Note: Descriptions are shown in the official language in which they were submitted.
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t
TNF LIGANDS
This application is a divisional application of the co-pending
application 2,105,534, filed September 3, 1993.
FIELD OF THE INVENTION
The present invention relates to ligands to Tumor Necrosis Factor
receptors (TNF-Rs) which inhibit the effect of TNF but not its
binding to the TNF-Rs, as well as to ligands.interacting with
other receptors of the TNF/NGF receptor family.
. H~ACKGROUND OF ~~NTION
Tumor necrosis factor (TNF) is a pleiotropic cytokine, produced
by a number of cel-1 types, mainly by activated macrophages. It
is one of the principal mediators of the immune and inflammatory
response. Interest in its function has greatly increased,
recently, in view of evidence of the involvement of TNF in the
pathogenesis of a wide range of disease states, including
endotoxin shock, cerebral malaria and graft-versus-host
reaction. Since many of the effects of _TNF are deleterious to
. the organism, it is of great interest to find ways of blocking
its action on host cells. An evident target for such intervention
are the molecules. to which TNF has to bind in order to exert its
effects, namely the TNF-Rs. These molecules exist not only in
cell-bound, but also in soluble forms, consisting of the cleaved
extra-cellular domains of the intact receptors (see Nophar et
al., EMBO Journal, ,~t10):3269-78, 1990). The soluble receptors
maintain the ability to bind TNF, and thus have the ability to
block its function by competition with surface receptors.
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CA 02470670 2004-06-11
Another method of TNF inhibition based on the principle of
competing with cell-bound molecules, is the use of antibodies
recognizing TNF receptors and blocking the ligand binding.
The cell surface TNF-Rs are expressed in almost all cells of the
body. The various effects of TNF, the cytoi~oxic, growth-promoting
and others, are all signalled by the TNF receptors upon the
binding of TNF to them. Two forms of these receptors, which
differ in molecular size: 55 and 75 kilodaltons, have been
described, and will be called herein p55 and p75 TNF-R,
respectively. It should be noted, however, that there exist
publications which refer to these receptors also as p60 and p80.
The TNF-Rs belong to a family of receptors which are involved in
other critical biological processes. Examples of these receptors
are the low affinity NGF receptor, which plays an important role
in the regulation of growth and differentiation of nerve cells.
Several other receptors are involved in the regulation of
lymphocyte growth, such as CDw40 and some others. Another member
of the family is the FAS receptor also called APO, a receptor
which is involved in signalling for apoptosis and which, based on
a study with mice deficient, in its function, seems to play an
important role in the etiology of a lupus-like disease. Herein,
this family of receptors is called "TNF/NGF receptor family". .
One of the most striking features of TNF compared to other
cytokines, thought to contribute to the pathogenesis of several
diseases, is its ability-to elicit cell death... The cell-killing
2
CA 02470670 2004-06-11
activity of TNF is thought to be induced by the p55 receptor.
However, this p55 receptor activity can be assisted by the p75
receptor, through a yet unknown mechanism.
European Patent Publication Nos. 0,398,327 and 0,412,486 disclose
antibodies to the soluble TNF-Rs. These antibodies were found to
recognize the soluble TNF-Rs and to inhibit the binding of TNF to
the TNF-Rs on the cell surface. Monovalent Flab) fragments
blocked the effect of TNF, while intact antibodies were observed
to mimic the cytotoxic effect of TNF.
SUI~iARY OF THE INVENTION
The present invention provides a ligand to a member of the TNF/NGF
receptor family, which binds to the. region of the C-terminal
cysteine loop of such a receptor. This region includes the amino
acid sequence cys-163 to thr-179 in the p75 TNF-R or a
corresponding region in another member of the TNF/NGF family.
Preferably, the receptor is the TNF-R, in particular the p75 TNF-
R.
One such ligand includes the amino acid sequence for the CDR
region of the heavy chain of monoclonal antibody No. 32, shown in
Fig. 11 or the amino acid sequence for the CDR region of the light
chain of this antibody shown in Fig. 12.
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Another such ligand includes the amino acid sequence for the CDR
region of the heavy chain of monoclonal antibody-no. 70 shown in
Fig. 11.
Yet another such ligand includes the amino acid sequence for the
CDR region of the heavy chain of monoclonal antibody no. 57,
shown in Fig. 11.
The above antibodies are called herein, for simplicity's sake,
"group 32" antibodies.
In another aspect of the invention, the ligands comprise the scEv
of a group 32 antibody.
The ligands may comprise e.g. proteins, peptides, immunoadhesins,
antibodies or other organic, compounds.
The proteins may comprise, for example, a fusion protein of the
ligand with another protein, .optionally linked by a peptide.
linker. Such a fusion protein can increase the retention time of
the.ligand in the body, and thus may even allow the ligand-
protein complex to be employed as a latent. agent or as a vaccine.
The term "proteins" includes muteins and fused proteins, their
salts, functional derivatives. and active fractions.
The peptides include peptide bond replacements and/or peptide
mimetics, i.e, pseudopeptides, as known. in the art ('see e.g.
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Proceedings of the 20th European Peptide Symposium, ed. G. Jung,
E. Bayer, pp. 289-336, and references therein), as well as salts
and pharmaceutical preparations and/or formulations which render''
the bioactive peptides) particularly suitable for oral, topical,
nasal spray, ocular pulmonary, T:V. or subcutaneous delivery,
depending on the particular treatment indicated. Such salts,
formulations, amino acid replacements arid pseudopeptide struc-
tures may be necessary and desirable to enhance the stability,
formulation, deliverability (e.g. slow release, prodrugs), or to
improve the economy of production, as long as they do not
adversely affect the biological activity of the peptide.
