Note: Descriptions are shown in the official language in which they were submitted.
~ Wo 95/16775 2 1 7 8 9 5 0 PCT/US94/14297
TIJMOR CEL~ FUSIONS AND METIIODS
FOR USE OF SUC~ TI~MOR CELL FUSIONS
FIELD OF TI~E INVENTION
The present invention relates to products and
methods useful for tumor immunotherapy.
BA~cvuNJ OF T/IE lNV~~
A major objective in the field of tumor
immunotherapy is the development of strategies for
enhancing tumor immunogenicity, with potential
application~ for both tumor prevention and cure. To date,
various product~ and methods useful for enhancing tumor
immunogenicity have been reported. ~owever, the methods
described below are not admitted to represent prior art to
the pending claims.
In general, tumors that arise de novo are poorly
immunogenic, thereby escaping host antitumor responses
(Hewitt et al., 33 Br. J. Cancer 241, 1976) .
Methods that have been described f or enhancing
tumor immunogenicity include: (1) using mutagen or drug
- treatment (Van Pel and Boon 79 Prnc~ ~atl. Acad. Sci. USA
4718 , 1982 and Frost el al . , 159 !:r. Ex~ . Med. 1491, 1984);
(2) transfecting with a foreign gene encoding an exogenous
antigen E:uch as influenza hemagglutinin (Fearon et al, 38
Cancer Res. 2975, 1988); (3) reducing the expression of
certain molecules in a tumor that regulate its
diiferentiation state (Tyk~ ;nqk; ~i Ilan, 259 Science 94,
1993); (4) transferring a gene expressing a lymph~-k;n,o
into a tumor, for example, interleukin-2 (Fearon et al.,
... . .. ~
Wo 95/16775 PCTiU594/l4297
21 78950
60 Cell 397, l9g0), interleukin-4 (Tepper et al. , 57 Cell
503, 1989, Golumbek et al., 254 Science 713, 1991),
interleukin-6 (Mullen et al./ 52 Cance~ Re~. 6020, 1992),
interleukin-7 (McBride et al., 52 Cancer Res. 3931, ls92);
(5) transferring a gene e~ressing a cell surface-
associated costimulator into a tumor, for example, B7
(Chen et al., 71 Ç~Ll 1093, 1992; and Townsend et al., 259
Science 368, l9g3); (6) transferring a gene expressing
major histocompatibility complex protein into a tumor
cell; ~7) transferring a gene eæpressing a protein that
,onh~nr~F: the expression =of ~ a ma~or histocompatibility
complex protein in a tumor cell; and (8) transferring a
gene expressing a heat shock protein into a tumor cell
(Luckacs et al ., 178 J. EXP . Med . 343, 1993 ) .
~ ~Antigen-presenting cells (APC) provide molecular
signals including signals mediated by APC-derived soluble
cytokines and APC-derived cell surface costimulators such
as: (1) B7 (Linsley et al_, 87 Proc. Natl. Acad. Sci.
U.S.A. 5031, lg90); (2) ICAM-I (van Seventer et al., 144
.J. Immunol. 4579, 1990); (3) VCAM-I (van Seventer et al.,
174 ~. EYr~. Med. 901, 1991); (4) L~A-3 (van Seventer et
al., 21 Eur. J. Immunol. 1711, 1991); and (5) fibronectin
(Shimizu et al., 145 ~. Tmml~nnl.l 59, 1990; Nojima et al,
172 J. EXP . Med. 1185 , 1990 ; and Davis et al ., 145 ~.
Tmml-nnl. 785, 1990) .
A tumor~ cell, once appropriately modif ied
through genetic manipulation, can itself function as an
APC (Chen et al., 71 Cell 1093, 1992; Townsend et al., 259
Science 368, 1993; Tykocinski & Ilan, 259 Science 94,
1993).
S~IARY OF TIIE LNV~N~L~)N
The pre~ent invention provides products and
methods useful ~or~ ~nh~nci nr tumor immunogenicity. More
specifically, the present invention provides a cell fusion
~ Wo 95/16775 2 1 7 8 9 5 0 PCT/US94/14297
i
product, and methods of using the cell fusion product to
enhance tumor immunogenicity.
The general usefulness of this technology
relates to the prevention and treatment of various
diseases, including cancer. The disea~e may be present in
any animal, including a human. This technology further
embraces a wide range of utilities ;nrll~tlin~ ;n~llrln~ the
production of antibodies in vitro. The present invention
may also be used to induce the production of antibodies in
a variety of animals, including humans.
It has surprisingly been discovered that the
immunogenicity of a tumor cell can be remarkably enhanced
by fusing a membrane of a tumor cell to a membrane from a
another cell with greater immunogenic potential than the
tumor cell. ~rhe phrase "cell fusion partner" is used to
describe any other cell with a greater immunogenic
potential than the tumor cell. Membrane extracts or whole
cells may be fused in the pre~ent invention.
