Note: Descriptions are shown in the official language in which they were submitted.
WO91/04745PCT/US90/05105
~67 ~83
COMPOSITIONS FOR CELL ADHESION IN~}8ITION -
AND METHODS OF USE :
This is a continuation-in-part of United States
Serial No. 07/413,332, filed September 27, 1989.
:',; '
5~acXaround o~ the I~vention
Field of the Inventlon
This invention relates to compositions that
transiently and reversibly dissociate the blood-brain
barrier. More particularly, the invention relates to
compositions that dissociate tight junctions between
brain capillary endothelial cells that constitute the
physiological barrier between the general circulation
and the brain.
et~led Doscri~tion of Related Art
15The entry of drugs from the blood stream to the
central nervous system (CNS), i.e., the brain and
spinal cord, is restricted by the presence of high
resistance tight ~unctions between brain capillary
cells and by the apparently low rate of transport
across these endothelial cells (Betz, A.L., et al.,
Ann. Rev. Phvsiol., 48:241 (1986); Pardridge, W.M.,
An~. Rev. Pharmacol. Toxicol., 28:25 (1988)).
The tight junctions of the blood brain barrier
(BBB) prevent diffusion of molecules and ions around
the brain capillary endothelial cells. The only
-~stances that can read.ly pass from the luminal core
o. the capillary to the ablumi- l tissues that surround
; the capillary are those molecuies for which selective
transport systems exist in the endothelial cells, as
well as those compounds that are lipophilic (i.e.,
; hydrophobic). In contrast, drugs, peptides and other
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WO91/04745 PCT/US90/05105
~oO3
molecules that are neither lipophilic nor transported
by specific carrier proteins are barred from entry into ~-
the brain, or their rates of entry are too low to be
useful, thereby imposing a severe limitation upon the ~ -
physician's ability to treat CNS disorders
pharmacologically.
The carrier-mediated transcellular transport
system mentioned above may have limited usefulness for
therapeutic modalities under some circumstances.
10 Transcytotic transport, in general, involves, first, -
the binding of molecules to specific carrier proteins ;
on the surface of endothelial cells, and, second, the
delivery of such molecules across the endothelial
cells. Limitations on the usefulness of such a system
for treatment of CNS disorders are based on the
following considerations: (1) physiological carrier
proteins may not function efficiently, or at all, with
non-physiological drugs; (2) even where function
occurs, the rate of transport of therapeutic agents
will be limited by the rate of transport of the
carrier; (3) the overall capacity of cerebral capillary
endothelial cells to transport any therapeutic
macromolecules may be simply too low to achieve
therapeutic levels of certain drugs in the brain; and
(4) once therapeutic macromolecules enter endothelial
cells, depending on their nature, they might be
delivered to any number of organelles, including
lysosomes that contain a wide variety of hydrolytic
enzymes. For these reasons, creating drug delivery
systems that do not rely upon transcytosis will clearly
be advantageous.
As tight junctions between brain capillary
endothelial cells constitute a major part of the BBB, j ~-
the possibility of modifying these junctions has been
considered. It has been found that tight junctions,
;':~" '
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W091/04745 PCT/US90/051~5
3 2067183 :-
including those of the BBB, can be disrupted by
hyperosmotic solutions administered intra-arterially.
For example, Polley et al., W089/04663, published
June l, 1989, disclose the osmotic disruption of the
interendothelial structure of the BBB by the intra-
arterial administration of hypertonic solutions of
mannitol, arabinose or glycerol as a means of
introducing into the brain genetic material.
Similarly, hyperosmotic solutions of urea have also
been used to alter the BBB (Bowman, P.D. et al., Ped.
Res., 16:335A (1982)).
Other chemical agents have been reported to
disrupt endothelial or epithelial cell tight junctions
when administered intravenously, including:
7-fluorouracil (MacDonell, L.A., et al., Cancer. Res.,
38:2930 (1978)), degradation by membrane enzymes
(Vincent, P.A., et al., Exp. Mol. Path., 48:403 (1988);
Diener, H.M., et al., J. Immunol., 13S:537 (1985)),
aluminum salts (Zigler, Z.Y., et al., IRCS Med. Sci.,
20 12:1095 (1984)), histamine (Meyrick, B., et al., ~a~ -
Luna Res., 6:11 (1984)), thrombin (Siflinger-Birnboin,
A., et al., Microvasc. Res., 36:216 (1988)), phorbol
esters (Shiba, K., et ~1., Ex~. Cell Res., 178:233
(1988)), and neutralization of the luminal anionic
charge (Hart, M.M., J. Neuro~athol. EXD . Neurol.,
46:141 (1987)). ~ -
Although the above-listed modalities may disrupt ;~
tight junctions and thereby increase permeability of
the B8B, problems attendant upon their use make them
less than desireable. For example, intra-arterial
perfusion with hyperosmotic solutions involves surgery,
and this cannot be repeated on a regular basis.