Besides substitutions, three particular forms of peptide mimetic
and/or analogue structures of particular relevance when desig-
nating bioactive.peptides, which have to bind to a receptor while
risking the degradation by proteinases and peptidases in the
blood, tissues and elsewhere, may be mentioned speci.~fically,
illustrated by the following examples: Firstly, the inversion of
backbone chiral centres leading to D-amino acid residue
structures may, particularly at the N-terminus, lead to enhanced
stability for protealytical degradation without adversely
affecting activity. An example .is given.iri the paper "T.ritriated
D-alai-Peptide T Binding", Smith C.S. et al., Drug Development
Res. 15, pp. 371-379 (1988). Secondly; cyclic. structure for
stability, such as N to C interchain i.mid~es and lactames (Ede et
al. in Smith and Rivier {Eds.) "Peptides: Chemi,stxy and Biolagy",
Escom, Leiden {1991), pp. 268-270), and sometimes also receptor
binding may be enhanced by forming cyclic: analogues. An example
CA 02470670 2004-06-11
1 ,
of this is given in "Confirmationally restricted thymopentin-like
compounds", US Pat. 4,457,489 (1985), Goldstein, G. et al.
Thirdly, the introduction of ketomethylene, methylsufli:de or
retroinverse bonds to replace peptide bonds, i.e, the interchange
of the CO and NH moieties are likely to emhance both stability
and potency. An example of this type is given in the paper
''Biologically active retroinverso analogues of thymopentin",
Sisto A. et al in Rivier, J.E. and riarshall, G.R. (eds)
''Peptides, Chemistry, Structure and Biology", Escom, Leiden
(1990). PP~ 722-773).
The peptides of the invention can be synthesized by various
methods which are known in principle, namely by chemical coupling
methods (cf. Wunsch, E: "Methoden der organi.schen Chemie", Volume
15, Band 1 + 2, Synthese von Peptiden, th:i.me Veriag, Stuttgart
(1974), and Barrany, G.; Marrifield, R.B.: "The Peptides", eds.
E. Gross, J. Meienhofer, Volume 2, Chapter. l., pp. 1-284, Academic
Press (1980)), or by enzymatic coupling methods (cf. Widmer, F.
Johansen, J.T., Carl.sberg Res: Gommun., Vo1.44, pg.~ 37=46 (1979),
and Kullmann, W.: "Enzymatic Peptide Synth.esis'° CRC Press Inc.
Boca Raton, F1. (1987), and Widmer, F., Johansen, J..T. in
"Synthetic Peptides in Biology and Medicines:, eds. Alitalo, R.,
Partanen, P., Vatieri, A., pp.79-86, Elsevie~r, Amsterdam {1985)),
or by a combination of chemical and enaymat:ic methods if this is
advantageous for the process design and economy.
A cysteine residue may be added at both t:he amino and carboxy
terminals of the peptide, which will allow t;he cyclisation of the
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peptide by the formation of a di-sulphide bond.
Any modifications to the peptides of the present invention which
do not result in a decrease in biological activity are within the
scope of the present invention.
There are numerous examples which illustrate the ability of
anti-idiotypic.antibodies (anti-Id Abs) to an antigen to function
like that antigen in its interaction with animal cells and
components of cells. Thus, anti-Id Abs to a peptide hormone
antigen can have hormone-like activity and interact specifically
with a mediator in the same way as the receptor does. {For a
review of these properties see: Gaulton, G.N. and Greane, M.I.
1986. Idiotypic mimicry of biological receptors, Ann. Rev.
Immunol. Vol. 4, pp. 253-280; Sege K. and Peterson, P.A., 1978,
Use of anti-idiotypic antibodies as cell surface receptor probes,
Proc. Nati. Acad. Sci. U.S.A., Vol. 75, pp. 2443-2447).
It is expected from this functional similarity of anti-Id Ab and
antigen, that anti-Id Abs bearing the internal image of an.
antigen can induce immunity to such an antigen. (See review in
Hiernaux, J.R., 1988, Idiotypi.c vaccines and infectious diseases,
Infect. Immun., Vol: 56, pp. 1407-1413).
It is therefore possible to produce anti-id~:otypic antibodies to
the peptides of the present invention which will have similar
biological activity.
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CA 02470670 2004-06-11
~ ,
Accordingly, the present invention also provides anti-idiotypic
antibodies to the peptides of the present invention, the
anti-idiotypic antibody being.capable of inhibiting TNF toxicity,
but not its binding to the receptor.
The individual specificity of antibodies resides in the
structures of the peptide loops making up the Complementary
Determining Regions (CDRs) of the variable domains of the
antibodies . Since in general the amino acid sequence of the CDR
peptides of an anti-Id Ab are not identical to or even:similar to
the ~ami.no acid sequence of the peptide antigen from which it was
originally derived, it follows that peptides whose amino acid
sequence in quite dissimilar, in certain contexts, can take up a
very similar three-dimensional structure. The concept of this
type of peptide, termed a "functionally equivalent sequence" or
mimotope by Geyson is known. (Geyson, H.M, et al, 1987,
Strategies for epitope analysis using peptide. synthesis., J.
Immun. Methods, Vol. 102, pp. 259-274).