It is believed that fusion of a membrane from a
tumor cell with a membrane from a cell fusion partner
changes the capacity of the tumor cell to activate
specific T-cell responders in the host immune system so
that an immune response can be mounted against that tumor
cell. In support of this, it has been discovered that the
introduction of a hybrid cell comprising a tumor cell
fused to a cell fusion partner not only reduces tumor
growth, but also the growth of normal tumor cells (i . e . .
non-fused tumor cells) within the same host.
It is further believed that the cell fusion
30 partner contributes relevant tumor-specific antigens to
the hybrid cell and that the cell fusion partner
contributes cell sur~ace costimulators, soluble cytokines,
MHC molecules, and other undefined molecular factors to
the hybrid cell These contributions in aggregate result
in a highly antigenic and immunogeni~ phenot~pe for the
hybrid cell. In turr~, the hybrid cell can be used a~ an
WO 95116775 PCTrUSs4/14297 ~
21 7895~
effective cellular vaccine. Xowever, this proposed theory
is not meant to act as a limit to any alternative theories
or mechanisms of carrying out the invention.
The pre~:ent inventlon is based upon the finding
that enha~ced ~ n; city can be induced by fusing a
cell membrane from~a tumor cell with a cell membrane from
a cell fusion partner, preferably one capable of effective
antigen presentation and T-cell activation. Through such
cell fusion, the full complement of molecular factors~that
are normally produced by APC' s and that are required for
effective T-cell activation are combined with the full set
of potential tumor antigens associated with a particular
tumor. Moreover, through the choice of fusion partners,
it is possible to tailor the=nature of the anti-tumor T-
cell immune response. ~ :
Thus, a broad means has been discovered by which
the antigenicity or immunogenicity of any tumor ce~l can
be increased by fusion with a membrane from another cell
with greater immunDgenic potential than the tumor cell.
Given this discovery and the methodology provided herein
(in which examples :of this discovery are provided), it is
now straightforward for those in the art to screen any
particular target tumor cell~to determine whether fusion
with any particular cell fusion partner will enhanc~ the
tumor cell' 8 1 n~n; city.
Thus, in a first aspect, the invention features
methodæ for inducing immunity against a tumor cell by
providing to a patient a cell fusion product.
By "cell fusion product" is meant a cell
membrane from a tumor cell ~fused to a cell membrane~from
another cell that has a greater immunogenic potential than
the tumor cell. O~e type of=~ cell fusion product ~is a
hybrid cell. .3y 'Thybrid cell" is meant a cell which is
derived from the fusion of two parental cells, and it is
either the direct fusion product or a daughter cell that
is derived by cell division from the original fusion
Wo 95/16775 ~ 1 7 ~ q 5 PCTIUS94/14297
product. A hybrid cell rrnti:l;nq one or more molecular
components of each of its parental cells . By "parental "
cell is meant either c~ t of the hybrid cell and
includes a tumor cell or another cell with greater
immunogenic potential than a tumor ce ~ l.
By ~provide~ is meant any method that resulte in
the presence of a cell fusion product in a patient. The
step of ~'providing" can be performed in a variety of ways
including either administering a cell fusion product that
was formed ex vivo or fusing the mem~oranes in vivo.
By " immunity" is meant the state of being
refractory to a specific disease, which is mediated by the
immune system or a state of not being susceptible to the
invasive or pathogenic affects of potGnt;~lly infective
microbes or to the affects of potentially toxic antigenic
substances . By " immune response" is meant the response of
the whole or part of an immune system of an organism.
This response could include the activation of r-~ r or
humoral systems, including B-cells, and T-cells.
By "tumor" is meant a collection of cells,
usually dysfunctional, due to abnormal proliferation.
Benign tumors are not life threatening, e.g., warts.
Malignant tumors are potentially lethal cancers. All
tumor types may be treated using the methods of the
present invention, since cell fusion is not dependent upon
any particular cell phenotype. The tumor cell may be
autologous, heterologous, cultured, primary, or
metastatic .
By ~autologous" is meant that a tumor cell is
- 30 from the patient to be treated, or from another patient
having a common major histocompatibility phenotype. sy
"primary~ is meant that a tumor cell from the organ of
tumor origin in the patient to be treated is used. It
also means a primary cultural cell, as distinct from a
cell line. By ~metastatic" is meant that the tumor cell
is prolif erating at sites distant ~rom the organ o~ tumor
Wo 9~16775 PCTnJSs4/l4297
2 ~ 78 q50
origin. By "heterologous" is meant that a tumor cell~from
another patient is used. Clinicians and others skilled in
the art are able to identify patients in need of treatment
using procedur~s that are well known and routine in the
art. Procedures for~ obtaining a tumor cell from such a
patient and for culturing such a cell are also well known
and routine in the art. By "patient" is meant any animal,
including a human, with a tumor
By " fusing" is meant a process whereby the cell
membranes are combined into a single membrane and a cell
fusion product is ~ormed. The fusing may be performed by
directly in~ecting ~ the cell fusion partner into a tumor
mass in a patient. This may further involve identifying
a patient in need of tumor therapy, obtaining a tumor cell
f rom the patient, and culturing the tumor cell . The
fusion step may involve more than two fusion partners.