Further, concentrated sugar solutions may not be
innocuous, and might be expected to have undesirable
side effects. In addition, the aforementioned chemical
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WO 91/04745 ~6~ ~3 PCT/US90/0~10~
agents may not be useful for the treatment of chronic
neurological disease, their effects on tight junctions
are not always reversible, and, as they all are
themselves powerful drugs, there is always the danger
that their use will compromise the patient's health
generally. For example, 7-fluorouracil is a powerful
inhibitor of pyrimidine synthesis, and thus nucleic
acid biosynthesis, in animals cells.
Thus, an important need still exists for means
which transiently and reversibly disrupt tight
junctions of the BBB in order that administered drugs
can reach the brain from the general circulation, and
which have no undesirable side effects of their own in
the subject.
Attem~ts have been made to disrupt cell-cell ~ -
adhesion by modifying the protein(s) responsible for
such adhesion, collectively referred to as "cell
adhesion molecules" (CAM). One class of CAM is termed
"cadherin". "Cadherin" is the term applied to a family
20 of glycoproteins found in most kinds of mammalian ~
tissues and thought to be responsible for Ca2~- -
dependent cell-cell adhesion, (Takeichi, M.,
Development, 102:639 (1988)). Three subclasses of
cadherin have been identified, namely, E-cadherin (from
epithelial tissues), P-cadherin (from placental
tissues), and N-cadherin (from neural tissues)
(Yoshida-Noro, C., et al. Dev. Biol., 101:19 (1984);
Nose, A., et al., J. Cell Biol., 103:2649 (1986);
Hatta, K., et al., Nature, 320:447 (1986)).
The different cadherins exhibit distinct tissue
distribution patterns (Takeichi, U., (1988) above).
E-cadherin, which was found to be distributed
exclusively in epithelial cells of various tissues
(Hatta, K., et al., Proc. Nat'l. Acad. Sci. (USAl,
82:2789 (1985); Takeichi, 1988, above), appears to be -
.. ... . ~ .. .... .. . . ...... . . . . . .. . ... . . . .. . .. .. . . ... .
.
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WO91/04745 PCT/US90/05105
5 2067183 ~ :~
identical to uvomorulin (Hyafil, F., et al., Cell,
21:927 (1986)), chicken liver-cell adhesion molecule
(L-CAM, Gallin, W.J., et al., Proc. Nat. Acad. Sci.
(USA~, 80:1038 (1983)), and cell-CAM 120/80 (Damsky,
C.H., et al., Cell, 34:455 (1983)) in terms of
biochemical properties (Cunningham, B.A., et al., Proc.
Nat. Acad. Sci. (USA), 81:5787 (1984)) and tissue
distributions (Thiery, J.-P., et al., Dev. Biol.,
102:61 (1984)).
N-cadherin, which is expressed in various neural
tissues including astrocytes (Hatta, K., et al., Devel.
Biol., 120:215 (1987); Matsunega, M., et al., Nature,
334:62 (1988); Tomaselli, X.J., Neuron, 1:33 (1988)), `
shows 92% amino acid sequence homology between
15 mammalian and avian homologs, shows from 40 to 50% -;~
similarity to epithelial E-cadherin and to placental
P-cadherin of the same species, but was immunologically
not cross-reactive with other cadherins within the same -~
animal ~Miyatani, S., Science, 245:631 (1989)).
Placental P-cadherin has also been cloned, and the
deduced amino acid sequence of this glycoprotein was
found to exhibit about 58~ homology with epithelial
E-cadherin ~Nose, A., et ~1., EM~O J,, 12:3655 (1987)).
Subsequent to the September 27, 1989 filing of the
parent application, Heimark, et al. (Heimark, R.L., et
al., J. Cell Biol., 110:1745 (1990) reported on the
identification of a Ca2~-dependent cell-cell adhesion
molecule in aortic endothelial cells.
Although each of the aforelisted cadherins
displays unique immunological and tissue distribution
specifications, all have features in common: (1) a
requirement for Ca2~ for cell adhesion function; (2)
protection by Ca2~ from proteolytic cleavage; (3)
similar numbers of amino acids, i.e., from about 723 to
about 822; (4) similar masses, i.e., about 124 kdal.