Moreover, the three-dimensional..structure~ and function of the
biologically active peptides can be simulated by other compounds,
some not even peptidic in nature, but which nevertheless mimic
the activity of such peptides. This fie7_d is summarized in a
review by Goodman, M. (1990), (Synthesis , Spectroscopy and
computer simulations in peptide research, Proc. 11th American
Peptide Symposium published in Peptides-Chemistry, Structure and
Biolocrv, pp. 3-29; Eds. Rivier, J.E. and Marshall, G.R. Publisher
Escom).
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It is also possible to produce peptide and non-peptide compounds
having the same three-dimensional structure as the peptides of
the present invention. These "functionally equivalent structures"
or "peptide mimics" will react with antibodies raised against the
peptide of the present invention and may also be capable of
inhibiting TNF toxicity.
Accordingly, a further embodiment of the present invention
provides a compound the three-dimensional structure of which is
simil-ar as a pharmacophore to the three-dimensional structure of
the peptides of the present invention, the compound being
characterized in that it reacts with antibodies raised against
the peptides of the present invention and that the compound is
capable of inhibiting TNF toxicity.
More detail regarding pharmacophores can lbe faund in Berlin et
al., p. 150, Polinsky et al., p.,287, and Smith et al., p. 485,,
in Smith and Rivier (eds.) "Peptides: Chemistry and Biology",
Escom, Leiden {1991).
All of the molecules {proteins, peptides, ~~tc.) may be produced
either by conventional chemical methods, as described herein, or
by recombinant DNA methods.
The inventi.on.also provides DNA molecules encoding the.ligands
according to the invention, vectors containing them and host
cells comprising the vectors and capable of expressing the
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_ CA 02470670 2004-06-11
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ligands according to the invention.
The host cell may be either prokaryotic or eukaryotic. -
The invention further provides DNA molecules hybridizing to the
above DNA molecules and encoding ligands having the same
activity.
The invention also provides pharmaceutical compositions
coinprisirig the -above ligands which are useful for treating
diseases induced. or Caused -by, the effects of TNF, either
endogenously produced or exogenously administered.
BRIEF .~?ES~RI~T~C~N OF T~, ~G~RE~
Fic~t~,re 1 shows a diagrammatic illustration of the bacterial
constructs used for determining the sequence to which antibodies
of the 32 group bind.
Figure 2 shows an example of the Western blotting analysis
technique by which the binding of the antibodies to the
constructs shown in Figure 1 have been determined.
Figures ~ & 4 show the competition of synthetic peptides whose
sequences contain the region of the epitope recognized by the
monoclonal antibodies of the 32 group, or parts of it, with the
binding of an antibody of this group to a construct comprising
part of TBP-II in which this epitape is present. -
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Figure 5 shows the nucleotide and deduced amino acid sequences of
the p75 receptor. TBP-II and transmembrana:L domains are boxed and
shaded. The region recognized by the group 32 antibodies is
underlined.
Figure 6 shows the pattern of protection of HeLa p75.3 cells {as
hereinafter defined) from TNF cytotcrxicity by different
monoclonal antibodies against p75 TNF-R, and fragments thereof.
Figure 7 shows the effects of a monoclonal antibody against TBP-I
arid -several against TBP-II on the extent o:~ killing of U937 cells
by TNF.
Figures 8a and 8b show the effects of monoclonal antibody 70 and
Fab fragments thereof on the binding of T1~1F to HeLa p75.3 cells
and U937 cells, respectively.
Figure 9 shows a comparison of the effects of the antibody 32
with other antibodies against the p75 TNF-R on TNF binding to
HeLa p75.3 cells.
Figure 10 shows dissociation of TNF from HeLa p75.3 cells in the
presence and absence of antibody no. 70 and its monovalent Fab
fragment.
Figure 11 shows the nucleotide and deduced amino acid sequences
for the CDR region of the heavy chain:> of three monoclonal
antibodies of the 32 group.
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CA 02470670 2004-06-11
Figure 12 shows the nucleotide and deduced amino acid sequences
for the CDR region of the light chains of monoclonal antibody No.
32.
Figure 13 shows the sequence homology between several members of
the TNF/NGF receptor family.
DETAILED D~SCR:~'BTION OF TF~E INVENTION
TNF, as stated above, is a cytokine which initiates its effect on
cell ':function by binding to 'two specific cell ~ surface receptors
the 'p55 arid p75 receptors. Binding of antibodies to the
extracellular domain of these receptors can interfere with its
effect. However, as shown in a number of studies, antibodies
binding to the extracellular domain of the receptors can also
txigger 'the.effects of TNF by inducing aggregation of the p55
receptors, as well as by inducing aggregation of the p75
receptors. (Engeimann, et al: J. Biol. Chem., 'Vo. 265, No. 24,.
pp. 14497-14504, 1990; and unpublished data)
We have found that certain antibodies binding to one particular
region in the p75 receptor are not mimetic but rather inhibitory
to the signalling for the cytocidal effect by this receptor.
This, in spite of the fact that when binding .to this region,
these antibodies do not block-TNF binding, but rather increase it
to some extent.
The present invention reveals that this region recognized by
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these antibodies which we call the 32 group, is the region
extending between the two C-terminal cysteines in the
extracellular domain of the p75 receptor, plus an additional
amino acid, thr179. This region fvr simplicity's sake, is called.
"cysteine loop" throughout this specification.
