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Increased Production of Specific Antibodies by Presentation of the Antigen Determinants with Covalently Coupled Lipopetide Mitogens.

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of added CF,=CCIY to greatly accelerate the reaction. For
example, in diglyme, l a reacts with C6H50K so slowly
that only trace amounts of 2a can be detected after 24 h,
but when 5 mol-% CF,=CCIF (relative to C5H50K)is added, 2a (15%) and 3a (3%) are formed in 20 min.
The reactions described here afford a facile route to ethers, one of whose alkyl groups is fully halogenated; such
ethers are not readily available via other preparative methods. More interestingly, these observations are the first
good evidence for a chlorophilic attack on C-CI bonds by
oxygen nucleophiles.
Received: June 10, 1985;
revised: July 26, 1985 [Z 1345 IE]
German version: Angew. Chem. 97 (1985) 867
[I] B. Haley, R. N. Haszeldine, B. Hewitson, A. E. Tipping, J. Chem. Soc.
Perkin Trans. 11976. 525; V. 1. Popov, V. N. Boiko, L. M. Yagupolskii, J .
Fluorine Chem. 21 (1982) 365; C . A. Postovoi, L. T. Lantseva, Yu. V. Zeifman, Izu. Akad. Nauk SSSR. Ser. Khzm. 1982. 2531; P. Bey, J. P. Vevert,
U. V. Dorsselaer, M. Kolb, J . Org. Chem. 44 (1979) 2732; 1. Rico, D.
Cantacuzene, C. Wakselman, J. Org. Chem. 48 (1983) 1979; A. E. Feiring,
ibid. 48 (1983) 347; D. J. Burton, J. L. Hahnfeld, Fhmrine Chem. Reu. 8
(1977) 119.
121 Xing-ya Li, He-qi Pan, Xi-kui Jiang, Tefrahedron Leu. 25 (1984) 4937.
[3] Xing-ya Li, He-qi Pan, Xi-kui Jiang, Huaxue Xuebao (Acta Chim. Sin.)42
(1984) 297.
14) A. E. Platt, B. Tittle, J. Chem. SOC. C 1967. 1150; A. W. Frank, C. F.
Baranauckas, J . Org. Chem. 30 (1965) 3970; M. Schlosser, Helu. Chim.
Acta 58 (1975) 2515; P. Johncock, Synthesis 1977, 551.
151 The reaction of lc with C,HIONa in butanone to give C,HIO-CFZCCllH in 30% yield has been mentioned by E. T. McBee and R. 0. Bolt
[lnd. Eng. Chem. 39 (1947) 4121. W e have reexamined this reaction and
found that it is initiated by a carbanion. Further evidence for this conclusion has also been obtained by a study of the reactions of C,H,ONa with
the less reactive substrate l a in the presence of pinacolone.
161 J. L. Roberts, Jr., T. S. Calderwood, D. T. Sawyer, J . Am. Chem. Sac. 105
(1983) 7691.
17) M. Hudlycky: Chemistr.v qf Organic Fluorine Compounds. 2nd ed., Wiley
New York 1976, p. 559.
[S] All the ‘H-NMR, I9F-NMR, MS spectra, and elemental analyses are in
good agreement with the designated structures of the products.
191 R. D. Chambers: Fluorine in Organic Chemistry, Wiley, New York 1973:
a) p. 99; b) p. 150.
Increased Production of Specific Antibodies
by Presentation of the Antigen Determinants
with Covalently Coupled Lipopeptide Mitogens
The initiating process in antibody formation is the endocytosis and processing of antigens by leukocytes and their
subsequent “presentation” on cell surfaces, the molecular
details of which are largely unknown. Our concept is based
on the idea of employing for the immunization a chemically unequivocally defined low molecular conjugate such
as 2 in which the carrier-antigen-adjuvant principles are
coupled with one another. Such a conjugate was designed
as a potential synthetic vaccine in connection with the
“Pam,Cys-antigen-helix” system.”’ Our conjugate differs
in essential points from the high molecular polylysine conjugates with antigens and muramyl dipeptidel’] as well as
from other adjuvant^^^' that were merely admixed but cannot perform any special carrier function. Most of the systems developed so far require multiple antigen administrations in vivo.
