Note: Descriptions are shown in the official language in which they were submitted.
2~)169~
POLYPEPTIDE-ANTIBODY CONJUGATE FOR
INHIBITING CELL ADHESION
Description
- ~-
Technical Field -
The present invention relates to a
polypeptide-antibody conjugate comprising an integrin~
binding polypeptide operatively attached to an
antibody molecule that immunoreacts with adhesitory -
cell surface antigens and thereby inhibits cell
adhesion. Also contemplated is a therapeutic method
wherein a polypeptide-antibody conjugate of the ~-
present invention is used to inhibit cell adhesion- ,
dependent processes such as tumor growth and platelet
aggregation.
Background
Cell adhesion is a critical process in a --~
variety of cell-cell and cell-extracellular matrix
interactions including tumor growth and platelet
aggregation. Therefore, agents that inhibit cell
adhesion are of therapeutic use at least to inhibit ~;
tumor growth or to inhibit platelet-mediated processes
such as blood coagulation.
Cell adhesion generally involves an
interaction between cell surface receptors and
specific matrix proteins. Rouslahti et al., Science
238:491-497 (1987). Of lnterest are those receptors ~ `-
that specifically bind to matrix proteins at sites ~;~
that have as a part of their structure the tripeptide ~ ;
amino acid residue sequence Arg-Gly-Asp (RGD). Such
receptors are known in the art as RGD-direct~d
adhesion receptors and include the fibronectin
~ 35 receptor (FNr), the vitronectin receptor (VNr), the '~
`~ platelet receptor known as GPIIb/IIIa and the collagen
:~ :
2~01~
receptor. Recently, additional RGD-directed adhesion
receptors were described on M21 human melanoma cells,
Cheresh et al., J. Biol. Chem., 262:1434-1437 (1987),
and on human endothelial cells, designated ECr.
5 Cheresh et al., Proc. Natl. Acad. Sci. USA, 84:6471-
6475 (1987).
RGD-containing polypeptides derived from
matrix protein sequences have been shown to inhibit
the ability of RGD-directed adhesion receptors to
10 interact and adhere to a matrix protein-containing
substrate. Pytela et al., Pro. Natl. Acad. Sci. USA,
82:5766-70 (1985~; and Pytela et al; Science,
231:1559-62 (1986).
RGD-containing polypeptides have also been
15 used to inhibit cell adhesion to a matrix protein-
containing substrate in vitro. United States Patent
Nos. 4,614,517, 4,517,686, 4,578,079, 4,683,291,
4,792,525; Pierschbacher et al., Nature, 309:30-33
(1984); Pierschbacher et al., Proc. Natl. Acad. Sci. ~-
USA, 81:5985-88 (1984); Hayman et al., J. Cell Biol., ~-
100:1948-54 (1985); Haverstick et al., Blood, 66:946-
52 (1985); Ginsberg et al., J. Biol. Chem., 260:3931-
36 (1985); Armant et al., Proc. Natl. Acad. Sci. USA,
83:6751-55 (1986); and Ouaissi et al., Science, ;~
234:603-7 (1986). One RGD-containing polypeptide was
; shown to inhibit murine melanoma cell adhesion and to
also inhibit colonization of mouse lung tissue n ~;
vivo. Humphries et al, Science, 233:467-70 (1986). ;
i A similar in vivo inhibition of lung tissue ~ ~ ~
colonization was described using a polypeptide derived ~ ~-
from the laminin matrix protein sequence. Iwamoto et -~
al., Science, 238:1132-1134 (1987). Both RGD-
containing polypeptides and other cell adhesion ~ ~;
inhibiting laminin-derived polypeptides were described
that contained the pentapeptide amino acid sequence
;~,
6~
. -
3 ~-
Tyr-Ile-Gly-Ser-Arg (YIGSR), thus indicating that RGD-
containing polypeptides are not the only candidates
for inhibition of cell adhesion.
Inhibition of cell adhesion has also been
demonstrated using monoclonal antibodi~s that
immunoreact with a molecule present on the surface of
cells that undergo cell adhesion. For example, cell
adhesion-inhibiting antibodies were described that
immunoreact with RGD-directed adhesion receptors such
as GPIIb/IIIa, tPytela et al., Science, 231:1559-62
(1986); Charo et al., J. Biol. Chem., 262:9935-38
! (1987)], a 140 kilodalton fibronection specific
receptor [Brown et al., Science, 228:1448-51 (1985)],
the cell substrate attachment antigen (CSAT) [Horwitz
et al., J; Cell Biol., 101:2134-44 (1985)], and the
human melanoma cell receptor [Cheresh et al., J. Biol.
Chem., 262:17703-11 (1987)].
Cell adhesion inhibiting-antibodies have
also been described that immunoreact with adhesitory
cell antigens where the antigens are not themselves
RGD-directed adhesion receptors. Yor example,
monoclonal antibodies that immunoreact with
disialoganglioside GD2 or GD3 were shown to inhibit
tumor cell adhesion to various matrix protein- -
containing substrates including collagen, vitronectin,
laminin and fibronectin. Cheresh et al., J. Cell
Biol., 102:688-96 (1986).
In the medical arts, it is seen from the
foregoing that RGD-containing polypeptides and other -
polypeptides derived from matrix protein primary
sequences are useful for inhibiting cell adhesions,
particularly adhesion of tumor cells or platelets.
However, the systemic use of adhesion-inhibiting
polypeptides is limited because it produces the side
effect of systemically and indiscriminately inhibiting
2~1~9~i2
normal cellular adhesive events.
Brief summarY of the Invention
It has now been discovered that the cell
adhesion-inhibiting effects of the polypeptides
described herein can be directed (targeted) to
particular tissues by means of their conjugation to
specific antibodies so that the polypeptides can
disrupt specific undesirable cell adhesion events
without significantly effecting normal adhesive
events.
The present invention provides an RGD-
antibody comprising an antibody molecule operatively
attached to an integrin-binding polypeptide. The
antibody combining site of the RGD-antibody
immunoreacts with an adhesitory cell surface antigen
and the polypeptide has an amino acid residue sequence ~ ;
of about 5 to about 50 residues in length that ~ -
includes a sequence having the formula: -RGD-; more ;~
preferably the formula: -GRGDSP-, and most preferably
the formula: -CGGAGAGRGDSP-.
In addition, it is preferred that the
antibody molecule of the contemplated RGD-antibody
immunoreacts with an adhesitory cell surface antigen.
Preferred adhesitory cell surface antigens are
disialoganglioside GD2, disialoganglioside GD3,
chondroitin sulfate proteoglycan, vitronectin
receptor, endothelial cell receptor or collagen
receptor. Preferably, the antibody molecule is ;
produced by a hybridoma selected from the group
consisting of 1418, 142A, 126, MB3.6, LM609 and LMl42.
;~; In preferred embodiments, a contemplated
RGD-antibody immunoreacts with an antigen present on
the surface of platelets or tumor cells.
The present invention also contemplates a
'. ' '~' ~
:: '' " '
20~
method for inhibiting the attachment of an adhesitory
cell to an RGD-containing matrix comprising
administering to a subject a therapeutically effective
amount of an RGD-antibody of the present invention.
Preferably, the adhesitory cell is a tumor cell or
platelet and therefore the method contemplates the
inhibition of tumor cell attachment or platelet
aggregation, respectively.
.In another embodiment, the present invention
contemplates a YIGSR-antibody comprising an antibody
molecule that immunoreacts with an adhesitory cell
surface antigen, operatively attached to a laminin
receptor-binding polypeptide having an amino acid
residue sequence of about 5 to about 50 residues in
length that includes a sequence having the formula: -
YIGSR-. More preferably the polypeptide has the ;
formula~
YIGSR,
CDPGYIGSR, or
RGDSGYIGSR.
Further contemplated is a chimeric antibody
that contains at least one hybrid protein molecule
having an antibody combining site-forming fragment
fused to at least one integrin-binding polypeptide, in
which the hybrid protein molecule forms an antibody
combining site that immunoreacts with a surface
antigen of an adhesitory cell (adhesitory cell surface
antigen). In a preferred embodiment the integrin-
binding polypeptide comprises an amino acid residue
sequence of about 5 to about 50 residues in length
that includes a sequence having the formula: -RGD-,
and more preferably the formula: -GRGDSP-.
The present invention also contemplates a
recombinant DNA molecule that encodes a hybrid protein
molecule comprising:
-` 2~
a) a first DNA segment encoding an
antibody combining site-forming fragment, and
b) a second DNA segment encoding an
integrin-binding polypeptide that is operatively
linked in phase to the first segment, wherein the
recombinant DNA molecule is capable, when present in
an appropriate expression vector, of expressing a
hybrid protein molecule having an antibody combining
site that immunoreacts with an adhesitory cell surface
antigen. ~ ~
Brief Description of the Drawinas -~ ~:
Figure 1 is a composite figure comprising
two panels (A and B) that graphically illustrate the
effects after 20 minutes of a polypeptide-antibody
conjugate on the attachment of M21 cells to microtiter
wells, each well having been coated with either von
Willebrand factor (Panel A) or fibrinogen (Panel B).
3H-leucine-labeled M21 cells were first immunoreacted
with various indicated concentrations of either Mab
142A-conjugate (open boxes) or control activated Mab
! 142A (closed boxes) prior to reaction with the
polypeptide. The antibody treated cells were then
plated onto the coated microtiter wells and maintained
25 for 20 minutes to allow the cells to adhere to the
wells as described in Example 4. The results are -~
expressed as the total number of cells bound, that
number being a function of the cell-associated label,
expressed in couhts per minute (cpm x10-3), bound to
30 the matrix protein at each indicated Mab
concentration.
Figure 2 is a composite figure comprising
two panels (A and B) that graphically illustrate the ;~
effects after 90 minutes of a polypeptide-antibody `
35 conjugate on the attachment of M21 cells to microtiter ~ ~
2016~6~,
" .
wells, each well having been coated with either von
Willebrand factor (Panel A) or fibrinogen (Panel B).
Reagents and methods were as described for Figure 1,
and data are reported as in Figure 1.