The present invention also provides the nucleotide sequences and
deduced amino acid sequences in the CDR of the heavy chain of the
three antibodies belonging to this group, r.~amed 32; 57 and 70. A
remarkable similarity between the sequence of amino acids in the
CDR-of the heavy chain of the .32 and 70 antibodies was found,
indicating that the sequence of amino acids in the CDR of the
heavy chain ~of these two antibodies is. close to the optimum
necessary for binding to the antigen. In addition, the invention
also provides the nucleotide sequence and the deduced amino acid
sequence of the light 'chain. of antibody 32. Based on these
sequences, small molecular weight compounds, peptides or mimetic
compounds which will inhibit the function of the p75 receptors
cari be defined.
In evidence that such small compounds can indeed achieve this and
that there is no need for aggregation of receptors, which
antibodies are known to be able to do, it was found that also
Flab) monovalent fragments of the antibodies of the 32 group
inhibit signalling for toxicity by the p75 receptor when they are
triggered. by TNF.
In view of these findings, as well as the close similarity of the
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0
receptors in this particular family, this-invention relates also
to agents which bind to the C-terminal cysteine loop ~in the
extracellular domain of the various other members of the TNF/NGF
receptor family and modulate the function of the other receptors,
similarly to the modulation of the funcaion of TNF. In this
receptor family, the localization of cysteine in the extra-
cellular domain and the spacing is highly conserved. Certain
members of this family, e.g. CDw40, exhibit particularly high
similarity to the p75-receptor. Particularly in such receptors,
agents binding to these xegions are expected to have effects
similar to the effect of the 32 antibodies on the p75 receptor.
As stated above, the ligands according to the invention may
comprise proteins, peptides, immunoadhesins, antibodies or other
organic compounds.
Proteins may be isolated from cellular extracts, e.g, by ligand
affinity purification employing a molecule having an amino acid
sequence substantially corresponding to the above-mentioned
stretch as ligand.
Peptides may be prepared by synthetizin.g first target peptides
which correspond to the amino acid stretch of the TNF-R found in
accordance with the invention to bind the ligands which inhibit
the effects of TNF. Thereafter, peptide libraries axe screened
for other ligands which bind thereto. The peptides which bind to
these regions are further screened for those which also bind to
TNF-R. Finally, the peptides capably of high affinity binding
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CA 02470670 2004-06-11
with both the target peptides and the TNF-R, are screened for the
ability of the peptide to perform the desired biological
activity.
In a similar manner, a variety of organic molecules, including
drugs known for other indications, are screened for their
ability to bind to the amino acid stretch found to be critical
for inhibiting the effects of TNF.
In addition to the organic. molecules, also broth of biological
matter such as~~bacteria culture. products, fungi~culture products,
eukaryotic culture products and crude cytokirie preparations are
screened with the amino acid target peptides described above.
Molecules obtained by this screening are then further screened
for their ability to perform the desired bi.oiogical function.
Alternatively, molecules are designed which spatially fit the
quaternary structure of the amino acid stretch in the receptor.
The active molecules obtained by the above procedures, inasfar as
they are biological substances, can also be prepared by
biotechnological approaches. In this way, massive production of
these molecules will be made possible. Peptides may either be
pxoduced by known peptide synthesis methods or using expression
vectors containing DNA sequences encoding them. Other molecules,
if produced in an enzymatic way, can be made by producing the
enzymes involved in the appropriate cultured cells.
15.
CA 02470670 2004-06-11
Pharmaceutical compositions containing the .l.igands of the present
invention may be employed fox antagonizing the effects of TNF in
mammals.
Such compositions comprise the ligands according to the invention.
as their active ingredient. The pharmaceutical compositions are
indicated for conditions such as septic shock, cachexia, graft-
versus-host reactions, autoimmune diseases such as rheumatoid
arthritis, and the like. They are also indicated for counter-
acting e.g. an overdose of exogenously administered TNF.
The pharmaceutical compositions according to the invention are
administered depending on the condition to be treated, via the
accepted ways.of administration. For example, in the case of
septic shock, intravenous administration will be preferred. The
pharmaceutical compositions may also be administered
continuously, i.e. by way of infusion, or orally. The formulation
and dose will depend on.the condition to be treated, the route of
administration and the condition and th.e body weight of the
patient to be txeated. The exact dose will be determined by the
attending physician.
The pharmaceutical compositions according .to the invention are
prepared in the usual manner, for example by mixing the active
ingredient with pharmaceutically and physiologically acceptable
carriers and/or stabi3izers and/or excipients, as the case may
be, and are prepared in dosage form, e.g. by iyophilization in
dosage vials.
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CA 02470670 2004-06-11
f a
As used herein the teran "muteins" refers to analogs of the
proteins, peptides and the like in which.one or more of the amino
acid residues of the protein found to bind are replaced by
different amino acid residues or are deleted, or one or more
amino acid residues are added to the original sequence, without
changing considerably the activity of tl~e resulting product.
These muteins are prepared by known synthesis and/or by site-
directed mutagenesis techniques, or any other known technique
suitable therefor.
The term "fused protein" refers to a poly;?eptide comprising the
ligands or a mutein thereof fused with another protein which has
an extended residence time in body fluids" The ligands may thus
be fused to another protein, polypeptide or the like, e.g. an
immunoglobulin or a fragment thereof.
The term "salts" herein refers to both sa:Lts of carboxyl groups
and to acid addition salts of .amino graups of the ligands,
muteins and fused proteins thereof. Salts of a carboxyl group may
be formed by means known in the art and include inorganic salts,
for example, sodium, calcium, ammonium, ferric or zinc salts, and
the like, and salts with organic bases as those formed, for
example, with amines, such as, triethanalamine, arginine or
lysine, piperidine, procaine and the like. Acid addition salts
include, for example, salts with minera:L acids such as, for
example, hydrochloric acid or sulfuric acid, and salts with
organic acids such as, for example, acetic acid or oxalic acid.