We have, for the first time, covalently coupled a B-cell
mitogen, which is an excellent carrier and concomitantly a
strongly active adjuvant, with a synthetic antigenic determinant. To d o this we used the synthetic lipopeptide Npalmitoyl-S-[(2RS)-2,3-bis(palmitoyloxy)propyl]-cysteiny~serine (Pam,Cy~-Ser),[~I
which constitutes the N-terminal
of the lipoprotein from the outer membrane of Escherichia
c ~ l i . The
~ ~ ’covalently bonded conjugate of Pam,Cys-Ser
and antigen exhibits pronounced amphiphilic properties.
They guarantee, on the one hand, a stable anchoring of the
three fatty acid residues in the lipid layer of the cell membrane (Scheme I ) and, on the other, that the usually
stronger polar antigen (hapten) is ‘presented’ in the outer
hydrophilic layer of the membrane. A decision on the pos-
synthesis of the
antigen determinants
addition of the
Fmoc-Sar( t Bu)-OH
I Pam3Cys-Ser( tBu) 1 -Am-Leu-Leu-Glu(0
By Giinther Jung,* Karl-Heinz Wiesmiiller,
Gerhard Becker, Hans-Jorg Biihring, and
Wolfgang G. Bessler
In the conventional methodology for the production of
antisera against antigenic determinants (epitopes) of a protein, synthetic partial sequences are coupled with high-molecular carriers such as serum allfumin; subsequently, immunization is carried out with addition of Freund’s adjuvant. The reproducibility, both of the preparation of the
conjugate as well as of the immunization, is frequently unsatisfactory. With the concept described herein, long immunization times, multiple administrations, high titers of
undesired antibodies, and serious inflammations of the immunized animals can be avoided.
0 V C H VerlagsgeseN~chaftmbH. 0 - 6 9 4 0 Wernherm. 1985
Prof. Dr. G. Jung, Dipl.-Biochem. K.-H. Wiesmiiller, Dr. G. Becker
lnstitut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-7400 Tiibingen (FRG)
Dr. H.-I. Biihring
FACS-Labor, Medizinische Universitatsklinik
Auf dem Schnarrenberg, D-7400 Tiibingen (FRG)
Prof. Dr. W. G. Bessler
Arbeitsbereich Mikrobiologie und Immunologie der Universitat
Auf der Morgenstelle 28, D-7400 Tiibingen (FRG)
CFSCOOH, thioanlsole. 2h
in vitro
in vivo
specific antibodies
against EGF-R 516-529
Scheme 1. Production o f antibodies using the covalently bound carrier and
adjuvant Pam,Cys-Ser. Fmoc = fluorenylinethoxycarbonyl; rBu =tert-butyl;
PS-DVB = styrene-divinylbenzene (1%) copolymer with 4-(hydroxymethy1)phenoxymethyl anchor groups; EGF-R= receptor of the epidermal
growth factor (cf. synthetic example).
0570-0833/85/10l0-0872 S 02.50/0
Angew. Chem. Int. Ed. Engl. 24 (1985) No. 10
sible micellar/monomeric distribution of the conjugate in
the cell culture medium can probably be made by spinand fluorescence-labeling.
Since the activating effect of the lipoprotein is brought
about by its N-terminal moiety,[‘] in all conjugates which
contain the Pam3Cys-Ser or analogues its immunostimulating action remains preserved. Pam,Cys-Ser-antigen-conjugates can be routinely prepared in the last step in Merrifield syntheses of partial sequences (Scheme 1). Preferably
an alkoxybenzyl alcohol-resin as well as N*-fluorenylmethoxycarbonyl-, rert-butyl-, and tert-butoxycarbonyl protective groups are used in the peptide segment.[” Since
Pam3Cys is stable towards trifluoroacetic acid,lB the conjugate can be cleaved from the carrier without problem free
of protective groups. The conjugate, which is usually readily soluble in aqueous organic systems or liposomes, can
be chromatographed and can be characterized unequivocally by all analytical methods including N M R spectroscoPY.