Detailed Description of the Invention
A. Definitions
Amino Acid: All amino acid residues
identified herein are the natural L-configuration. In
keeping with standard polypeptide nomenclature, J.
Biol. Chem., 243:3557-59 (1969), abbreviations for ::.:~ :
amino acid residues are as shown in the following
Table of Correspondence: . . ,
TABLE OF CORRESPONDENCE
SYMBOL AMINO ACID ;:;~
1-Letter3-Letter
Y Tyr L-tyrosine
G Gly glycine
F Phe L-phenylalanine
M Met L-methionine
A Ala L-alanine
; S Ser L-serine
I Ile L-isoleucine
L Leu L-leucine
T Thr L-threonine
V Val L-valine
P Pro L-proline
K~ Lys L-lysine ~ -
H His L-histidine
:~ Q Gln L-glutamine
:~ E Glu L-glutamic acid
W Trp L-tryptophan
R Arg L-arginine
D Asp L-aspartic acid :: :~
.
'- 20169~
N Asn L-asparagine
C Cys L-cysteine ;~
It should be noted that all amino acid residue ; ;~
seauences are represented herein by formulae whose
left to right orientation is in the conventional
direction of amino-terminus to carboxyl-terminus.
Furthermore, it should be noted that a dash at the ~ `~
beginning or end of an amino acid residue sequence
indicàtes a bond to a further sequence of one or more
amino acid residues up to a total of about fifty
residues in the polypeptide chain. ~; ;
Polypeptide and Peptide: Polypeptide and
peptide are terms used interchangeably herein to
designate a linear series of no more than about 50
amino acid residues connected one to the other by
peptide bonds between the alpha-amino and carboxyl ;~
groups of adjacent residues. --
Protein: Protein is a term used herein to
designate a molecule having a primary structure
comprised of a linear series of greater than 50 amino
acid residues connected one to the other as in a
polypeptide. Of course, both a protein and a ;
polypeptide can further include intramolecular
disulfide and other bonds that cause the protein or
the polypeptide to assume a non-linear configuration
when in solution. ;~;
B. Polvpeptide-AntibodY Conluqates
1.~ Backar~und
The polypeptide-antibody conjugate of the
present invention comprises in its most broad
embodiment an antibody molecule operatively attached
to an integrin-binding polypeptide. The attached
antibody molecule immunoreacts with an antigen present
on the surface of an adhesitory cell, and the
20169~?.
g ,
integrin-binding polypeptide inhibits cell adhesion.
The terms adhesitory cell, integrin-binding
polypeptide and cell adhesion are discussed and
defined hereinafter.
The combination of antibody molecule and
polypeptide as disclosed herein produces a cell
adhssion-inhibiting molecule because, upon
immunoreaction of the antibody with an adhesitory cell
surface antigen, the antibody situates the attached
integrin-binding polypeptide into sufficient proximity
with cell surface integrin molecules so that the
polypeptide can interact with and be bound by (or
bound .o) the integrin, thus actiny as an antagonist
and inhibiting the ability of the bound integrin to
participate in adhesion processes.
In view of the utility of an integrin-binding
polypeptide to inhibit cell adhesion, the combination
of antibody molecule and polypeptide disclosed in the
present invention provides the benefit of targeting
the inhibitory effect to those cells that contain a
particular adhesitory cell surface antigen. Without
targeting, topical or systemic application of an
integrin-binding polypeptide can indiscriminately
inhibit adhesion of all cells exposed to the
polypeptide, including adhesitory cells present in
normal tissues. Therefore the benefit of localizing
cell adhesion-inhibiting e~fects to specific
adhesitory cells can be readily appreciated by one
skilled in the art of cell adhesion.
The phrase "cellular adhesion" refers to a
process by which an adhesitory cell attaches (adheres)
to another cell's surface or to a tissue substrate.
; The adhesion process occurs by a specific interaction
between adhesitory cell surface protein receptors,
called integrins, and matrix protein ligands that
21~69~
,-''
. : ,; ,:-
include the RGD-tripeptide as a part of their amino ~-
acid residue sequences. Thus, adhesitory cells, as
used hereIn, are those cells that adhere to biological - ~
surfaces by means of the specific interaction ~ -
(receptor/ligand complex formation) between an
integrin and RGD-containing matrix proteins. RGD-
containing matrix proteins are also referred to as
adhesion protein ligands and include fibronectin, ~ -
vitronectin, fibrinogen, von Willebrand factor,
laminin, thrombospondin, osteopontin, collagens and
the like.
An adhesitory cell is also characterized as any
cell that contains on its surface one or more of the
cell surface receptors known as integrins. Integrins
are also referred to as RGD-directed adhesion
receptors or cytoadhesins, and include vitronectin
receptor, fibronectin receptor, collagen receptor,
platelet glycoprotein GPIIb/IIIa, human endothelial
cell receptor and the like.
Exemplary adhesitory cells include platelets and -
numerous types of tumor cells including neuroblastoma,
small cell carcinoma, adenocarcinomas of the lung,
colon or breast, melanoma, astrocytoma, glioma,
squamous cell carcinoma and the like.
An adhesitory cell surface antigen refers to any
antigen present on the surface of an adhesitory cell.
"Antigen" is used herein to refer to any molecule that
immunoreacts with and is bound by an antibody molecule
to form an immunoreactant.~ Thus an adhesitory cell
surface antigen can be any molecule on an adhesitory
cell surface that immunoreacts with an antibody
molecule while the antigen is present on the surface
of the cell.
Exemplary adhesitory cell surface antigens
3S include the disialogangliosides GD2 and GD3, tumor
.. . .
~ :':: '~:
2~ 9~2 ~ ~
cell surface antigens, platelet surface antigens,
chondroitin sulfate proteoglycans, cell surface
receptors such as integrins, and the like.
2. Antibody_Molecules
Antibody molecules to be used as a component of
the present invention include any antibody molecule
that is capable of immunoreacting with an adhesitory ~
cell surface antigen to form an immunoreaction product ;
(antigen-antibody complex).
The phrase "antibody molecule" in its various
grammatical forms as used herein contemplates both an
intact immunoglobulin molecule and an immunologically
active portion of an immunoglobulin molecule, i.e.,
molecules that contain an antibody combining site or
paratope. ~
An "antibody combining site" is that structural ~ ~`
portion of an antibody molecule comprised of heavy and
light chain variable and hypervariable regions that
- specifically binds antigen. `
Exemplary antibody molecules are intact
immunoglobulin molecules, substantially intact
immunoglobulin molecules and those portions of an
immunoglobulin molecule that contain the paratope,
including those portions known in the art as Fab,
Fab', F(ab')2 and F(v).
Fab and F~ab')2 portions of antibody molecules
are prepared by the proteolytic reaction o~ papain and
pepsin, respectively, on substantially intact antibody ;~
molecules by mebhods that are well known. See for ! '
example, U. S. Patent No. 4,342,566 to Theofilopolous
;~ and Dixon. Fab' antibody molecule portions are also
well known and are produced from F(ab')2 portions
followed by reduction of the disulfide bonds linking
the two heavy chain portions as with mercaptoethanol,
;~ 35 and followed by alkylation of the resulting protein
.;, .~ ,.
: ; ~ .~ :
2 1~ 9~
.
12
mercaptan with a reagent such as iodoacetamide. i
The term "antibody" in its various grammatical
forms is used herein to refer to a composition `
containing a plurality of antibody molecules, e.g., an
antiserum. An antibody containing intact antibody
molecules is preferred. Particularly preferred is a
monoclonal antibody.
The phrase "monoclonal antibody" or "Mab" in its
various gra~natical forms refers to an antibody
containing only one species of antibody combining site
capable of immunoreacting with a particular antigen
and thus typically displays a single binding affinity -
for that antigen. A monoclonal antibody can therefore
contain a bispecific antibody molecule having two
antibody combining sites, each immunospecific for a
different antigen.
A preferred monoclonal antibody is characterized - :
as containing, within immunologically detectable
limits, only one species of antibody combining site
capable of immunologically binding (immunoreacting
with) an adhesitory cell surface antigen.
Particularly preferred are monoclonal antibodies
that immunoreact with disialogangliosides GD2 or GD3,
vitronectin receptor, fibronectin receptor, human
endothelial cell receptor, chondroitin sulfate
proteoglycan or platelet glycoprotein GPIIb/II~Ia.
Table 1 lists monoclonal antibodies having
immunospecificities useful in the present invention.
Those Mabs are listed by the name utilized in a
publication, by the reported American Type Tissue
Collection (ATCC) accession number of the hybridoma
that produces the antibody and by the antigen
indicated with which the Mab reportedly reacts. A
- . -
citation to a discussion of each Mab and its
immunospecificity is provided by the footnote to the
: ~-
13
listed antigen. Hybridomas producing monoclonal
antibodies functionally equivalent to those in Table 1
can be made by techniques well known in the art
including those described herein.
Table 1
Exemplary Monoclonal Antibodies That Immunoreact
With An Adhesitory Cell Surface Antigen
Mab ATTC No. Antigen
1418 HB 9118 disialoganglioside GD2
142A - disialoganglioside GD2
126 HB 8568 disialoganglioside GD23
MB3.6 HB 8890 disialoganglioside GD34 ~ ~
llC64 - disialoganglioside GD35 ~;
R24 - disialoganglioside GD3
9227 - chondroitin sulfate
proteoglycan7
LM609 HB 9537 vitronectin receptor8
LMl42 ~ vitronectin receptor9
HT29/26 HB 8247 colon cancer glycoprotein
gp291o
T16 HB 8279human bladder tumor
glycoprotein gp48
14 ~ -
1115-NS-l9-9 HB 8059 colorectal carcinoma
monosialoganglioside12 -~
13 ~
CTHB-5 HB 135 complement C3d receptor
OXT 11 CRL 8027 rosette receptors14
Anti-HL~15 _ histocompatibility (HLA)
lQ
T~ql HB 169 leukocyte (leu-5)
antigen16 ~
C7 CRL 1691 low density lipoprotein ~
receptOrl7 ~.