: . , . , ~,, ,,, , . . ' . ' ' ' '' ' "' " ' " " '" ' ' , ' ,: ' ' .: ' ' ' '
WO91/04745 ~ PCT/US90/05105
: 6
for the glycoprotein; (5) substantial interspecies
(S0%-60%) overall sequence homology with interspecies
homologies increasing to about 56% to 99% in the
cytoplasmic region of the protein, suggesting that they
constitute a gene family (Nose, A., 1987; Miysysni, D.,
et al., 1989); and (6) a common mechanism o~ action,
namely, homophilic binding of cadherins on one cell to
similar cadherins on the adjoining cell.
CAMs independent of Ca2~ are also known, for
example, the 125K glycoprotein of Urushihara et al.
(Urushihara, H., et al., Cell, 20:363 (1980)); N-C~M
(Rutishauser, U., Nature. Lond., 310:549 (1984));
Ng-CAN (Grunet, M. et al., Proc. Nat'l. Acad. Sci. ;~
(VSA), 81:7989 (1984)); Ll (Rathjien, F.G. et al.,~
J., 3:1 (1984)); G4 (Rathjien, F.G. et al., J. Cell
Biol., 104:343 (1987)); and platelet glycoprotein `
PECAM-1 (CD 31) (Newman, P.J., Science, 247:1219
(1990)). Ca2t-independent CAMs are known to exhibit
certain properties of the Ca2'-dependent CAMs. Thus, 1!"~
N-CAM and N-cadherin both promote retinal neurite
outgrowth on astrocytes (Neugebauer, R.M., et al., J.
Cell Biol., 107:1}77 (1985)), and on Schwann cells
~Bixby, J.L. et 81-, ~ Cell Biol., 107:353 (1988)).
Monoclonal antibodies raised against epithelial
E-type cadherins such as uvomorulin are known to
disrupt the adhesion O r several cell types, including -
embryo cells, cultured teratocarcinoma cells, ~ ~
- hepatocytes, and MDCK kidney epithelial cells (Ogou, ~ -
S.-I., et al., J. Cell Biol., 97:944 (1983); Yoshida-
Noro, et al., (1984), above: Shirayoshi, Y., et al.,
Cell Struct. Funct., 11:285 (1986); Gallin, et al.,
(1983), above; Vestweber, D., et al., ENBO`J., 4:3393
`~ (1985); Johnson, M.H., et al., J. Embrol. Exp.
Mor~hol., 93:239 (1986); Gumbiner, B., et al., J. Cell .
Biol., 102:457 (1986)).
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WO91/04745 PCT/US90/05105
20~7183
However, prior to the present discoveries
disclosed in the parent applications cadherins had not
been found in brain capillary or other endothelial
cells (see, Takeichi, et al. (1988), above). Further,
the CAMs of microvascular endothelial cells had not yet
been identified, nor had such molecules been localized
specifically to brain capillary endothelial cells.
Thus, until the present invention no means were known
for transiently and reversibly disrupting tight
junctions between microvascular endothelial cells,
including those of the BBB, based upon ~ attack upon
the CAM's of such cells that are responsible for tight
junction formation and maintenance.
It has been hypothesized that the cadherins
contain a common cell adhes-3n recognition (CAR)
sequence. The CAR sequences of several cell and
substratum adhesion molecules are known. Martin, G.R.,
et al., Ann. Rev. Cell Biol., 3:57 (1987) ; Ruoslahti,
E., et al., ~çience, 238:491 (1987). In general, CAR
sequences are composed of at least three amino acid
residues. The most rigorously investigated CAR
sequence is RGD which is found in laminin, fribronectin
and other basement membrane components that are
responsible ~or the binding of cells to the substratum.
Blaschuk, et al., in a paper to be published
subsequent to the filing of the present application
~Blaschuk, 0., et al., J. Mol. Biol., in press,
(1990)), disclose the presence of three potential
cadherin CAR sequences in the first extracellular
domains of liver CAM, E-, P-, and N-cadherin, namely,
PPI, GAD and HAV. Blaschuk, et al. (Blaschuk, O., et
al., Develop. Biol., 139:227 (1990)), also disclosed
recently that synthetic peptides containing the HAV
sequence inhibited two biological processes (compaction
of 8-cell-stage mouse embryos and rate of neurite
W09l/04745 ~ PCr/US90/~SI~S
outgrowth on astrocytes) that are known to be mediated
by cadherins. Effective peptides in these assays were --~
LRAHAVDVNG and AHAVSE; PPI-containing peptides were
without effect. However, Blaschuk et al. provide no
guidance for determining the regions flanking the HAV
tripeptide that are critical for cell-cell adhesion.