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CA 02470670 2004-06-11
"Functional derivatives'° as used herein cover derivatives of the
ligands and their fused proteins and muteins, which may be
prepared from the functional groups which occur as side chains on
the residues or the N- or C- terminal groups, by means known in
the art, and are included in the invention as long as they remain
pharmaceutically acceptable, i.e. they do not destroy the
activity of the ligand and do not confer toxic properties vn
compositions containing it. These derivatives may, for example,
include polyethylene glycol side-chains wh_Lch may mask antigenic
sites and extend theresidence of the ligands in body fluids.
(7ther derivatives include aliphatic esters of the carboxyl
groups, amides of the carboxyl groups by reaction with ammonia or
with primary or secondary amines, N-acyl derivatives of free
amino groups of the amino acid residues formed with acyl moieties
(e. go alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives
of free hydroxyl groups (for example that of Beryl or threonyl
residues) formed with aryl moieties.
The invention is illustrated by the following non-limiting
examples:
EXAMPLE l: Determinat,~on of the_ region of 'the p75 receptor which
is recocrnized by the group ~2 antibodies
In example 5 of the main application, a number of constructs were
prepared by expression in E. coli, and it was concluded that
the epitope recognized by antibody no. 32 maps between amino
acids 125-182.
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CA 02470670 2004-06-11
We have now prepared further constructs and the complete list of
constructs examined, as well as their relationship to the ;
structure of the soluble p75R are shown in Fig. 1. Constructs
recognized by the antibodies of the' 32 group are listed in bold
numbers and illustrated as solid lines. Those not reacting. with
these antibodies are listed in thin numbers and illustrated by
broken lines. All constructs are identified by their N- and
C-terminal amino acid residues.
Figure 1_, above the diagrammatic illustration of the constructs,
show, the amino acid sequence of part of the p75 TNF-R, the
regions corresponding to the soluble form oaf the receptor and the
transmembranal region being boxed. Amino acid residues conserved
between man and mouse are underlined.
The Western blotting analysis. shown in. Figure 2, of the binding
of the groug 32 antibodies to some of the constructs shown in
Figure 1 was carried out as in Example 5 of the main application..
EXPZE 2L Competition fc~ bindina to the e~xtracellular domain of
th 7 T -R etwee ro a tib di s a d s t eti a id s
A number of synthetic peptides whose sequences correspond to
various parts of the region on the TNF-R suspected to be the
group 32 epitope were synthetized (residues 160-179, 162-179,
163-179, 165-179 and 157-179). The peptid~a were examined in an
ELISA test for their ability to compete for the binding to the
antibodies of the 32 group.
i
i
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CA 02470670 2004-06-11
A bacterially produced construct corresponding to amino acids 3
to 180 of the p75 TNF-R (p75 construct in P'ig. 3) was applied, at
the indicated concentrations, to PVC plates precoated with
antibody 32 followed by application of rabbit antiserum to TBP-II
(p75 soluble TNF-R). The amount of rabbit antiserum bound to the
plate was determined by applying goat antiserum against rabbit
immunoglobulin, coupled to horse-radish peroxidase and enzymatic
assessment of the amount of goat immunoglobulin bound to the
plate. Figure 3 shows the data of an experiment in which a
synthetic peptide corresporiding~to amino acid residues 163 to 179
was found to compete for the binding.
Figure 4 shows the data of an eXperiment in which a fusion
protein of maltose binding protein (MBP) with the sequence of
amino acids extending from 125 to 192 of the p75 receptor was
used to coat PVC plates at a concentration of 10~,g/mi, then the
No. 32 McAb was applied at a concentration of 2~,g/ml 'together
with the indicated concentrations of different peptides:
DW16 - amino acids 165-179
DW18 -armno acids 163-179
DW19 - amino acids 162-279
DW21 - amino acids 160-179
Thereafter, the reaction was developed by .adding goat anti-mouse
coupled to horseradish peroxidase. As shown in Fig. 4, marked
~.nhibition of fusion protein recognition by monoclonal antibody
No. 32 was observed only with the three peptides covering the
whole epitope.
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EXAMPLE ~: Mutational'study of the 32 epitone
Replacing cysteine 178 with alanine in a recombinant peptide
whose sequence corresponds to amino acids 3 to 181, made this
protein unrecognizable by the 32 group antibodies. This finding
suggests that in order to be recognized by these antibodies, the
two cysteines in the group 32 epitope region must be free to
interact with each other; i.e. that the structure recognized by
the antibodies is a loop. In. support of this notion, we found
that reduction of the peptide with dithiothreitho7: prior to SDS
PAGE and Western blotting analysis somewhat decreased the
effectivity of its recognition by the 32 group antibodies, and
reduction by dithiothreithol followed by alkylation with
iodoacetimide made it completely unrecognizable by the
antibodies.
EXAMPLE 4: Effects of various ,~ntiboc.~'te~ and fragments thereof on
TNF toxicity
{a) In order to compaxe the~funetion of the 32 group antibodies,
not only to.antibodies which bind to the.receptor upstream to
the 32 epitope region (as must of the anti-TBP-II antibodies
are expected to), but also to antibodies that bind to the
receptor downstream to that epitope region, we immunized mice
with a chimeric construct corresponding to the region
extending downstream to the 32 epitope {amino acids 181 to
235; the "stalk" region), linked to MBP. The rabbits
developed antibodies which bound to the chimera with which
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CA 02470670 2004-06-11
they were immunized as well as to the intact p55 TNF
receptor.. These antibodies were affinity purified by binding
*
to the chimeric protein, linked to an Affi-gel 10 column, and
tested for effect on TNF function and binding: {The affinity
purified antibody preparation was termed "318").