BBM HB 28 B-2 microglobulin18
-:
1 Cheresh et al., Cancer Res., 44:5112-18 (1986).
Prepared as described in Example 1.
3 U.S. Patent No. 4,675,287.
2S
;; 4 Cheresh et al., Proc. Natl. Acad. Sci. USA,
82:5155-59 (1985).
5 Cheresh et al., J. Cell Biol., 102:688-96 (1986).
6 U.S. Patent No. 4,507~391
7 Bumol et al., Proc. Natl. Acad. Sci. USA, 79:1245-
, :;
49 (1982).
3S
`~ 8 Cheresh et al., J. Biol. Chem., 262:17703-11
(1987~
9 Cheresh et al., J. Biol. Chem., 262:17703
(1987).
u.s. Patent No~ 4,579,827.
European Patent Application No. 84102517.4,
publication No. 0 118 891, published September 19,
1984.
~ ' ~:--
12 U S. Patent No. 4,471,057-
' 13 Weis et al., Proc... Natl. Acad. Sci. USA, 81:881-
885 (1984). ~;~
4 U.s. Patent No. 4,364,937~
5 ATTC Cataloque of Cell Lines and Hybridomas, 6th
edition, 1988, at pg. 283 lists over 27 different
Mabs that immunoreact with numerous HLA antigens.
16 Grumet et al., Human Immunol., 6:63-73 (1983).
7 Goldstein et al., ~ell, 30:715-24 (1982).
18 Parham et al., Eur. J. Immunol, 9:536-45 (1979). ~ `
Antibodies or monoclonal antibodies that
immunoreact with adhesitory cell surface antigens can ; -~
be obtained from a variety of commercial vendors. ;;
Alternatively, antibodies can be prepared by
immlmization techniques well known in the art. The
preparation of monoclonal antibodies and their
subsequent purification sufficient for use in the
present invention are well known. Typically, a ~ -
monoclonal antibody is prepared from a hybridoma
culture supernatant by separating the supernatant from
~; cultured hybrldoma cells to form a cell-free
~ . :: . :::
~01f~9~
16
monoclonal antibody molecule-containing solution.
Alternatively, a monoclonal antibody can be prepared
from ascites by introducing a hybridoma cell, as by
injection, into the peritoneal cavity of a mammal such
as a mouse and later harvesting the resulting
peritoneal exudate (ascites tumor fluid) from the
mouse by well known techniques. See, for example, H.
Zola, Monoclonal Antibodies: A Manual of Techniques,
CRC Press, Inc. (1987).
Additionally, a monoclonal antibody can be
produced by recombinant DNA methodologies in which a
monoclonal antibody molecule-encoding gene is cloned
and manipulated into a suitable expression medium for
production of a recombinantly produced antibody
molecule. Monoclonal antibody production by
recombinant methods is described in more detail below
under Section D entitled "Chimeric Antibodies".
Particularly preferred are the monoclonal
antibody molecules 142A, 1418, 126, MB3.6, LM142, and
LM609, as disclosed in more detail herein. Hybridomas
that produce monoclonal antibody molecules 1418, 126,
MB3.6 and LM609 were deposited pursuant to Budapest
Treaty requirements with the American Type Tissue
Collection (ATCC), Rockville, MD, on June 4, 1986, May
~5 28, 1984, August 16, 1985 and September 15, 1987,
respectively, and were assigned the accession numbers
HB 9118, HB 8568, HB 8890 and HB 9537, respectively.
3. Polypeptides
An integrin-binding polypeptide useful in the
present invention is a polypeptide having an amino
acid residue sequence of about 5 to about 50 residues ;
in length, and exhibiting the property of
competitively inhibiting cellular adhesion. A useful
polypeptide binds to an integrin expressed on the
surface of an adhesitory cell. It also inhibits
` ~'`'`.'''
;~Q 3L6~
17
(antagonizes) cellular adhesion between an adhesitory
cell and a surface that contains a matrix protein.
Many polypeptides are known to have such
adhesion inhibiting properties. See, for example,
Rouslahti et al., Cell, 44:517-518 (1986);
Pierschbaaher et al., Nature, 309:30-33 (1984~;
ouaissi et al., Science, 234:603-607 (1986~; Iwamoto
et al., Science, 238:1132-1134 (1987); Haverstick et
al., Blood, 66:946-952 (1985); and U.S. Pat. Nos.
4,578,079, 4,792,525, 4,614,517, 4,517,686 and - ~
4,683,291. (The teachings of the art cited herein are ;
incorporated by referencel.
Adhesion-inhibiting conditions for integrin~
binding polypeptides are well known in the art.
Exemplary conditions are described in Example 4 and in
the teachings cited immediately above.
Typically, integrill-binding polypeptides contain ;
an amino acid residue sequence that corresponds to a
portion of the sequence of an adhesion protein. This
polypeptide sequence is present in that portion of the ; --
adhesion protein that participates in contacting the `~
integrin to which it binds when adhesion occurs. This
contacting portion of the adhesion protein has been
;~ referred to as the cell recognition site. For many of
the characterized adhesion proteins, the recognition
site includes the amino acid tripeptide RGD. See, for
; example, Rouslahti et al., Science, 238:491-497
(1987)-
The abilit~y of a~ integrin-binding polypeptide
to bind to an integrin, and to inhibit cellular ~ ;
adhesion, is quantified by means well kno~-~n in the
art. Exemplary means are set forth in detail in
Examples 3 and 4. Further exemplary means can be -
found in the disclosures cited above that describe -
exemplary integrin-binding polypeptides.
.. ...
, .. .
, .: ~. .
, - :''~-
2~ 9~
18
A preferred integrin-binding polypeptide has an -~
amino acid residue sequence that includes residues ~ ;~
represented by the formula: -RGD-. Exemplary
polypeptides are represented by the formulas:
VTGRGD,
GRGDS, and
RGDSPASSKP.
Preferably, the polypeptide sequence includes
amino acid residues represented by the formula:
-GRGDSP-. Exemplary polypeptides are represented by
the formu~as:
AVTGRGDSP,
GRGDSP, and
CGGAGAGRGDSP.
For a polypeptide having more residues than the
above-recited included sequences, it is preferred that
any additional residues be selected such that the
entire resulting polypeptide has a sequence that
corresponds to a portion of the amino acid residue
~equence of fibronectin. The sequence of an 11.5
kilodalton fragment of fibronectin that contains the
RGD sequence is described by Pierschbacher et al., J.
Biol. Chem., 257:9593-97 (1982).
Where a polypeptide-antibody conjugate contains
the tripeptide -RGD- as a part of its integrin-binding
polypeptide, it is referred to herein as an RGD-
antibody. -
An additional preferred integrin-binding ~
polypeptide has an~amino acid residue sequence that ! ' ".
includes residues represented by the formula: -YIGSR-.
For a polypeptide having more residues than this
included sequence, it is preferred that any additional
residues be selected such that the entire resulting
polypeptide has a sequence that corresponds to a
portion o~ the amino acid residue sequence of the Bl
19
chain of laminin. The sequence of the laminin Bl
chain is described by Sasaki et al., Proc. Natl. Acad.
Sci. USA, 84:935-39 (1987).
Where a polypeptide-antibody conjugate contains
the pentapeptide -YIGSR- as a part of its integrin-
binding polypeptide, it is referred to herein as a
YIGSR-antibody.
Exemplary polypeptides suitable for use in a
YIGSR-antibody include polypeptides represented by the
formulas
YIGSR,
CDPGYIGSR, and
RGDSGYIGSR. ~ ;
An integrin-binding polypeptide to be utilized
in a polypeptide-antibody conjugate of the present -
invention can be synthesized by any suitable method ;
known to those skilled in the polypeptide art, ~ ;
including recombinant DNA techniques. Thus, synthèsis
can be by exclusively solid-phase techniques, by -~
partial solid-phase techniques, by fragment
condensations or cleavages, or by classical solution
addition.
The polypeptides are preferably prepared using ~ ~
the solid-phase Merrifield-type synthesis for reasons ~ ;
of purity, freedom from undesired side products, ease
of production and the like, such as that described by ~ ;~
Merrifield, J. Am. Chem. Soc., 85:2149 (1964),
although other equivalent chemical syntheses known in ~;;
the art can also be~uséd, such as the syntheses of
Houghten, Proc. Natl. Acad. Sci. USA, 82:5132 (1985). ~ ~
A summary of the many techniques available can also be ~ ;
found in J. M. Steward and J. D. Young, "Solid Phase ` ~-
Peptide Synthesis", W. H. Freeman Co., San Francisco,
1969; M. Bodanszky, et al., "Peptide Synthesis", John - ~ :
Wiley & Sons, Second Edition, 1976; and J. Meienhofer,
', :.
~.
,.
j96~
"Hormonal Proteins and Peptides", Vol. 2, p. 46,
Academic Press (New York~, 1983, for solid phase
peptide synthesis, and E. Schroder and K. Kubke, "The
Peptides",-Vol. 1, Academic Press (New York), 1965,
for classical solution synthesis.
4. PolYpeptide to Antibody Attachment
The polypeptide-antibody conjugate of the
present invention contains an integrin-binding
polypeptide that is operatively attached to a before-
discussed antibody molecule that immunoreacts with an
- adhesitory cell surface antigen. -
As used herein, the term "operatively attached"
means that the polypeptide and the antibody molecule
are physically associated by a linking means that does
not significantly interfere with the ability of either
of the linked groups to function as described herein.
This ability is not significantly interfered with
where the number of polypeptides operatively attached
` is limited so that each antibody molecule has linked
to it from 1 to about 30 polypeptides. It is
preferred that from about 8 to about 12 polypeptides
axe linked per antibody molecule, more preferably
about 10 polypeptides per molecule. ~ ~ -
Methods for controlling the number of
polypeptides that are linked (conjugated) to an
antibody molecule are well known in the art and
typically depend upon the particular linkage chemistry
utilized. Typically, those methods rely on
controlling the ratio of polypeptide to antibody ! ~ ,,~,`'':
molecule in the linkage reaction mixture or the time - :
allowed for the linkage reaction to occur, or both. ;~ -
An exemplary linkage method is described in Example 2.