In the BBB disrupting peptides of the present invention
detailed below, we have observed that the mere presence
of the HAV sequence in a small cadherin-derived peptide
is not the sine g~3 non for a composition effective to
prevent cell-cell adhesion. Indeed, it should be
emphasized that neither Blaschuk et al. nor any other
publication known to the present inventors suggest that
cadherin sequences containing HAV or SHAVS sequences
would be effective in opening tight junctions and
piercing blood brain barriers formed by E-cadherins in
brain microvascular endothelial cells.
'
8UMMARY OF THE INVENT~ON
It has now been discovered that molecules
homologous to, and immunologically related to, cadherin
cell adhesion molecules are present on brain and non-
brain microvascular endothelial cells, such that
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WO91/04745 i PCT/US90/05105
2067 ~83
g . ~
junctions between such endothelial cells can be
reversibly opened so as to permit passage of
therapeutic drugs by the use of polypeptide and
antibody compositions that compete with such cell
adhesion molecules for binding to such cells.
It is therefore an object of this invention to
provide the identity of microvascular endothelial cell
adhesion molecules.
Another ob;ect of this invention is to provide DNA
sequences of genes, and plasmids containing same,
coding for the expression of all or a cell-binding
portion of microvascular endothelial cell adhesion
molecules.
Yet another object of this invention isito provide
means to identify those sequences of cell adhesion
molecules responsible for the tight binding of
adjoining endothelial cells.
A further object is to provide therapeutic
compositions comprising polypeptides derived from cell
adhesion molecules that reversibly disrupt cell-cell
adhesion.
Stlll another object o~ this invention is to
provide therapeutic compositions comprising polyclonal ~;
or monoclonal antibodies or fragments thereof directed
against endothelial cell adhesion molecules, or against
polypeptides representing cell binding regions thereof,
that reversibly disrupt endothelial cell-cell adhesion.
Yet another object of this invention is to provide
therapeutic formulations comprising therapeutic drugs
conjugated with blood-brain barrier-disrupting
compositions of this invention, that are capable of
entering the central nervous system following
~ disruption of the blood-brain barrier.
;~ These and other objects of this invention will
35 become clear by reference to the following description ~ -
':
.
WO91/04745 ,1,Q,6~3 Pcr/usso/uslns
of the invention and to the appended claims. -
DESCRIPTION OF THE DRAWINGS
Figure l-illustrates the partial cDNA sequence for
bovine endothelial cell adhesion molecule homologous to
chicken N-cadherin.
Figure 2 illustrates the partial cDNA sequence for
bovine endothelial cell adhesion molecule homologous to
mouse P-cadherin.
Figure 3 illustrates the cDNA sequence for the
MDCK cell adhesion molecule homologous to mouse
E-cadherin.
Figure 4 illustrates the restriction sites in the
bovine endothelial cell N- (4-1 to 4-5) and P-cadherin
(4-6 to 4-8) cDNA sequences and in the MDCX E-cadherin
(4-9 to 4-14) cDNA sequence.
Figure S shows the staining of a mouse brain thin
section by an antibody raised against a fusion protein
derived from amino acids 9-96 of MDCK E-cadherin
containing an HAV region.
Figure 6 is a repeat of the experiment of ~ig. 5,
except that the antibody was raised against the entire
E-cadherin molecule.
Figure 7 illustrates the effects of an 18-mer HAV~
containing polypeptide on the resistance of tight ~;
junction monolayers of MDCK epithelial cells.
Figure-8 illustrates the effects of 11-mer and
18-mer HAV-containing polypeptides on the resistance of
tight junction monolayers MDCK epithelial cells.
Figure 9 illustrates the effects of 11-mer and 18-
mer HAV-containing polypeptides on the resistance of
tight-junction monolayers of brain microvascular ~ -
endothelial cells.
,
.
WO91/04745 PCT/US90/05105
- 206718~ ~ ~
11
DETAILED DESCRIPTION OF THE INVENTION
It has now been discovered that cell adhesio~
molecules with characteristics of cadherins are present
on the surfaces of brain capillary endothelial cells -
and of microvascular endothelial cells of non-brain -
origins. The present invention is based on the
discovery that a polypeptide composition comprising
cell binding domains of endothelial cell adhesion
molecules may compete against such molecules for
binding to such cells, such that by this means the
junctions between such cells could be reversibly
opened, thereby permitting penetration by therapeutic
agents. The present invention also discloses that
polyc onal or monoclonal antibodies (or fragments
thereof) raised against endothelial cell adhesion
molecules or cell-binding domains thereof may also
compete for endothelial cell surface binding sites,
and, by this means, reversibly disrupt junctions
between endothelial cells, thereby permitting entry
into the central nervous system of therapeutic agents.