As example we cite the use of our concept for the generation of specific antibodies against the receptor of the epidermal growth factor (EGF-R).‘’’ The extracytoplasmic region 5 16-529 was selected by computer-supported epitope
search, built u p by Merrifield synthesis, and finally coupled with Fm~c-Ser(tBu)-OH.‘~~
After cleavage from the resin, the analytically pure conjugate 2 was administered,
without further additives, intraperitoneally to mice in a single dose. High titers of specific antibodies (IgM and IgG)
against the tetradecapeptide EGF-R 516-529 could be established by ELISA assays[’] after two weeks (Fig. 1). The
vital point is that no significant antibody titers were obtained by the tetradecapeptide alone in control experiments. A conventional immunization by a tetradecapeptide-serum-albumin conjugate required multiple antigen
administration with addition of Freund’s adjuvant, in order to achieve comparable antibody production. With the
in vitro
in vivo
b c d e
b c d e
Fig. I . Formation of specific antibodies against the tetradecapeptide EGF-R
5 16-529. In-vivo experiment: Control (a), immunization (single i.p. 0.2 Fmol,
groups of 3-5 mice (Balb/c)) [9] with the conjugate Pam,Cys-Ser-(EGF-R
516-529) 2 (b), with the tetradecapeptide EGF-R 516-529 (c), with the adjuvant Pam3Cys-Ser alone (d), and with a mixture of ECF-R 516-529 and
Pam,Cys-Ser (e). 14 days after the immunization the serum antibody titer
was determined by ELISA (enzyme-linked immunosorbent assay) [9]. As a
measure of the antibody production (specific antibody titer against EGF-R
516-529 coupled to bovine serum albumin), in each case the mean values 2
standard deviation of three measurements ( O D at 405 nm) are quoted; the
values of the serum albumin-specific antibody ( O D 0.000-0.034) are subtracted from these values.-In-vitro experiments: The experiments were carried
out in microtiter plates (Falcon Plastics, Los Angeles) at a cell density of
2.5 x 10’ cells/mL (37”C, 5% COX,5 days) [6c]. Concentration of the reagents
used: 5 x lo-’ mM.
Angeu, Chem. In!. Ed. Engl. 24 (1985J No. 10
Pam,-Cys-Ser-antigen conjugate 2 we were already able to
prepare monoclonal antibodies that were detectable in cell
culture supernatants and as ascites.
Pam,Cys conjugates are also strongly immunogenic in
cell cultures1” (Fig. 1). Hence, conventional and monoclonal antibodies against weakly immunogenic compounds
can also be obtained in a rapid and elegant way by in-vitro
immunization. The antibody titers shown in Figure 1 reflect the well-known finding that only a minor immune response can be elicited by primary in-vitro immunization.
The advantages of our concept are: simple preparation
of chemically unambiguously defined antigen-adjuvantconjugates in any desired amount; in contrast to other conjugates single administration without multiple boosting;
high efficiency in vivo and in vitro. A considerable saving
of experimental animals, often a complete elimination of
in-vivo immunizations, and a drastic saving of time, especially for gene-technological studies are evident. Experiments with other antigens are presently being carried out
in mouse cell and human cell cultures to demonstrate the
general applicability of our system.
Synthetic example
Following the usual stepwise synthesis (Merrifield synthesis with N“-Fmoc/
(tBu)-protection with dicyclohexylcarbodiimide/hydroxybenzotriazole(DCC/
HOBt) and symmetrical anhydrides) of the EGF-R segment 516-529 1101.
