215 mAb Colo205 Pos
Ra~i Neg
SEA-C242 mAb Colo205 Pos
Raji Pos
C242 mAb Colo205 Pos
Raji Neg
SEA-anti-Thy-1.2 mAb EL-4 Pos
anti-Thy-1.2 mAb EL-4 Pos
SEA Colo205 Neg
Ra~i Pos
control Colo205 Neg
Ra~i Neg
EL-4 Neg
. , .
Cells were incubated with the vario~s additives of control
(P~S-~SA) for 30 minutes on ice, washed and processed as
descrlbed below. The staining of C215 mAb and C242 mAb bound
to Colo205 cells and anti-Thy-1.2 bound to EL-4 cells was
detected using FITC labelled rabbit anti mouse 1 g. The
staining of SEA to Ra;i cells was detected using a rabbit
anti-SEA sera followed by a FITC-swine anti rabbit 1 g. The
staining ~ SEA-C215 mAb con~ugate to Colo 205 and Ra~i
cells was detected utilizing the above described procedures
for C215 mAb and SEA. FACS analysis was performed on a FACS
star plus from Becton and Dickinson. Staining with secona
and third steps only was utilized to define the background.
.. ': .
: ':
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58
208~ Table 2
CD4 and CD8 CTLs lyse colon carcinoma cells presenting the
C215-SEA conjugate.
~ cytotoxicity
EffectorA) Targetcontrol SEA C215-SEA
CD4 SW620 2 5 50
CD4 Ra~i 0 41 43
CD8 SW620 0 1 23
CD8 Ra;i 2 72 68
A) The CTLs (SEA-3) were used at effector to target ratios
of 30:1 in the absence (control) or presence of SEA and
C215-SEA at 1 ,ug/ml.
~. .. .
~ . ~. ". -. .. . ~ . . . .