In order to obtain compositions useful for
disrupting tight junctions betw~en mlcrovascular
endothelial cells, the cell adhesion molecules
responsible for such junctions were identified.
The endothelial cell cadherins disclosed herein
exhibit one or more of several characteristics of E-,
P- and N- cadherins, incIuding: characteristics of a
transmembrane integral protein, with cytoplasmic,
hydrophobic plasma membrane, and extracellular regions;
intraspecies DNA sequence homologies of greater than
about 50% for the entire molecule; immunological cross-
reactivity with antibodies raised against non-
endothelial cell cadherins; and containing cell-binding
~- domains. "Immunologically related to" means that these
cadherin-like molecules cross-react with antibodies
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WO91/04745 PCT/US90/05105
~Q6~3 12 ~;~
raised against non-endothelial cell cadherins.
E-cadherin-like molecules were localized in brain
by immunofluorescence. Cryostat sections of mouse
brain were labeled with a rabbit antibody prepared
against E-cadherin, and then with fluorescein
isothiocyanate-conjugated goat anti-rabbit
immunoglobulin. There is clear labeling of a capillary
in brain sections as shown by immunofluorescence
microscopy. Endothelial cells in liver and kidney were
not stained by this procedure.
cDNAs coding for the expression of bovine
microvascular endothelial cell (BMEC) cadherins were
cloned and sequenced as described below, and the
partial sequence of N-cadherin and P-cadherin are
disclosed herein in Figures l and 2, respectively. In
addition, as MDCK dog kidney epithelial cells are known -
to employ E-cadherin to form high resistance tight
junctions, and as the present invention discloses that ;~
brain capillary endothelial cell adhesion molecules i
include E-type cadherin, the DNA of this cadherin was
also cl~ned; its complete DNA seguence is disclosed
herein (Fig. 3).
N-, P- and E-cadherin-type clones described herein
were deposlted in the American Type Culture Collection
on September 26, 1989, and were assigned the following
accession numbers:
W091/04745 PCTtUS90/05}05
- ~ 20671~ -
13 ~ -
Clone Designation Accession No.
N-cadherin-type clones
pUCl9-bNCad lOA 40667
pUC19-bNCad 39A 40669
P-cadherin-type clones
pUC18-bPCad 3B-10 40668
pUC19-bPCad 9B 40670
E-cadherin-type clones
pBluescript MDCKECad 45-3OE 40671
The cloning of cadherins was accomplished by
taking advantage of the fact that the cadherins
characterized thus far are transmembrane glycoproteins,
the cytoplasmic domains of which are highly conserved,
that is, are highly homologous.
Two degenerate oligonucleotides flanking the
42-amino acid coding region in the cytoplasmic domain
were selected to serve as primers for polymerase chain
reaction ~PCR) using either BMEC cDNA or MDCK cDNA as
templates. The PCR reactions were carried out
essentially according to Saiki, R. K. et al., Science,
239:487 (1988), which is incorporated herein by
rererence.
The cloned PCR products ~rom each cell type were
sequenced essentially according to the method of
Sanger, F. et al., Proc. Nat'l. Acad. Sci. (USA),
74:5463 (1977), which is incorporated herein by ~ -
reference.
It was discovered that BMEC cadherins are of two ~
types - one homologous to chicken N-cadherin (neuronal : -
type, see, e.g., Hatta, K., et al., J. Cell Biol.,
106:873 (1988)) and the other homologous to mouse ~ -
P-cadherin (placental type, see e.g., Nose, A., et al.,
(1987) above). It has also been found that there are
two species of cadherins ln MDCK cells - one homologous
.. .. . . .
W09l/04745 PCT/US90/05105
to mouse E-cadherin (see, e.g., Nagafuchi, A., et al.,
Nature, 329:341 (1987)) and the other homologous to
mouse P-cadherin (Nose, et al. (1987), above).