Because antibody molecules are proteins
themselves, the techniques of protein conjugation or
coupling through activated functional groups is ;~
~: - ,,:
: ~:
,
: : :
Z~169~2
21
particularly applicable to operatively attached the
polypeptide to the antibody molecule. See, for - ~-
example, Aurameas, et al., Scand. J. Immunol., Vol. 8
Suppl. 7:7-23 (1978); U.S. Pat. Nos. 4,493,795, and
4,671,g50.
One or more additional amino acid residues can
be added to the amino- or carboxyl-termini of the
synthetic polypeptide to assist in binding the ;
polypeptide to a carrier. Cysteine residues added at
lo the amino- or carboxyl-termini of the synthetic ~ ~;
polypeptide have been found to be particularly useful
for forming linkages via disulfide bonds. However,
other methods well known in the art for preparing
conjugates can also be used. Exemplary additional
linking procedures include the use of Michael addition
reaction products, dialdehydes such as glutaraldehyde,
Klipstein et al., J. Infect. Dis., 147:318-326 (1983) -~
and the like, or the use of carbodimide technology as~ ;
in the use of water-soluble carbodimide to form amide -~
links to the antibody molecule.
It is preferred that the linking means be a
covalent coupling between a cysteine terminus on the
polypeptide and the epsilon amino group of a lysine ;~
residue present in the monoclonal antibody molecule.
As ;s also well known in the art, it is often
beneficial to bind a synthetic polypeptide to its ;
antibody molecule by means of an intermediate, linking
group. As noted above, glutaraldehyde is one such
linking group. However, when cysteine is used, the
int~rmediate linking group is preferably an _-
maleimidobenxoyl N-hydroxy succinimide (MBS) or 4-
(maleimidomethyl)-l-cyclohexane carboxylic acid N-
hydroxysuccinimide ester(NHS), as was used herein.
Additionally, MBS can be first added to the
antibody molecule by an ester-amide interchange
9~
~ 22
reaction as disclosed by Liu et al., siochem.~ 80: 690
(1979). Thereafter, the addition can be followed by
addition of a blocked mercapto group such as
thiolacetic acid (CH3COSH) across the maleimido-
double bond. After cleavage of the acyl blocking
group, a disulfide bond is formed between the -
deblocked linking group mercaptan and the mercaptan of
the added cysteine residue of the synthetic
polypeptide.
In addition, site directed coupling reactions
can be carried out so that the attached polypeptide
does not substantially interfere with the
immunoreaction of the antibody molecule with the
adhesitory cell surface antigen. See, for example,
Rodwell et al., Biotech., 3:889-894 (1984).
The polypeptides utilized in this invention can
contain additional residues at either terminus for the ;~
.. ..., ..:-:
purpose of providing a "spacer" by which the integrin-
binding polypeptide can be operatively attached to the
antibody. The use of a spacer will thereby extend the
polypeptide further out from the site of linkage upon
the antibody molecule than if the polypeptide were
attached without a spacer. ;~ `
Amino acid residue spacers are usually from 1 to
about 50 residues in length, more often 2 to 10
residues and do not necessarily comprise sequences
that correspond to the sequence of an adhesion
protein.
A preferred spacer polypeptide contains the
amino acid residue sequence CGGAGA operatively ;~
attached by its carboxyl-terminal alanine through a
peptide bond to the amino terminal residue of the
integrin-binding polypeptide. More preferably, the
spacer polypeptide when attached to an integrin-
binding polypeptide comprises~th~ sequence
23 :
CGGAGAGRGDSP.
A polypeptide utilized in the present invention
can be connected together to form a polymer (synthetic
multimer) comprising a plurality of polypeptide
repeating units. Such a polymer typically has the
adv~ntage of increased exposure of the cell
recognition site for the potential binding to
integrins.
A polymer for use in this invention can be
prepared by synthesizing a polypeptide as discussed
-- before, and including a cysteine residue at both the
amino- and carboxyl-termini to form a "diCys-
terminated'l polypeptide. After polypeptide synthesis,
in a typical laboratory preparation, 10 mg of the
diCys polypeptide (containing cysteine residues in un-
oxidized form) are dissolved in 250 ml of 0.1 M
ammonium bicarbonate buffer. The dissolved diCys-
terminated polypeptide is then air oxidized by
stirring the resulting solution gently for a period of
about 18 hours in the air, or until there is no ~;
detectable free mercaptan by the Ellman Test. [See
Ellman, Arch. Biochem. Biophys., 82:70-77 (1959).] ;
The polymer so prepared contains a plurality of
the synthetic polypeptide repeating units that are
randomly bonded together by cysteine residues. Such
polymers typically contain their polypeptide repeating
units bonded together in a head-to-tail manner as well
as in head-to-head and tail-to-tail manners; i.e., the -
amino-termini of two polypeptide repeating units can ;
be bonded together through a single cysteine residue
as can two carboxyl-termini since the linking groups
at both polypeptide termini are identical.
Alternatively, the polypeptides can be connected
in numerous branched polymer configurations to provide -~
a similar advantage as above for the polymer that
24
contains linear repeating units.
In a further embodiment, it is contemplated that
different integrin-binding polypeptides can be -
operatively attached to a single antibody molecule.
For example there can be two or more different
polypeptides that are independently linked as
monomers, polymers or branched polymers to the ~
antibody molecule by the same linkage means descri~ed - -;,
above for the polymers. In this embodiment each
polymer is comprised of different integrin-binding
c polypeptide monomers.
- C. Therapeutic Methods and Compositions
The present invention contemplates a
polypeptide-antibody conjugate and compositions
containing said conjugate wherein said conjugate
comprises an antibody molecule operatively attached to
an integrin-binding polypeptide, said antibody
molecule being capable of immunoreacting with an
adhesitory cell surface antigen and said polypeptide
being a polypeptide described herein having adhesion
inhibiting properties.
Preferably, the polypeptide-antibody conjugate
(- is an RGD-antibody in which said conjugate contains a
polypeptide comprising an amino acid residue sequence
of about 5 to about 50 residues in length that
includes a sequence having the formula: -RGD-, and
more preferably having the formula: -GRGDSP-.
In another embodiment the polypeptide-antibody
conjugate is a YIGSR-antibody in which said conjugate ~;
contains a polypeptide comprising an amino acid
residue sequence of 5 to about 50 residues in length ~ ~-
that includes a sequence having the formula: -YIGSR-.
The present invention further contemplates that
a polypeptide-antibody conjugate of the present
invention and compositions containing said conjugate
96~:
can be used in a method to inhibit the attachment of
adhesitory cells to an adhesion protein-containing
surface.
The polypeptide-antibody conjugate of the
present invention can be used in a pharmaceutically
acceptable composition that, when administered to a
subject in a therapeutically effective amount, is
capable of inhibiting the ability of an integrin
molecule, present on adhesitory cells, to bind to an
RGD-containing matrix protein (adhesion protein).
That inhibition of integrin is believed to result in
the decreased capacity for attachment (adhesion) by
cells containing an integrin molecule to other
adhesion protein-containing cell surfaces or tissue
substrates in the body. Thus, in vivo administration ~;
of a polypeptide-antibody conjugate composition of the
present invention can be used to inhibit cell adhesion
in a specific manner depending upon the choice of
antibody molecule to be included in the conjugate as
disclosed further herein.
For example, the use of a polypeptide-antibody
conjugate containing a monoclonal antibody molecule
( that immunoreacts with the disialoganglioside GD2, a
molecule present on a variety of tumor cell types, ' ~`
inhibits tumor cell adhesion and tumor metastasis.
This embodiment is effective to inhibit growth and ;
metastasis of tumor cells because those events depend
on tumor cell attachment, which attachment is ;;
specifically inhibitediby the adhesion-inhibiting ;
polypeptide presènt in the conjugate that is targeted
to those tumor cells having GD2 as a cell surface
antigen by means of the anti-GD2 antibody present in -~
the conjugate.
The use of a polypeptide-antibody conjugate ~ ;~
containing a monoclonal antibody that immunoreacts ;
...: .
::; ~
., ~ :-
--` 20~
26
with a platelet surface antigen is also contemplatedto inhibit platelet adhesion. It is preferred that
the monoclonal antibody immunoreact with the platelet
glycoprotein GPIIb/IIIa, a platelet aggregation- i
dependent molecule. This use is effective to inhibit
blood coagulation because coagulation depends in part
on the ability of platelets to aggregate by means of
cell adhesion.
Other cell adhesion inhibitory uses of the
claimed polypeptide-antibody conjugate will be
apparent to one skilled in the cell adhesion arts and
depend upon the selection of specific monoclonal
antibodies.
It is therefore seen that a polypeptide-antibody
conjugate of the present invention is useful in a
variety of methods for inhibiting cell adhesion and
for inhibiting the processes that are dependent upon a
particular cell's ability to adhere to matrix
proteins.
Thus, the present invention contemplates a ~ -
method for inhibiting the attachment of an adhesitory
cell to an RGD-containing matrix comprising
administering to a subject a therapeutically effective
amount of a polypeptide-antibody conjugate described
herein. Preferably the conjugate is an RGD-antibody
or YIGSR-antibody.
Where the adhesitory cell is a tumor cell, the
invention contemplates a method for inhibiting tumor
cell adhesion and tumor metastasis.
Where the adhesitory cell is a platelet, the
invention contemplates a method for inhibiting
platelet adhesion, platelet aggregation and platelet~
dependent blood coagulation.
The preparation of therapeutic compositions
which contain polypeptide-antibody conjugate molecules
r~ 20~69~
as active ingredients is well understood in the art.
Typically, such compositions are prepared as
injectables, either as liquid solutions or
suspensions, however, solid forms suitable for
solution in, or suspension in, liquid prior to
injection can also be prepared. The preparation can
also be emulsified. The active therapeutic ingredient
is often mixed with excipients which are ~;
pharmaceutically acceptable and compatible with the
active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol,
or the like and combinations thereof. In addition, if
desired, the composition can contain minor amounts of
auxiliary substances such as wetting or emulsifying
agents, or pH buffering agents which enhance the
effectiveness of the active ingredient. ~
As used herein, the phrase "pharmaceutically ;~ ~;
acceptable" refers to molecular entities and
compositions that do not produce an allergic or
similar untoward reaction, such as gastric upset,
dizziness, and the like, when administered to a
subject.