(b) All monoclonal anti-TBP-II antibodies as well as the affinity .
purified antistalk antibodies were tested for effect on TNF
toxicity in clones of, the epitheloid l3eLa cells which were
made to over-express the p75-receptors ~(by their transfection
Swith the p75 receptor's cDNA. We called the particular over
expressing clone used in the experiments presented here, HeLa
p75.3). The only antibodies found to inhibit TNF function
were the antibodies of the group 32 epitope; that; in spite
of the fact that they do not inhibit, but somewhat increase
TNF.binding to the receptor (Figs. S and 9). Two of the other
anti-TBP-II antibodies (No. 67 of Figs. 6 and 9 and number
81) had very little effect on TNF binding to the receptor or.
on TNF toxicity. All other monoclonal anti-TBP-II antibad~.es
somewhat potentiated the cytocidal effect of TNF .even though
competing with TNF binding {e.g. antibody 36 of Figs 6 and
9). The "anti-stalk" antibodies had very little effect on TNF
binding or function {Figs. 5 and 9). Applying the anti-stalk
antibodies on the cells together with antibodies of the 32
group did not interfere with the inhibitory effect of the
latter on TNF function.
(c} The same ganel of antibodies was tested for effect on the
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killing of the myelocytic U937 cells by TNF. As opposed to
the mimetic effect of anti-TNF receptor antibodies in the
IieLa cells, neither anti-p55 nor anti-p75 receptor antibodies
were found to be mimetic to the cytocidal effect of TNF on
the U937 cells under the. conditions of the experiment carried
out. Having no ability to mimic the effect of TNF, all
monoclonal antibodies which evmpete for TNF binding either to
the p75 receptor, {e.g. antibodies 14, 31 and 36 of Figure 9)
or to the p55 receptor (e.g. antibody number 18 of Figure 7)
are inhibitory to the TNF effects. Antibodies which had no
effect on TNF binding to the receptors {e.g. number 67 of
Figure 9) had no effect on TNF function (Figure 6). The 32
group antibodies were unique in having an ability to inhibit
TNF function in this cell without having any inhibitory
effect on TNF binding. The antibodies actually enhanced the
binding of TNF to these, cells, much more so than in the HeLa
p75:3 cells (Figure 8). The inhibitory effect of the 32 group
antibodies was additive to that of antilbodies which block TNF
binding to the p55 receptor (e.g. the combination 18/32 in
Figure . 7 ) .
F~ArmLE 5 ~ Effect of crroup, ~~2 antibodies and Fab monav 1~ ant
fracrments thereof on t~c~,issociati~n of TNF from the T~tF-Rs
In order to explore the .mechanism by which the 32 group
antibodies cause an indrease in TNF binding, we compared the xate
of TNF dissociation from HeLa p75.3 tails in the presence and
absence of these antibodies.
23
CA 02470670 2004-06-11
Radiolabelled TNF was added to confluent HeLa p75.3 cells and the
cells were incubated for 2 hr on ice . Unbound ligand was washed
away and l ml of binding buffer containg 500 ng/ml of cold TNF
was applied into quadruplicate wells for the indicated periods of
time on ice. Thereafter, the wells were washed once again with
cold PBS, and amount of residual li.gand was determined by
measuring radioactivity of cells detached from. plates by
incubation with PBS/EDTA solution. The antibodies were applied
throughout the assay at a concentration of l.Uwg/ml.
As illustrated in Fig. 10, both these antibodies as well as their
F(ab) monovalent fragments caused a decrease in the rate of TNF
dissociation from the receptors. Besides providing a possible
explanation for the way in which these antibodies affect TNF
binding to its receptors, this finding indicated an additional
application for this effect. Salable forms of the p75 TNT-Rs or
of the p55 receptor or of any other member of the TNF/BGF.
' receptor fami.iy in which a conformational change as that imposed
by the 32 group antibody will occur, will serve as better
inhibitors of the respective agonist.
E L 6: Dete t'o o nuc eo i a se ces an de uc d
amino acid seciuences in the CDR of the heavy chains of monoclor.~
antibodies32. 57 and~Q~~rou~?3? anti died and in the CDR of
the light tKappa~ chain of antibody 32
In order to determine the nucleotide sequences of the CDR of the
heavy chains of antibodies 32, 57 and 70, total RNA was isolated
24
CA 02470670 2004-06-11
by the Promega protocol from the respective h.ybridoma cells, with
the use of guanidinium thioisocyanate. First strand cDNA
synthesis on this RNA was performed with the use of AMV reverse
transcriptase and eithex~oligo(dT)1.5-18 or an oligonucleotide
complementary to the constant region of the heavy chain of murine
IgG as a primer. The cDNA was used as a template for PCR,
applying a partially degenerate 5'-Primer. 40 cycles of PCR were
carried out. PCR products with the size of about 350 by were
purified electrophoretical:ly and, cloned into the. Bluescript*
vector. Clones having inserts of the right size were sequenced.
Double-stranded cDNA .of the CDR region of'the light chain of
antibody no. 32 was synthesized in a similar ~c~ranner.