The PCR products were then used as probes to
isolate the BMEC and MDCK cadherin cDNA clones as
follows. A cDNA library was constructed essentially
according to Gubler et al. (Gubler, U. et al., Gene,
25:263 (1983), which is incorporated herein by
reference), using poly (A) RNA isolated from either
BMEC or MDCK cells. The cDNA was ligated via EcoRI
adaptors into gtlO arms (BMEC) or ZAPR (from ,
Stratagene, Inc., La Jolla, CA) vector arms (MDCK).
cDNA libraries containing 5 x 105 - 1.5 x 1o6
independent cDNA clones were screened using
radiolabeled PCR products (Benton, W.D. et al.,
Science, 196:180 (1987), which is incorporated herein
by reference). Northern blot analysis (Maniatis, T. et
al., "Molecular Cloning: A Laboratory Manual", Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1982) may be used to determine whether each cDNA
species cloned hybridizes to a single mRNA species, as ;
well as the tissue distributions of each cDNA species.
cDNA clones for each cadherin were sequenced by
the method of Sanger et al. (1977) above.
The partial restriction maps for each cDNA clone
based on their sequences are shown in Fig. 4. Some of :
these restriction sites were confirmed by restriction
enzyme digestions, including Hind III, Pst I, Kpn I,
Bgl II for N-cadherin; Pvu II, Sac I and Pst I for
30 P-cadherin; Pst I, Pvu II, BamH I, and Sac I for ,
E-cadherin.
In order to test whether the cloned E-cadherin -
cDNA contains all the information necessary for -
- cadherin function, full-length E-cadherin cDMA joined
to a suitable promoter may be introduced into mouse
. . .
WO9l/04745 PCT/US90/OSlOS
20671~`3
L-cells that have very little endogenous cadherin
activity (Nagafuchi, et al. (1987), supra). To test
for expression of E-cadherin ir. transfectants derived
- from the introduced cDNA, transfected L-cells may be
tested for Ca2~-dependent aggregating activity. The
extent of this aggregating activity should be closely
correlated with the amount of E-cadherin expressed
tTakeichi, M. (1988), su~ra). This same technique may
be used for testing cDNAs encoding bovine endothelial
N- and P-cadherins, according to the method of Hatta,
et al. ~Hatta, K., et al. (1988), supra).
In order to identify cell binding domains in, for
example, MDCK E-type cadherin, L-cells may be first
transfected as above with a cDNA of a size sufficient
to cause Ca2~-mediated aggregation of transfectants. A
series of deletion mutants comprising truncated cDNA
species missing different regions of the extracellular
domain may be prepared by restriction enzyme digestion
and proper end filling or exonuclease digestion to make
the deletions in the proper coding frames. These
deletion mutants can then be tested for their ability
to express in ~-cells a protein causing Ca2~-dependent
aggregation. By correlating a loss of aggregation with
deletion of particular fragments, the regions important
for cell binding may be determined. A variety of
polypeptides corresponding to binding regions of
cadherins, as deduced from the nucleotide sequences of
deleted cDNA, may be synthesized chemically using an
automated peptide synthesizer such as that of Applied
Biosystems, Inc., Foster City, CA, or expressed by
recombinant DNA methods. Effective polypeptides may be
of varying lengths, depending upon the natures of
junctions being disrupted and the cell adhesion
molecule present.
'
: - . ; , :,, .................. - .: , ,
:, , -:, :~:: ,:- ~:
WO91/0474s PCT/US90/05105
~ ' , , .
~- 16
Nucleotide, and corresponding amino acid,
sequences of cadherins may be analyzed to detect - -
homologous regions. Applying this technique to bovine
endothelial cell N- and P-cadherins and to epithelial ~-
cell E-cadherin, we have determined that, in the amino
acid 80 region of each of these cadherins, there is
conserved a triplet HAV (His-Ala-Val) region. We have
deduced that this HAV region may be a common cell `
adhesions recognition (CAR) sequence.
We have chemically synthesized the following
polypeptides, each of which containing the HAV
sequence:
,'~' '
6-mer(78-83~ NH2-SHAVSS-CONH
11-mer(76-86) NH2-LYSHAVSSNGN-CONH2
1517-mer(74-90) NH2-YILYSHAVSSNGNAVED-CONH2
18 mer(69-86) NH2-EQIAKYILYSHAVSSNGN-CONH2
20-mer(71-90) NH2-IAKYILYSHAVSSN~NAVED-CONH2
and have tested each for efficacy in opening brain :
endothelial cell tight junctions in the BBB model
disclosed in copending United States application Serial
No. 07/413,274, and also on kidney epithelial cell
tight ~ucntions..