A pqlypeptide-antibody conjugate can be
formulated into the therapeutic composition as
neutralized pharmaceutically acceptable salt forms.
Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of ;
the polypeptide-antibody conjugate molecule) and which ~
are formed with inorgànic acids sùch as, for example, ~ ~ ;
hydrochloric or phosphoric acids, or such organic ~ ~
. . .
acids as acetic, oxalic, tartaric, mandelic, and the
like. Salts formed with the free carboxyl groups can ;
also be derived from inorganic bases such as, for
.- : . ~, -:
example sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as
:
~ . ' '
' "
201~9G2
28
isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine, and the like.
The therapeutic polypeptide-antibody conjugate
molecule-containing compositions are conventionally
administered intravenously, as by injection of a unit
dose, for example. The term "unit dose" when used in
reference to a therapeutic composition of the present
invention refers to physically discrete units suitable
as unitary dosages, each unit containing a
predetermined quantity of active material calculated
to produce the desire therapeutic effect in
association with the required diluent; i.e., carrier,
or vehicle.
Therapeutic polypeptide-antibody conjugate-
containing compositions can also be administeredtopically, as by means of a salve or ointment, or by
application of a packet or patch containing the -
composition in a formulation suitable for diffusion of
the active ingredient into the contacted tissue as is
well known.
The polypeptide-antibody conjugate compositions
administered typically contain 0.1-95% of active
ingredient, preferable 25-70
The compositions are administered in a manner
compatible with the dosage formulation, and in a
therapeutically effective amount. The quantity to be
administered depends on the subject to the treated,
capacity of the subject's system to utilize the active
ingredient without adverse side affects, and degree of
inhibition of interaction desired between an integrin ~`
and an adhesive protein. ~ ;
The phrase "therapeutically effective amount" -
when used herein refers to an amount of a polypeptide-
antibody conjugate sufficient to measurably inhibit a
par~icular cell's ability to adhere to an adhesion
;~016~6~ ~
29
protein-containing substrate. Typically, that ability
is measured in vitro by an adhesion assay, such as .s
described in Example 4. ;
Measuring for a therapeutically effective amount
by means of the in vitro adhesion assay depends on the
type of cell to be treated, which in turn depends on
the particular antibody molecule contained in the
polypeptide-antibody conjugate. Thus, the cells to be
utilized in the adhesion assay are of a cell type that
contain an adhesitory cell surface antigen with which
the antibody molecule immunoreacts, preferably the
same cell type as is to be treated by the use of the
contemplated composition, and more preferably the
cells utilized are a population of cells obtained from
the subject to be treated.
A therapeutically effective amount is an amount
sufficient to cause a decrease in the cell's ability
to adhere in the in vitro adhesion assay by at least ~ ~-
10 percent, and preferably by about 50 percent. That
amount can be expressed as an effective concentration
as determined by the in vitro assay. Thus, a ;;
therapeutically effective amount can be expressed as
that amount sufficient to deliver an effective -
concentration to the subject's blood.
Precise amounts of active ingredient required to
be administered depend on the judgment of the
practitioner and are peculiar to each individual.
However, suitable dosage ranges are of the order of
0.1 to liO milligrams, preferably one to several
milligrams, of active ingredient per kilogram
bodyweight of subject per day, and depend on the route
of administration. Suitable regimes for initial
administration and booster shots are also variable,
but are typified by an initial administration followed
by repeated doses at one or more hour intervals by a
~,
.,.~. . . . ... . . . . . . . ... . . . . .
i~
subsequent injection or other administration.
Alternatively, continuous intravenous infusion
sufficient to produce effective intravascular
concentrations are contemplated, preferably
intravascular concentrations of about ten nanomolar to
about ten micromolar.
D. Chimeric Antibodies
The present invention also contemplates a
chimeric antibody that is capable of immunoreacting
with an adhesitory cell surface antigen and inhibiting
integrin-mediated cell adhesion.
A "chimeric antibody" as used herein refers to a
type of antibody molecule that contains the amino acid
residue sequence of an integrin-binding polypeptide of
the present invention as a part of the antibody
molecule's primary structure, eg., amino acid residue
sequence. As an antibody molecule, a chimeric
antibody is comprised of heavy and light chains, at ~ `
least one chain of which is a hybrid protein molecule ~ ~ -
. .
having an antibody combining site-forming fragment
that is fused to an integrin-binding polypeptide. ;~
An antibody combining site-forming fragment as ~-
used herein refers to a linear series of amino acid
residues that correspond to the variable region ;~
portion of either a heavy or light chain of an ;
antibody molecule that contributes to the structure ~
known as the antibody combining cite. ~`
A hybrid protein molecule refers herein to a
molecule that ca~tains a continuous length of amino
acid residues comprising a first length corresponding
to an antibody combining site-forming fragment fused
to a second length corresponding to an integrin-
binding polypeptide.
The term "fused" refers to the manner in which
the fragment and the polypeptide are associated and is
~ 201~9G~:
- 31
used to distinguish a chimeric antibody from a
polypeptide-antibody conjugate in which the ;~
polypeptide is associated with the antibody molecule
by operative attachment. For a hybrid protein
molecule, the fusion refers to the peptide bond
between adjacent amino acid residues, one adjacent
residue being the carboxyl-terminal residue of the ` ;~
first length and the other adjacent residue being the
amino-terminal residue of the second length.
Although a chimeric antibody typically contains
a single integrin-binding polypeptide component fused
to each antibody combining site-forming fragment, it
is not to be construed as so limited. For example, a
hybrid protein can contain one antibody combining
site-forming fragment that is fused to one or more
integrin-binding polypeptides. Further, the included
polypeptides can be polymers comprised of repeating or
different integrin-binding polypeptides, or both.
In preferred embodiments, the integrin-binding ; ~ -
2C polypeptide portion of a chimeric antibody is the same ;
polypeptide as described herein as a component of the
polypeptide-antibody conjugate. -~
A hybrid protein can additionally contain more
than two lengths, such as where the first and second
lengths are followed by a third length of amino acid
residues in which the amino-terminal residual of the
third length is fused to the carboxyl-terminal residue
of the second length. The third length can have an
amino acid se~uence that corresponds to a second
integrin-binding polypeptide, a portion of an antibody
molecule or a portion of a third protein molecule
unrelated to the first two.
In a preferred embodiment a third length has an
~mino acid sequence that corresponds to a portion of
the Fc region of an antibody molecule. Typically the
2~ ~9~ ~
resulting hybrid protein in this embodiment has a
structure.in which the integrin-binding polypeptide
comprising the second length is inserted into an
intact (native) heavy chain of an antibody molecule so
as to interrupt the native heavy chain molecule and
produce a first length that includes an antibody
co~bining site-forming fragment and a third length
that includes a portion of the Fc region.
From the foregoing it can be seen that the
location of the interrupting second length within the ;-~
antibody heavy chain amino acid residue sequence can - -
vary along the length of the Fc portion of the native
heavy chain. In preferred embodiments, the site of
the interruption by the second length will reside
nearer to the carboxyl terminus than to the hinge
region that makes the amino terminus of the Fc region.
More preferably, however, the second length is fused
to the carboxyl terminus of the heavy chain of an
antibody molecule. ;~
The general structure of antibody molecules, and ~-~
the terminology used herein to identify the heavy and
light chains, the hinge region and the Fc region of
antibody molecules (immunoglobulins) are well known.
See, for example Paul et al., "Fundamental
Immunology," fourth printing, Raven Press, N.Y. ~
(1984), at pages 7 and 150. `
The combination of an antibody combining site
and an integrin-binding polypeptide in the chimeric
antibody provides the same uses and benefits as
described herein above for a polypeptide-antibody
conjugate.
Therefore the present invention contemplates a
chimeric antibody that contains at least one hybrid
protein molecule having an antibody combining site-
forming fragment fused to at least one integrin- `
X0~1 ~,9~
33
binding polypeptide such that the hybrid protein
molecule for~s an antibody combining site that is
capable of immunoreacting with an adhesitory cell
surface antigen and thereby inhibiting cell adhesion. ~-
In preferred embodiments, a chimeric antibody
contains a polypeptide having an amino acid residue
sequence of about 5 to about 50 residues in length
that includes a sequence having the formula: -RGD-,
and more preferably includes residues represented by
the formula: -GRGDSP-.
A preferred chimeric antibody additionally
contains an antibody combining site-forming fragment
portion of an antibody molecule whose primary amino
acid residue sequence corresponds to the amino acid
residue sequence of a monoclonal antibody as described ;~
herein. Exemplary monoclonal antibodies are shown in ;
Table 1. A preferred chimeric antibody contains an
antibody combining site-forming fragment whose amino
acid residue sequence corresponds to the amino acid ~;
residue sequence present in the monoclonal antibody
Mab 142A.
The preparation of chimeric antibodies of the
present invention can be accomplished using methods
well known that involve the cloning of immunoglobulin
genes, the subsequent manipulation of the cloned genes
to incorporate integrin-binding polypeptide encoding
sequences, the introduction o~ the manipulated genes
into a suitable expression vector and expression
medium, and the production of the resulting hybrid
protein molecules to form chimeric antibodies. The
above-recited preparative steps are routine
recombinant DNA cloning, manipulation and expression
procedures, and are described in more detail herein
below. The inventive aspect of a chimeric antibody
does not reside in the techniques for preparing a ;~-
~0~9~ :
34
chimeric antibody, but rather in the resulting
manipulated gene, and it's expressed antibody molecule
having a combination of integrin-binding polypeptide
fused to an antibody combining site that immunoreacts ;
with an adhesitory cell surface antigen.