The nucleotide sequences obtained by i~he dideoxy chain
termination method, and the amino acid sequences deduced
therefrom are shown in Figures 11 and 12. The CDR1, 2 and 3
regions are underlined. w
EXAMPLE 7: Preparationof scFv of_~he 3~c~rouo antibodies
The cloned variable regions of the heavy and light chains of the
monoclonal antibodies of the 32 group are linked with a linker of
15 amino acid length and introduced into a commercial expression
vector. The vector contains a promoter, e.g. lac, a leader
sequence e.g. pel-B, as well as a sequence encoding a small
peptide ("tag" peptide) against crhich a .monoclonal antibody is
commercially available. The plasmid is nowintroduced into E.
coli and the bacteria are grown to O.D: 0.5-1Ø Expression of
scFv is induced by addition of IPTG and growth is continued for
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CA 02470670 2004-06-11
another 6-24 hrs. The soluble scFv-tag complex is then isolated
from the culture medium by immunoaffinity purification using the
monoclonal antibody against the tag and then purified on a
metaloaffinity column.
Any scFv accumulating within the bacteria is purified by
isolating and repeatedly washing the inclusion bodies, followed
by solublization by e.g. urea or guanidinium and subsequent
renaturation.
Alternative possibilities are employing an oligohistidine as the
tag, using a stronger promoter instead of lac, i.e. T7,
constructing the vector without the leader sequence or
introducing a sequence encoding a "tail" of irrelevant sequences
into the vector at the 5' end of the scFv. This "tail" should not
be biologically active, since its only purpose is the creation of
a longer molecule than the native scFv, thus causing a longer
retention time in the body.
EXA~~E 8: .
Figure 13 shows the internal cysteine rich repeats in the
extracellular domains of the two TNF-Rs and their alignment with
the homologous repeats in the extracellular domain of the human
FAS, nerve growth factor receptor (NGF) anal CDw40, as well as rat
Ox40. The amino acid sequences (one letter symbols) are aligned
for maximal homology. The positions of the amino acids within
the receptors are denoted in the left hand margin.
26
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E 9s r at'on f ec 'n nt D A of a es co r's'
a ot'de s a es od' or t a dive a t'de d th
nnolecules and their expression
The peptides and other molecules can also be prepared by genetic
engineering techniques and their preparation encompasses all the
tools used in these techniques. Thus DNA mo7:ecules are provided
which comprise the nucleotide sequence coding for such peptides
and other biological molecules. These DNA molecules can be
genomic DNA, cDNA, synthetic DNA and a combination thereof.
Creation of DNA molecules coding for such peptides and molecules
is carried out by conventional means, once the amino acid
sequence of these peptides and other molecules has been
determined.
Expression of the recombinant proteins can be effected in
eukaryotic cells, bacteria or yeasts, using the appropriate
expression vectors. Any method known .in the art may be.
employed.
For example, the DNA molecules coding for the peptides or other
molecules obtained by the above methods are inserted into.
appropriately constructed expression vectors by techniques well
known in the art (see Maniatis, T. et al., Molecular Cloninq: A
Laboratory Manual, Cold .Spring Harbor Labozatory, Cold Spring
Harbor (1982)). Double-.stranded cDNA is 3.i.nked to plasmid vectors
by homopalymeric tailing or by restriction .inking involving the
use of synthetic DNA linkers or blunt-ended ligation techniques.
27
CA 02470670 2004-06-11
DNA ligases are used to ligate the DNA molecules and undesirable
joining .is avoided by treatment with alkaline phosphatase.
In order to be capable of expressing a desired biological
substance, i.e. a peptide or protein (hereinafter "protein~', for
simplicity's sake), an expression vector should comprise also
specific nucleotide sequences containing transcriptional and
translational regulatory information linked to the DNA coding for
the desired protein in such a way as to permit gene expression
and production of the protein. First, in order for the gene to be
transcribed, it must be ;preceded by a promoter recognizable by
RNA polymerase, to Which the polymerase binds and thus initiates
the transcription process. There are a variety of such promoters
in use, which work with different efficiencies (strong and weak
promoters). They are different for prokaryotic and eukaryotic
cells.
The promoters that can be used in the present invention may be,
', either constitutive, for example, the i.nt promoter of.
bacteriophage lambda, the bla promoter of i:he ~-lactamase gene of
pBR322, and the GAT promoter of the chloramphenicol acetyl
transferase gene of pPR325, etc., or iiaducible, such as the
prokaryotic promoters including the major right and left
promoters of bacteriophage lambda (P~ and PA), the trp, ~ecA,
lacZ, cI, ompF_ and c,~,al promoters of E;, coli, or the trp,-lac
hybrid promoter, etc. (click, B.R. (1,3$7) J.Ind.Microbiol.,
1_:277-282).
28
CA 02470670 2004-06-11
Besides the use of strong promoters to generate large quantities
of mRNA, in order to achieve high levels of gene expression in
prokaryotic cells, it is necessary to use also ribosome-binding
sites to ensure that the mRNA is efficiently translated. One
example is the Shine-Dalgarno (SD) sequence appropriately
positioned from the initiation codon and complementary to the
3'-terminal sequence of 16S RNA.
Far eukaryotic hosts, different transcriptional and translati.onal
regulatory sequences may be employed, depending on the nature of
the host. They may be derived from viral sources,, such as
adenovirus, bovine papilloma virus, Simian virus, or the like,
where the regulatory signals are associated with a particular
gene which has a high level of expression. Examples are the TIt
promoter of Herpes virus, the SV40 early promoter, the yeast gal4
gene promoter, etc. Transcriptional initiation regulatory signals
may be selected which allow for repression and activation, so
that expression of the genes can be modulated.