Polyclonal antibodies raised in rabbits and
monoclonal antibodies derived from hybridomas may be
generated against each of the chemically-synthesized
polypeptides by standard methods. (Harlow, E., et al.,
"Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988;
Goding, J.W., "Monoclonal Antibodies: Principles and
Practice", Academic Press, N.Y. 1986). In addition,
recombinant antibodies may be prepared. Fragments of
antibodies, e.g., Fc, Fab, F(ab)', may be prepared by
standard methods.
We have cloned and sequenced fusion proteins
derived from amino acids 9-96 of MDCK E-cadherin
WO91/04745 PCT/US90/05105
17 2Q~71~3
containing the HAV region. A polyclonal antibody
prepared against this fusion protein stained rat
(Fig.55) mouse brain sections as well as did an
antibody raised against the entire E-cadherin (Fig. 6).
A polyclonal antibody raised against a fusion protein
derived from amino acids 9-37 failed to stain brain
sections. These results indicate that the key cell-
binding domain of E-cadherin lies in the region of
amino acids 37-96.
The ability of CAM-derived polypeptides containing
cell-binding domains, and the corresponding polyclonal
and monoclonal antibodies, of the invention to disrupt
tight junctions may be tested in ln vitro and ln vivo
models of high resistance tight junctions and in animal
models. Monolayers of MDCK dog kidney epithelial
cells, that are known to contain high resistance tight
junctions (Gumbiner, B., J. Cell Biol., 102:457
~1986)), can be used to test for the ability of the
polypeptides.and corresponding antibodies of the
present invention to disrupt such tight ~unctions.
Polyclonal antibodies prepared as described above
may also be used in con~unction with Western blotting
(Old, R.W., et ~1., Prin~iples_~f Gene Manipulation, 3d
ed., Blackwell, Oxford, 1985, p. 10) and a variety of
tissue extracts in order to identify cell adhesion
glycoproteins in such extracts.
Another embodiment of the present invention is in
drug delivery systems. Conjugates between therapeutic ~ - -
drugs and agents that affect cell adhesion molecule
30 function in brain capillary endothelial cells may be ~ -
- used to deliver therapeutic drugs to the CNS. For
example, a polypeptide derived from a cell adhesion
molecule that contains within its amino acid sequence a
cell-binding domain, or antibodies thereto, may be
conjugated in biologically-active form to a therapeutic
:
:
WO91/0474s 6~3 PCT/US90/0510S
18
modality. Such conjugates may have the dual effect of
opening the BBB and delivering the therapeutic agent to
the brain side of the BBB. Delivery of therapeutic
drugs to the CNS, either alone or conjugated to agents
- 5 that disrupt cell-cell adheslon, may be accomplished by
administering such drugs to a subject either
simultaneously with or subsequent to the administration
of the agents of this invention that disrupt the tight
junctions of the BBB. Examples of therapeutic
modalities that may be delivered to the brain by the
cell adhesion disruption compositions of this invention
include Nerve Growth Factor, anti-Parkinsonian drugs,
and brain enzymes known to be missing in
sphingolipidoses, e.g., Tay-Sachs disease. Means of -
chemically conjugating protein or polypeptide carriers
to therapeutic agents such that the biological . -
integrity of the therapeutic agent is not compromised
and such that the therapeutic agent is readily cleaved
from the carrier by enzymes present on or within
endothelial cells (e.g., amidases, esterases,
disulfide-cleaving enzymes), are well known in the art. ~
It is also apparent that these therapeutic conjugates `
may be delivered to endothelial cells in encapsulated
form (e.g., in liposomQs) or as microsuspensions
stabilized by pharmacological excipients.
lt is known (Jain, R.K., J. Natn'l Cancer Inst., ~;
81:570 (1989)) that many solid tumors develop internal
barriers, including high pressure zones and collapsed -
blood vessels, that make it difficult for blood-borne
chemotherapeutic agents to reach the tumor's inner
core. The barrier problem is particularly troublesome
with therapeutic products drawn from the human immune - -
system, such as monoclonal antibodies conjugated with
chemotherapeutic agents, interleukin-2, interferon and
activated killer T-lymphocytes, because of their large
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.: - . ~. . . . . . : . ~, ", . - . ~ : ,.
wo 91~n4745 2 0 ~ 7 ~ ~ ~590/05105
.;;
19
size. Thus, in another embodiment of this invention,
compositions that disrupt the junctions between
endothelial cells, particularly the relatively small
peptides that contain one or more cell-binding regions
of cell adhesion macromolecules, may be used to enhance
drug delivery to tumors with depressed blood flow.