The cloning of immunoglobulin protein-encoding
genes, and their manipulation to form recombined DNA -
molecules that encoded antibody combining sites fused
to other heterologous protein fragments are generally
known methods. Once prepared, these recombined DNA
molecules are introduced into an expression medium
that produces assembled antibody molecules that are
capable of immunoreaction with the same specificity as -~
the antibody produced by the antibody producing cell
from which the immunoglobulin protein-encoding genes
were isolated. See, for example Roberts et al.,
Protein Enqineerinq, 1:59-65 (1986), Morrison,
Science, 229:1202-07 (1985), U.S. Patent No.
4,474,893, and published patent application Nos.
EP 0125023, EP 0239400 and Wo 89/00999, --
For the isolation of an immunoglobulin protein-
encoding gene in preparation of a chimeric antibody,
the gene can be obtained from any cell that produces
an antibody molecule that immunoreacts with an
adhesitory cell surface antigen. A preferred cell is
a hybridoma cell. Hybridomas can be prepared as
~; disclosed herein, or obtained from commercial sources.
Exemplary hybridomas are listed in Table l.
For exampleslof general recombinant DNA cloning
methods, see Molecular Clonina, Maniatis et al., Cold
;~ Spring Harbor Lab., N.Y., 1982; DNA Cloninq, Glover,
ed., IRL Press, McLean, VA (1985). For the genomic
cloning and expression of immunoglobulin genes in
lymphoid cells, see Neuberger et al., Nature, 312:604-
8 (1984); Ochi, et al., Proc. Natl. Acad. Sci. USA,
~: '
' ', ' ~',`''-'.':
2~)16~6~:
~;
80:6351-55 (1987); and oi et al., Proc. Natl. Acad.
Sci. USA, 80:825-29 (1983). For cloning of
immunoglobulin genes from hybridoma cells, and their
expression in Xeno~us oocytes, see Roberts et al.,
Protein Engineerina, 1:59-65 (1986), and see Wood et
al., Nature, 314:446-9 (1985) for their expression in
yeast.
A DNA segment is included as a part of the above
described manipulated recombinant DNA molecule and ~ ~
that segment has a nucleotide sequence that encodes at ~ ~-
least a polypeptide whose amino acid residue sequence
corresponds to the amino acid residue sequence of an ~;
integrin-binding polypeptide of the present invention.
A DNA segment may further encode an antibody
combining site-forming fragment. The nucleotide
sequence of the DNA segment can correspond to the
sequence of an antibody combining site-forming
fragment of an immunoglobulin protein cloned as
described above, or the sequence can correspond, for
example, to known sequences. Exemplary antibody
combining site-forming fragment-encoding nucleotide
sequences are described by Kabat, et al. in "Sequences
of Proteins of Immunological Interest", 4th Edition,
National Institutes of Health, Bethesda, MD (1987).
The DNA segment that encodes an integrin-binding
pvlypeptide, an antibody combining siteforming
fragment, or both, can be synthesized by chemical
techniques, as by, for example, the phosphotriester
method of Matteuccil etlal., J. Am. Chem. Soc.,
103:3185 (1981). Once prepared the segment is
included n the recombinant DNA molecule that is being
manipulated to encode and express the entire hybrid ~ ~
protein molecule. ~ ~;
The present in~ention therefore also
contemplates a recombinant DNA molecule that encodes
`'',:: ,.''.
" ' ~: - .' `' '''
.. , , . . . , - - , . .. . .
2~)1696~
36
the hybrid protein disclosed herein and is useful for
the preparation of a chimeric antibody of the present
invention.
Thus in one embodiment the present invention
contemplates a recombinant DNA molecule that encodes a
hybrid protein molecule comprising a first DNA segment ~
encoding an antibody combining site-forming fragment - i
operatively linked in phase to a second DNA segment
encoding an integrin-binding polypeptide, such that
the recombinant DNA molecule is capable, when present
; in an appropriate expression vector, of expressing a
hybrid protein molecule having an antibody combining ~ -
site that immunoreacts with an adhesitory cell surface
antigen. ~ ~
A DNA segment, as is generally understood and ~ ~-
used herein, refers to a molecule comprising a linear
stretch of nucleotides wherein the nucleotides are
present in a sequence that encodes, through the ~ -
genetic code, a molecule comprising a linear sequence ;
of amino acid residues that is referred to as a
protein, a protein fragment or a polypeptide.
A particularly useful DNA segment is
characterized as including a DNA sequence that encodes
(l) an antibody combining site-forming fragment, or
(2~ a DNA sequence that encodes an integrin-binding
polypeptide.
A recombinant DNA molecule, as used herein,
refers to a molecule that contains two or more DNA
segments operataively linked to produce a third DNA
segment. Thus a recombinant DNA molecule (rDNA) is a
hybrid DNA molecule comprising at least two nucleotide
sequences (DNA segments) not normally found in nature. ;~
The phrase "operatively linked" indicates that
the two joined DNA segments are connected by a typical
phosphodiester bond normally found between adjacent
9~
37
nucleotide residues in a DNA sequence. Where the two
linked DNA segments each encode an amino acid residue
sequence of interest, the term further indicates that
the linkage maintains the reading frame of the joined
5 DNA segments so that the two encoded amino acid
residue sequences can be translated from the hybrid ~
DNA molecule in phase as a single larger hybrid -
protein.
A recombinant DNA molecule of the present
lo invention contains at lPast a DNA segment that encodes
an antibody combining site-forming fragment
operatively linked in phase to a DNA segment that
encodes an integrin-binding polypeptide to form a
hybrid protein encoding DNA segment. Typical
recombinant DNA molecules also contain a vector
operatively linked to the hybrid protein encoding DNA
segment.
As used herein, the term "vector" refers to a
DNA molecule capable of autonomous replication in a
cell and to which another DNA segment can be
operatively linked so as to bring about replication of
the attached segment. Vectors capable of directing
the expression of a hybrid protein-encoding DNA ~ ~:
segment are referred to herein as "expression
vectors". The recombinant DNA molecule having a
hybrid protein-encoding DNA segment operatively linked
to an expression vector-containing ~NA segment -
provides a system ~or expressing a hybrid protein
product in a medium compatible with the included
expression vector. ;
The choice of vector to which a recombinant DNA
molecule of the present invention is operatively
linked depends directly, as is well known in the art,
on the functional properties desired, e.g., protein ;
expression, and the host cell to be transformed, these
' ,~. '`'::
'`, '~ '
;~ 9~
38 ~ ~-
being lim~tations inherent in the art of constructing
and expressing recombinant DNA molecules. However, a
vector contemplated by the present invention is at
least capable of directing the replication, and
preferably also expression, of the hybrid protein-
encoding DNA segment included in recombinant DNA
molecule to which it is operativel~- linked.
In preferred embodiments, a vector contemplated
by the present invention includes a procaryotic
replicon, i.e., a DNA sequence having the ability to
direct autonomous replication and maintenance of the
recombinant DNA molecule extrachromosomally in a
~rocaryotic host cell, such as a bacterial host cell,
transformed therewith. Such replicons are well known
in the art. In addition, those embodiment that
include a procaryotic replicon also include a gene
whose expression confers drug resistance to a
bacterial host transformed therewith. Typical
bacterial drug resistance genes are those that confer
resistance to ampicillin or tetracycline.
Those vectors that include a procaryotic
replicon can also include a procaryotic promoter
capable of directing the expression (transcription and
translation) of the hybrid protein-encoding DNA
segment in a bacterial host cell, such as E. coli,
transformed therewith. A promoter is an expre~sion
control element formed by a DNA sequence that permits
binding of RNA polymerase and transcription to occur.
Promoter sequences!compatible with bacterial hosts are -
typically provided in plasmid vectors containing ~ ;~
convenient restriction sites for insertion of a DNA
segment of the present invention. Typical of such
plasmid vectors are pUC8, pUC9, pBR322 and pBR329
available from Biorad Laboratories, (Richmond, CA) and
pPL and pKK223 available from Pharmacia (Piscataway,
~6~
~ .
39
NJ).
Expression vectors compatible with eucaryotic
cells, preferably those compatible with vertebrate
cells, can also be used to form the recombinant DNA
molecules.of the present invention. Eucaryotic cell
expression vectors are well known in the art and are
available from several commiercial sources. Typically,
such vectors are provided containing convenient
restriction sites for insertion of the des~red DNA
segment. Typical of such vectors are pSVL and pKSV-10
(Pharmacia), pBPV-1/pML2d (International
Biotechnologies, Inc., New Haven, ~T) and pTDT1 (ATCC,
#31255).
In preferred embodiments, the eucaryotic cell
expression vectors used to construct the recombinant
DNA molecules of the present invention include a
selection marker that is effective in an eucaryotic
cell, preferably a drug resistance selection marker.
A preferred drug resistance marker is the gene whose
expression results in neomycin resistance, i.e., the
neomycin phosphotransferase (neo) gene. Southern et
al., J. Mol. Appl. Genet., 1:327-341 (1982). ;~ -
The use of retroviral expression vectors to form ~
the rDNAs of the present invention is also -
contemplated. As used herein, the term "retroviral
expression vector" refers to a DNA molecule that
includes a promoter sequence derived from the long
terminal repeat (LTR) region of a retrovirus genome.
In preferr~dlembodiments, the expre$sion vector
is typically a retroviral expression vector that is
preferably replication-incompetent in eucaryotic
cells. The construction and use of retroviral vectors
has been described by Sorge et alO, Mol. Cell. Biol.,
4:1730-37 (1984).
Also contemplated by the present invention are
: ''' ;"~'~
RNA equivalents of the above described recombinant DNA ~;
molecules.
EXAMPLES
The following examples are intended to
illustrate, but not limit, the present in~ention.
1. Preparation of Monoclonal Antibodies
The hybridoma cell line 1418, on deposit with
the ATCC as HB 9118, produces a monoclonal antibody
(Mab) that immunoreacts with the disaloginglioside
GD2. The hy~ridoma cell line 142A was isolated as one
of several isotype switch variants from a culture of
hybridoma 1418 using the floursecence activated cell
aorting (FACS) techniques described by Kipps et al.,
J. Ex~. Med., 161:1-17 ~1985). Hybridoma 142A
produces a monoclonal antibody that also immunoreacts
with GD2. Hybridoma 1418 and the FACS-isolated
- hybridomas were then characterized to determine the
isotype of their produced antibodies using the
following isotyping ELISA assay.