The DNA molecule comprising the nucleotide sequence coding for
the peptides or other molecules of the invention and the operably
linked transcriptional and translational regulatory signals is
inserted into a vector Which is capable of integrating the
desired gene sequences into the host cell chromosome. The cells
which have stably integrated the introduced DNA into their
chromosomes can be selected by also Lntroducing one or more
markers which allow for selection of host cells which contain the
expression vector. The marker may provide for prototrophy to an
29
CA 02470670 2004-06-11
a
auxotropic host, biocide resistance, e.g., antibiotics, or heavy
metals, such as copper, or the like. The selectable marker gene
can either be directly linked to the DNA gene sequences to be
expressed, or introduced into the same cell by co-transfecti.on.
Additional elements may also be needed for optimal synthesis of
single chain binding protein mRNA. These elements may include
splice signals, as well as transcription promoters, enhancers,
and termination signals. cDNA expression vectors incorporating
such elements include those described by Ok:ayama, H., (1983) Mol.
Cell Biol., 3:280.
In a preferred embodiment, the introduced DNA molecule will be
incorporated into a plasmid or viral vector capable of autonomous
replication in the recipient. host. Factors of importance in
selecting a particular plasmid or viral vector include: the ease
with which recipient cells that contain the vector may be
recognized and selected from those recipient cells Which' do not
contain the vector; the number of copies of the vector which are
desired in a particular host; and whether it is desirable to be
able to "shuttle" the vector between host cells of different
species.
Preferred prokaryotic vectors include plasmids such as those
capable of replication in E. coli, for example, pBR322, ColEl,
pSC301, pACyC 184, etc. (see Maniatis et al., (1982) op. cit.);
Bacillus plasmids such as pC194, pC221, pT1''?, etc. (Gryczan, T.,
the Molecular Bioloav of the Bacilli., Academic Press, NY (1982j);
Streptomyces plasmids including pIJ101 (K~endall, R.J. et al.,
CA 02470670 2004-06-11
Fk
(1987) J.Bacteriol. ,~G~:4177-83},; Streptomyces bacteriophages
such as ~C31 (Chater,- R.F. et al., in: Sixth I~t~,~nat~nal
;~rmnosium on Actinomycetales l~ioloav, {1986)), and Pseudomonas
plasmids (John, J.F., et al. (1986} Rev. Infect.Dis. $:693-704;
and Izaki, R. (1978} Jpn. J. Bacteriol., x:729-742}.
Preferred eukaryotic plasmids include BPV, vaccinia, SV40,
2-micron circle, etc., or their derivatives. Such plasmids are
well known in the art (Botstei.n, D., et a:L. (1982) Miami Wint.
Symp..,l~, pp. 265-274; Broach, J.R., in: T a Molecular Biology of
the Yeast Saccharomxces~ Life Cycle and Inheritance, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-470 (1981);
Broach, J.R., (1982} Cell, x$:203-204; Hollon, D.P., et al.
(1980) J. Clin. Hematol. Oncol., ,0:39-48; Maniatis, T., in:_Cell
~oloqy~ A Co~ngrehensive treatise. Vol. 3: Gene Expression,
Academic Press, NY, pp. 563-608 {1980)).
Once the vector or DNA sequence containing the constructs) has
been prepared for. : expression, the DNA construct { s } may be
introduced into an appropriate host cell by any of a variety
of suitable means: transformation, transiection, conjugation,
protoplast fusion, electroporation, calcium phosphate-
p~ecipitation, direct microinjection, etc.
Host cells to be used in this invention may be either prokaryotic
or eukaryotic. Preferred prokaryotic hosts include bacteria such
as E. cgli, Bacillus, Streptomyces, Pseudomonas, Salmonella,
Serratia, etc. The most preferred prokaryotic host is E. coli.
31
CA 02470670 2004-06-11
w
Bacterial hosts of particular interest include E. coli K12 strain
294 (ATCC 31446), E~ o~li X1776 (ATCC 31537), E. coli W3110 {F',
lambda , prototropic {ATCC 27325)), and other enterobacterium
such as Salmonella typhimurium or Serratia marcescens and various
Pseudomonas sgecies. Under such conditions; the protein will not
be glycosylated. The prokaryotic host must. be compata.ble with the
replicon and -control sequences in the expression plasmid.
Preferred eukaryotic hosts are mammalian cells; e.g. human,
monkey, mouse and Chinese hamster ovary (CT30) cells, because they
provide post-translational modifications to protein molecules
including correct folding or glycosylation at correct sites. Also
yeast cells can carry out post-translational peptide
modifications including giycosylation. A number of recombinant
DNA strategies exist which utilize strong gromoter sequences and
high copy number of plasmids which can be utilized for production
of the desired proteins in yeast. Yeast recognizes leader
sequences on cloned mammalian gene products and secretes peptides
bearing leader sequences (i.e. pre-peptides).
After.the.introduction of the vector, the host cells are grown in
a selective medium, which selects for the growth of veetor-
containing cells. Expression of the cloned gene sequences)
results in the production of the desired proteins.
Purification of the recombinant proteins is carried.out by any
one of the methods known for this purpose.
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CA 02470670 2004-06-11
Deposit Information
Hybridoma TBP-II 70-2 was deposited with the Collection National
de Cultures de Microorganismes; Institut Pasteur (CNCM? on
March 12,1990 and assigned No. I-928.
Hybridoma TBP-11. 32-5 was deposited with the CNCM on 1 Sept. 1993
and assigned No. I-1358.
33