Fmoc-Ser(tBu)-OH was incorporated as the last amino acid. After cleavage
of the Fmocgroup with piperidine/dimethylformamide (VMF) ( I : I , 15 min)
the resin-bound pentadecapeptide protected in the side chain by tBu (I g,
loading 0.5 mrnol/g) %’as coupled with PamXys-OH 4a (2 mmol, in DMF/
CH2CII ( I : I ) ) and DCC/HOBt ( 2 mmol, preactivated for 20 min at 0°C)
(16 h, then 2nd coupling, 4 h). The protected lipohexadecapeptide I was removed from the polymeric carrier with trifluoroacetic acid ( 5 mL) in the
presence of thioanisole (0.25 mL) within 2 h and thereby freed also from protective groups. Yield: 960 mg (76Oh) 2 xCF,COOH (correct amino acid analysis, no racemization).
Received: June 12, 1985;
supplemented: July 29, 1985 [ Z 1339 IE]
German version: Angew. Chem. 97 (1985) 883
CAS Registry numbers:
2, 98125-76-9; Pam,Cys, 87420-41-5; FmocSer(t-Bu)OH, 71989-33-8; AsnLeu,GluClyGluProArgCluPheValGluAsn,98104-60-0; SerAsnLeu2GluGlyGluProArgGluPheValGlu, 98 104-61-1: EGF, 62229-50-9.
[ I ] G. Jung: “Synthesis and Perspectives of New Adjuvant and Carrier Systems for Potential Application for Synthetic Vaccines”, in Proc. Int.
Symp. Synth. Antigens. Siena, October 29-30, 1984; Ann. Sclauo 1984,
No. 2, p. 191-208.
[2] a) M. Sela, Biopolymers 22 (1983) 419: b) R. Arnon, M. Sela, M. Parant,
L. Chedid, Proc. Natl. Acad. Sci. USA I 1 (1980) 6769; c ) L. Chedid in
Proc. Forum Peptides, C a p d’Adge, Sept. 1984, in press.
131 E. Lederer, J . Med. Chem. 23 (1980) 819.
[4] a) K.-H. Wiesmiiller, W. G. Bessler, G. Jung, Hoppe-Seyler’s Z . Physiol.
Chem. 364 (1983) 593, and references cited therein; b) G. Jung, C. Carrera, H. Briickner, W. G. Bessler, Liebigs Ann. G e m . 1983, 1608: c) W.
G. Bessler, R. B. Johnson, K.-H. Wiesmiiller, G. Jung, Hoppe-Seylerk 2.
Physiol. Chem. 363 (1982) 767: d) A. Lex, H.-M. Vordermeier, R. Sprenger, 6. Suhr, W. G. Bessler, Immunobiology 168 (1984) 71.
[5] V. Braun, Biochim. Biophys. Acla 415 (1975) 335, and references cited
161 a) F. Melchers, V. Braun, G. Galanos, J . Exp. Med. 142 (1975) 473: b) W.
G. Bessler, V. Braun, 2. Immunilaetsforsch. Exp. Klin. Immunol. 150
(1975) 193; c) K. Resch, W. G. Bessler, Eur. J. Biochem. 115 (1981)
(71 C. D. Chang, M. Waki, M. Ahmad, J. Meienhofer, E. D. Lomdell, J. V.
Haug, Int. J . Pept. Protein Res. I5 (1980) 59.
[XI A. Ullrich, L. Courreus, J. S. Hayflick, T. J. Hull, A. Grey, A. W. Tam, J.
Lee, Y. Yarden, T. A. Libermann, J . Schlessinger, J. Downward, E. L. V.
Mayse, N. Whittle, M. D. Waterfield, P. H. Seeburg, Nature (London)
309 (1984) 418.
(91 W. G. Bessler, B. Suhr, H:J. Biihring, C. P. Muller, K.-H. Wiesmiiller,
G. Becker, G. Jung, Immunobiology, in press.
[lo] H. J. Biihring, C . P. Muller, G. Becker, G . Jung, unpublished.
0 VCH Verlagsgesellschaft mbH. 0-6940 Weinheim, 1985
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production, mitogen, increase, antibodies, specific, determinants, antigen, presentation, covalent, lipopetide, coupled
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