It has been theorized that cancer cells -
metastasize by secreting soluble cadherins variously to
open tight junctions in cells that block their movement :
and to prevent their being bound to such cells. We
consider it likely that antibodies raised against these
cadherins, which are derived from extracellular domains
of the cadherins disclosed in this invention, may ~ ~
provide a therapeutic modality that inhibits or -
prevents cancer cell metastases.
In another embodiment, the compositions of this :~
invention may also be used to provide penetration for
chemotherapeutic agents of other well-known blood- ~ ~ :
tissue barriers, such as blood-testis barriers and
blood-retina barrlers. The latter barrier is known to
prevent the efficient transport of, for example,
administered antibiotics to the retina from the general
circulation. The cell adhesion disruptin~ compositions
of this invention may, thus, be used in conjunction
with the administration of antibiotics to treat retinal
infections.
The following examples are illustrative of several
embodiments of this invention, and should not be -
construed in any way as limiting the invention as
recited in the claims.
. EXAMPLE 1
EFFECTS OF HAV-CONTAINING POLYPEPTIDES
ON TIGHT JUNCTIONS OF MDCK EPITHELIAL
i AND BOVINE ENDOTHELIAL CELLS
' ,:
.
WO~1/04745 PCT/US90/OS105
The BBs model of copending U.S. Serial No.
07/413,332 was used to examine the effects of
polypeptides containing the HAV region on the tight
junctions of~monolayers of MDCX epithelial cells and
bovine capillary endothelial cells as determined by
resistance measurements across the monolayers.
The polypeptide was added to the cells either from
the apical side (top) or basolateral side (bottom), as
shown in the following sketch. ;
10APICAL
EPITHELIAL CELLS ENDOTHELIAL CELLS
Gut Side Blood Side
Blood Side Brain Side
BASOLATERAL
' '~
15 Figure 7 illustrates the effects of various
concentrations of the aforementioned 18-mer polypeptide
on resistance of MDCX epithelial cells. At the lowest
concentration tested, 0.5 mg/ml, resistance was
markedly decreased. The polyp~ptide was more effective
when added from the basolateral side, but at high
concentrations was quite effective even when added from
the apical side. These data indicate that the 18-mer
is effective in making tight junctions permeable. The
20-mer was similarly effective, and a 17-mer less
effective.
Figure 8 illustrates the effects of the
aforementioned ll-mer and 18-mer on MDCX cell
resistance when added from either the apical or
basolateral side of the monolayers. The concentration
of polypeptide was about 1 mg/ml. The ll-mer (as well
: . ~ ' ' ' ' ' ' ' :: . ` ' ~: ` - ....... ' ' ' ,: : :
- ~
WO91/04745 PCTtUS90/0510S
2067183 .... .. -
as the 6-mer data not shown) was virtually without -
effect. With the 18-mer, resistance was almost totally
abolished by about 6 hours, indicating disruption of
tight junctions. That the effect of the 18-mer is -
reversible is indicated by the "wash-out" experiment.
When the i8-mer was washed out of the MDCK cells at 6
hours, resistance recovered to a substantial extent
over the next 21 hours. This recovery was`particularly -
pronounced when the 18-mer had originally been added
_rom the basolateral side of the monolayers. The
20-mer produced results similar to those of the 18-mer,
and the 17-mer was effective, but somewhat less so.
Figure 9 illustrates the effect of 1 mg/ml of the
ll-mer and 18-mer on high resistance monolayer cultures
of brain endothelial cells (see copending United States
Serial No. 07/413,332 for method of preparation). As . --:
with MDCK cells, the ll-mer (and the 6-mer) failed to
reduce resistance values over a 48-hour period of
observation. In contrast, the 18-mer (as well as the
20-mer) decreased resistance values markedly when added
from either the basolateral or apical side, but the
effect of the polypeptide was more rapid and more
pronounced when it was added from the basolateral side; ;~
the 17-mer was less effective.
The conclusion of these experiments is that a
particular set of peptides (but not all peptides)
centered around the HAV region of E-cadherin are
effective in opening tight junctions of brain
endothelial cell blood-brain barriers, and also of
epithelial cells that form such junctions ("gut
barrier"~. Both the length and compositon of the amino
acid region flank;ng the HAV triplet thus appear to
play a role in the efficacy of such compositions.
While the aforementioned embodiments represent the ~ -
35 preferred embodiments of the invention, those skilled ~ -
''. ~ '.
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WO91/04745 PCTtUS90/05105
~ 22
in the art may, without undue experimentation, devise
other executions of the compositions and methods of use :'
of this invention without departing from the concept -
and spirit inherent therein. :., :
' ~ .
.... ~ ~ , . ., . . . , I '