Fifty microliter volumes (1:1000 dilutions in
PBS) of rabbit anti-mouse IgGl, IgG2a, IgG2b, IgG3 or
IgM (Southern Biotech Associates, Birmingham, AL) were
;~ plated per well of a flat-bottom polyvinyl chloride
microliter plate (Dynatech, Alexandria, VA) The
plates were then maintained overnight at 37C in a
drying oven. The dried plates were stored at 4C
until use. Prior to the ELISA assay, dried plates
were rehydrated by two washes of two minutes each with -~
wash buffer (lOImillimolar [mM] PBS, pH 7.4,
containing 0.1 percent polyoxyethylene [20] sorbitan
monolaurate [Tween 20] and 0.02 percent Thimerosal
[sodium ethylmercurithiosalicylate; Sigma, St. Louis,
M0]).
Culture supernatant from FACS-isolated
hybridomas were diluted 1:2 in wash buffer, containing
:
2~3~6~
41
0.1% BSA (bovine serum albumin), added at fifty ;
microliters per well and maintained at 4-C for 1 hour.
After 2 wash buffer rinses, fifty microliters (ul) of
horseradish peroxidase-labeled goat anti-mouse
immunoglobulin (Biorad Laboratories, Richmond, CA)
diluted 1:1000 were added to each well and maintained
at 4C for 1 hour.
The substrate used to assay bound peroxidase
activity was prepared just prior to use and consisted
of 400 micrograms/ml o-phenylenediamine (Sigma, St.
Louis, M0) in 80 mM citrate-phosphate buffer, pH 6.0,
containing 0.23 percent H202. After two final wash
buffer rinses, 50 ul of substrate solution was added -~
to each well and color was allowed to develop for 15 ;~
minutes in the dark. Color development was stopped by
adding 25 ul of 4 Normal (N) H2S04 to each well, and ~
the optical density at 492 nanometers (nm3 was -; ~;
measured with Multiskan ELISA Plate reader (Bio-Tek ~ -~
~ . . .
Instruments Inc., Burlington, VA).
Isotype designations were assigned to those ~
hybridoma-produced culture supernatants that generated - ~-
`~ an optical density at 492 nm that was at least 10
times the value obtained for a control non-reactiny
~; antibody. By this assay, Mab 1418 was verified to be ~ `~
an IgG3 isotype and Mab 142A was identified to be an
IgG2a isotype.
~; The monoclonal antibodies Mab 142A were prepared
by introducing a culture of hybridoma 142A cells into
the peritoneal cavity of~a Balbc/byj mouse (Scripps ! `
Clinic Vivarium, La Jolla, CA) and later harvesting
the resulting peritoneal exudate (ascites tumor fluid)
from the mouse by well known techniques. Mab 142A
antibody molecules present in the recovered ascites `~
fluid were further isolated by affinity chromatography
using protein-A Sepharose (Bio-Rad Laboratories,
.~: '`'"'""''`''';
~ 9~
42
Richmond, CA) at a ratio of 1.0 milliliters (ml)
ascites fluid per ml packed Sepharose beads according
to the methods recommended by the manufacturer with
the exception that the elution buffer was adjusted to
pH 5.0 before use. The Sepharose sluant was collected
in fractions and the protein content of the fractions
was determined using the BCA Protein Assay Reagent
available from Pierce Chemical Co. (Rockford, IL).
Peak protein-containtng fractings were pooled to form
purified Mab 142A.
In addition to Mab 142A, the following Mabs were
used to demonstrate the present invention, listed here
with their respective isotypes and sources denoted in
parentheses: Mab 9227 (IgG2a, Dr. D. Cheresh; Scripps
Clinic and Research Foundation, La Jolla, CA,
hereinafter Scripps), directed against chondroitin ~ -
sulfate proteoglycan of human melanoma cells [Cheresh
et al., J. Cell. Biol., 102:688-696 (1986); Harper et
al., J. Immunol, 132:2096-2104 (1984); and Bumol et
al., Proc. Natl. Acad. Sci. USA, 79:1245-1249 (1982)]; ~ `
and Mab KS14, a control antibody that does not
immunoreact with M21 melanoma cells but does
immunoreact with UCLA-P3 cells.
Additional purified monoclonal antibodies were
prepared by the same method as described above for Mab
142A except that hybridomas 9227 and KS14 were
utilized to form purified Mab 9227 and purified Mab
KS14, respectively.
2. Couplinq of ~olY~eptide to Monoclonal
Antibody
A three ml solution containing 14.4 milligrams
(mg) of purified Mab 142A in 100 millimolar (mM)
phosphate buffered saline (PBS; pH 7.8) was prepared
as described in Example 1. About 25 microliters (ul)
of a solution of NHS [75 mM 4-(maleimidomethyl)-1- -;
:
201G96~ --
43
cyclohexane carboxilic acid N-hydroxysuccinimide ester
in N-methylpyrolidone] was admixed with the prepared
Mab 142A-containing solution and the admixture was
maintained while stirring for 3 hours at room
temperature to form an activated Mab 142A-containing
solution. The activated Mab 142A- containing solution ~-
was then applied to a Sepharose G-25 column having a
bed volume of l.o mls an~ pre-equilibrated with 10 ~ -
mls, and the resulting eluant was monitored for
protein content and collected in fractions. Peak
fractions containing protein were pooled to form
activated Mab 142A.
A synthetic polypeptide having the amino acid
sequence Cys-Gly-Gly-Ala-Gly-Ala-Gly-Arg-Gly-Asp-Ser- ~ -
Pro (CGGAGAGRGDSP) was provided by Dr. M.D.
Pierschbacher (La Jolla Cancer Research Foundation, La
Jolla, CA) after having been synthesized by Peninsula
Laboratories (San Carlos, CA). Ten mg of the - ~;
polypeptide was admixed as a solid to the above~
prepared activated Mab 142A and the admixture was
maintained while stirring for 3 hours at room
temperature to form a reaction admixture. The
reaction admixture was then applied to a Sephadex
G-25 column having a bed volume of 1.0 mls that was
pre-equilibrated with 10 mls and the resulting eluant
was monitored for protein as before and peak fractions
containing protein were collected to form a -
polypeptide Mab 142A conjugate (Mab 142A-conjugate).
Polypeptide-Mab conjugates were also prepared by
the above procedure using purified Mab 9227 or control
Mab KS14 prepared as described in Example 1, in place
of purified Mab 142A to form Mab 9227-conjugate and
control Mab-conjugate, respectively.
Polypeptide-Mab conjugates can also be prepared ;
by the above methods of Examples 1 and 2 using the
~:
~ :.
~ 2~16~2
.
44
monoclonal antibodies produced by hybridoma 1418, 126,
MB3.6, 9227, LM142Or LM609.
3. Monoclonal AntibodY Coniuaate Bindinq
To Cultured Cells
M21 human melanoma cells were kindly provided by
Dr. D.L. Morton (University of California at Los
Angeles, hereinafter UCLA) and grown as a suspension
culture in growth medium tRPMl 1640 containing 2mM L-
glutamine and 50 mg/ml gentamicin sulfate; all from
GIBCO Laboratories (Grand Island, NY)] containing 10
percent fetal calf serum (FCS) at 37C in 7.5%
C0~92.5% air. UCLA-P3 human lung adenocarcinoma
cells were provided by Dr. Martin (UCLA) and grown as
a suspension culture as above for M21 cells.
Cultured M21 cells were washed twice in normal
saline and plated in a flat-bottom polyvinyl
microtiter plate (Dynatech, Chantilly, VA) at 5 x 104
cells per well in 50 ul in growth medium containing
10% FCS. Thereafter the plates were maintained at :
37C for about 12-18 hours to dry the plates and to
form dried ~21 plates. Dried UCLA-P3 plates were
similarly prepared using cultured UCLA-P3 cells.
Dried plates were rehydrated by two washes each
~ comprising first the addition of wash buffer tlOmM
;~ 25 PBS, pH 7.4, 0.1% Tween 20 (polyoxyethylenesorbitan
monolaurate) 0.02% thimerosal (sodium
ethylmercurithiosalicylate)], then the maintenance of
the plates at room temperature for two minutes, and
finally~the removal of the liquid by inverting the
plates.
Purified Mabs prepared in Example 1 and Mab- ;
~ conjugates prepared in Example 2 each were then
;~ serially diluted to the final concentrations indicated
in Table 2 using diluent buffer t1.10 (v/v) in wash
buffer containing 0.1~ BSA (bovine serum albumin)] and
~ ~ :
50 ul thereof was admixed into each well o~ the
rehydrated plates to form immunoreaction admixtures.
Th~ plates were maintained for one hour at 4-c on a
gyroshaker, emptied of their liquid contents by `
inversion and shaking, and then washed twice as
described above to isolate solid-phase bound ;
immunoreaction products. Afterwards, 50 ul of a
solution containing peroxidase-linked, goat anti-mouse
Ig (Tago, Burlingame, CA) diluted 1:1000 (v/v) in
diluent were admixed into each well of the plates and ~
maintained for l hour at 4C to allow formation of - ~ ~-
labeled immunoreaction products. The plates were
again emptied by inversion and shaking, washed twice
as described above, and 50 ul of freshly prepared
chromogenic substrate solution t400 ug/ml 0-phenylene
diamine (Sigma Chemical Co., St. Louis, MO), 80 mM
citrat~-phosphate buffer, pH 5.0, 0.012% (v/v) H202 ;~
(Eastman Kodak, Rochester, NY)] was admixed into each ~ ~-
well, The plates were then maintained in the dark for
30 minutes after which time 15 ul of 4N H2S04 was
admixed into each well. The optical absorbance at 492
nm (A492) of the resulting solutions in each well was -. ;~
then measured with a Multiscan ELISA plate reader
(Flow Laboratories, McLean, VA).
Results of studies of binding of purified Nab ;
;~ ("Free") and Mab-conjugate ("Conjugate") by
immunoreaction with adhesitory cell surface antigens
presant on M21 and UCLA-P3 cells are shown in Table 2, ;
,: ,:, .,, .:
below.
Table 2
`~ Binding of Polypeptide Monoclonal Antibody Conjugates
To Cells
A. M21 Cell
.
46 ~ 9~
Free~ _Coniuqatesb
[Mab]C 9227 142A Control 9227 142A Control
5.0 1.171 1.995 0.147 1.088 1.687 0~994 ::
l.o 1.146 1.602 0.114 0.985 1.758 0.951
0.5 1.142 1.590 0.071 1.023 1.786 0.963
0~1 1.101 1.402 0.032 0.929 1.471 0.554 --
0.05 0.510 0.105 0 0.408 0.078 0 - ~
0.005 0.261 0.015 ~s 0. 121 0.017 0 . ;
O O O O O O O
" . ' ,','.';
B. UCLA-P3 Cells :.~:
Ereea Coniuqatesb
[Mab]C 9227 142A Control 9227 142A Control -~
5.0 Ø090 0.256 1.754 0.234 0.345 1.858 : ~:
1.0 0.093 0.116 2.192 0.122 0.174 2.076 ;
0.5 0.077 0.149 2.127 0.126 0.155 1.996
0.1 0.022 0.081 1.815 0.070 0.085 2.047
0.05 0.030 0.085 0.241 0.001 0.114 0.335
0.005 0.056 0. 042 0. 103 0.050 0.070 0.148
;: 20 ~ 0.050 0.037 0.021 0.013 0.034 0.017
~:; a "Free" designates the use of indicated purified : :~
Mab's (Mab 9227, Mab 142A or Control Mab) prepared
as described in Example 1 and being free of
polypeptide conjugate.
b "Conjugate" designates the use of the ~ame Mab's as
indicated for "Free" except that the Mab's have
been operatively attached (conjugated) to ;
polypeptide CGGAGAGRGDSP as described in Example 2.
c "[Mab]" indicates the final protein concentration
of the serially diluted Mab (free) or Mab-conjugate
9~.~
47
(conjugate) preparation. ~-
Table 2 shows that both free and conjugated Mab ;
9277 were ~ble to immunoreact with chondroitin sulfate
proteoglycan present on M21 cells, as were the Mab
142A preparations able to immunoreact with ganglioside
GD2 on those same cells. For both of these Mabs, the
immunoreaction was only slightly decreased when using
Mab-conjugate as compared to free Mab. Similarly,
both free and conjugated control Mab KS14 were able to
immunoreact with the target antigen present on UCLA-P3
cells. These data indicate that the operative ;
attachment of an integrin-binding polypeptide to an
antibody molecule by the disclosed linking methods
does not significantly interfere with the Mab-
conjugates ability to immunoreact with its target
antigen when present on adhesitory cell surfaces. ~
Control Mab KS14 that does not immunoreact with M21 - ;
cells was observed to bind to M21 cells upon
polypeptide conjugation. Because this increased -
binding was observed only after formation of a Mab-
conjugate it is believed that the binding results from
; an interaction between the RGD-containing polypept-.de
present on the Mab-conjugate and the RGD-directed
adhesion receptors present on the M21 cells.
;~ It is not known why the Mab 9227 or Mab 142A
conjugates do not also show an increase in binding to
UCLA-P3 by the mechanism proposed above for Mab KS14
on M21 cells. However,l UCLA-P3 cells can have
3a significantly fewer RGD-directed adhesion receptors
than M21 cells as measured by the assay.
4. Monoclonal Antibody C~n~ ates Inhibit
Cell Adhesion In vitro
The following cell adhesion assay was performed to
characterize the ability of monoclonal antibody (Mab)
9~
48 -
conjugates to inhibit cell adhesion.
The M21 cells were grown in suspension culture in ~ ;
RPMI 1640 growth medium containing 10% fetal calf ;
serum ~FCS) at 37C with 7.5% C0~92.5% air. These ~;
cells were metabolically labeled in leucine-free
growth media containing 50 microcurie tuCi)/ml 3H
leucine (ICN, Irvine, CA) for 72 hours at 37C. ~
Thereafter labeled cells were washed by centrifugation ;
in growth medium containing 1% FCS to remove
unincorporated radiolabel forming 3H-leucine-labeled
M21 cells. -~
Approximately 5 x 103 3H-leucine labeled M21 cells -~
were resuspended in 100 ul growth medium containing
various concentrations of a Mab conjugate prepared as
described in Example 2 to form an immunoreaction
admixture. As controls, labeled cells were
resuspended in growth medium alone, or a control
antibody was used. The control Mab is known as
activated Mab and was prepared as described in Example
2.
The admixture was then maintained for 1 hour at 4DC
to allow formation of Mab-M21 cell immunoreaction
products. The cells were then washed by two cycles of
~ irst a low speed centrifugation at 400 x g followed
;~ 25 by resuspension of the resulting pellet using growth
medium containing 1% FCS. After the wash the cells
were resuspended in growth medium to form antibody
treated M21 cells. ~
The wells of polystyrene microtiter plates (96- ! ' ;'''~,
well; Flow Laboratories, McLean, VA) were coated with
matrix proteins (adhesion substrates) by admixing
solutions of PBS (pH 7.2) containing 5 ug/ml of an
adhesion substrate to a well. The wells were
maintained overnight at 25DC to allow adsorption of
the proteins onto the wells. Matrix proteins used
2~6~;% . ~-
- 49 ;
included fibrinogen, provided by Dr. E. Plow (Research
Institute of Scripps Clinic, La Jolla, CA hereinafter ~ -~
RISC); von Willebrand factor, provided by Drs. Z.
Ruggeri and T. Zimmerman (RISC); and human ;~
fibronectin, provided by Dr. M. Pierschbacher (La
Jolla cancer Research Foundation, La Jolla, CA, -
hereinafter LCRF).
Fifty ul of growth medium containing 5 x 103
antibody treated M21 cells were admixed in an adhesion
substrate-coated well to form an adhesion-reaction
admixture. The admixtures were maintained at 37C in
a humidified incubator for 20 minutes to allow cell
adhesion to occur, at which point the plates were
inverted to removed growth medium and non-adhered
cells. A duplicate set of plates were similarly
prepared and maintained for an adhesion period of 90
minutes. All wells were washed twice with 150 ml PBS
(pH 7~2) to assure removal of unattached cells. The -~
remaining attached cells were harvested by adding 100
ul of Trypsin/EDTA (lX; Gibco Laboratories, Grand ;~
Island, NY) to each well, incubating the cells at 37C
for 30 minutes and then removing the cells and
collecting tkem on glass fiber filters using a Skatron
automated cell harvester (Skatron Instruments,
Sterling, VA) according to instructions provided by
the manufacturer. The fiber filter disks were place
into vials containing 3 ml of liquid scintillation
cocktail and the amount of radioactive label present
determined as counts pçr;minute in a liquid
scintillation counter. Results were expressed as the
total number of cells (count per minute) that adhered
(bound) to the matrix protein at the designated
concentrations of added Mab-conjugate or control Mab.
As shown in Figures 1 and 2, M21 cell adhesion was
inhibited by Mab 142A conjugate during either a 20
' ~ ~ :
'~
. ~.
-
minute (Figure 1) or 90 minute (Figure 2) adhesion
period, when using either von Willebrand factor (vWF)
for fibrinogen (Fb) as the adhesion substrate.
Inhibition was observed at concentration less than 2
ug Mab conjugate per ml. Adhesion inhibiting potency -
(IC50) can be expressed as the concentration of Mab -
conjugate sufficient to effect a 50% decreased in
adhesion when compared to the adhesion obtained with
no added Mab conjugate. Thus, the ICso for Mab 142A
con~ugate during a 20 min adhesion time was 4 ug/ml
- for adhesion to vWF and 0.5 ug/ml for adhesion to Fb.
Similarly, the IC50 for the conjugate during a 90
minute adhesion time was 15 ug/ml for adhesion to vWF
and 0.5 ug/ml for adhesion to Fb.
Using the in viiro cell adhesion assay, essentially
as described above, soluble polypeptide CGGAGAGRGDSP
from Example 2 was added at various concentration to
the cell suspensions in place of the Mab 142A
conjugate. The results of those adhesion measurements ;~
is expressed as adhesion inhibiting potency (IC50) in ~ ;
Table 3, below.
Table 3
EFFECTS OF SOLUBLE OR MAB 142A-CONJUGATED ~:~
CGGAGAGRGDSP ON M21 CELL ATTACHMENT TO
FIBRINOGEN, VON WILLEBRAND FACTOR OR FIBRONECTINb
:
. SOLUBLE MAB 142A
TIME PEPTIDE (uM) CONJUGATE (uM)
,; , ~~.
VWF
20 min. 10 0.24
90 min. 55 0.91` ~
Fibrinoqen ~;
~, , ,
~ 35 20 min. N/Tc N/T
~ ' .;' ;`
V~t6~
51
so min. 2.7 0.03
Fibronectin
2D min. 106 >18
90 min. >500 >18
. ,
a Length of time cells were maintained for adhesion
to occur.
b Effects on cell attachment is expressed as a
micromolar (uM) concentration of polypeptide -
present in solution or on the conjugate sufficient
to cause a 50% decrease in adhesion when compared
to adhesion with no added polypeptide. -
c N/T indicates not tested.
Table 3 shows that at each adhesion time, 20 or 90 ~;
minutes, and for all adhesion substrated tested,
there was a significant reduction in the concentration
of polypaptide necessary to inhibit cell adhesion when
~; 20 the polypeptide was conjugated.
Thus although it is known that RGD-containing
polypeptides and other adhesion protein derived
polypeptides have the ability to inhibit cell
adhesion, the data in Table 3 shows that the adhesion
inhibition potency of these polypeptides was improved
by conjugation to an antibody that immunoreacts with
an adhesitory cell surface antigen.
The foregoing specification, including the specific
iembodiments and examples, it intended to be
illustrative of the present invention and is not to be
taken as limiting. Numerous other variations and
modificat;ons can be effected without departing from ;~
the true spirit and scope of the present invention.