The fusion step may also involve the use of a chemical
fusogen, such as polyethylene glycol, or electrofusion.
By "immunogenic potential" is meant the capacity
to activate specific T-cell responders in the immune
system or the ability to raise an immune response in an
animal, preferably a human. It is commonly found that
tumor cells have poor immunogenic potential relative to
APCs. By "anti-tumor response" is meant any response that
measurably reduces the size of a tumor, int-lllrl;ng the
complete destruction of the tumor ~
By "membrane extract" is meant an extract of a
cell enriched for membranes, but not necessarily
c~nl-il;n;n~ only membranes. Such an extract is chosen
3 0 because it will have the antigenic and immunogenic
properties necessary to induce an immune response to the
tumor cell in vivo or ex vivo. A cell membrane extract
may be derived fro~L an autologous or heterologous tumor
cell or another cell with greater immunogenic potential
than the tumor cell. The tumor cell or cell fusion
product may be treated, for~ example by irradiation, to
Wo 95/16775 2 1 7 5 0 PCT/lDS94/14297
reduce it6 ability to proliferate. By nmembrane~ is meant
a sheet, usually about lO nm thick and normally composed
of a bimolecular layer of lipid and protein, enclosing or
partially enclosing a cell, organelle, or vacuole. Cell
fusion products may be formed using less than an entire
membrane, for example, portions of membranes may be used.
In other aspects the invention features methods
for ;n~ ;ng immunity against a tumor cell by providing to
a patient either a tumor cell fused to an APC or tl~e fused
membranes of such cells. Examples of a conventional APC
include an activated B-cell, a dendritic cell, a
macrophage, an activated T-cell, or an endothelial cell.
Methods for identifying other cell ~usion partners that
can confer enhanced immunogenicity to a tumor cell are
defined herein. Preferred candidate cells are those for
which there is evidence of immunogenic potential.
In yet other aspects the invention f eatures
methods for inducing immunity against a tumor cell by
providing to a patient a tumor cell fused to an activated
B-cell with an artificial adhesin. By ~artificial
adhesin" is meant a genet cally engineered molecule that
is expressed through gene transfer, or through protein
transfer is exogenously coated, on the surface of a cell
and thereby promotes adhesion to another cell expressing
a molecule on its surface that can bind to the engineered
molecule . Examples of artif icial adhesins include a
glycosyl -phoshatidylinositol -modif ied polypeptide or a
biotin-lipid con~ugate, or other compounds with equivalent
properties .
In other aspects, the invention features methods
for ;n~ -;n~ immunity against a tumor cell by providing to
a patient a T-cell activated by contact with a cell fusion
product. The T-cell may be part of an immunoselected
subset such as CD8-positive T-cells. T-cells are known to
be critical mediators of anti-tumor immunity. In general,
T-cells are activated by cells collectively referred to as
Wo 95/16775 PCr/USs4/14297 ~
2178950
"antigen-presenting cells~ (APC) . The diverse cell types
that comprise this category share the ability to present
antigens, via their major histocompatibility molecules, to
the T-cell receptors on antigen-specific T-cells.
The preæent invention tliscloses the capacity of
a cell fusion product to stimulate anti-tumor T-cells.
This capacity can be utilized not only for ;n vivo
stimulation of T-cells, but also for çx vivo stimulation
of T-cells. Ex vivo 6timulation of a T-cell using a cell
fusion product can be used as a means of amplifying T-
cells with tumor specificity prior to infusion of such T-
cells into patie~ts. Methods are. well known in the art
for delivering T-cells into patients. Methods are well
known in the art for derivirLg T-cçlls from the peripheral
bloDd of cancer patients or lsolating infiltrating T-cells
directly from tumors and nonspe~ lly amplifying their
cell numbers using reagents such as interleukin-2.
Contacting such T-cells with a cell fusion product offers
a means for selectively amplifying the tumor-specific T-
cells out of the mixed T-cell populations at these~sites.
Once amplified, the T-cells can be re-infused into a
patient. It should be eviderLt frDm this that the same
patient can be coordinately treated with a cell fusion
product, as an active vaccine, along with ex vivo
amplified T-cells, as a passive vaccine. This combined
treatment maximizes therapeutic effects and is
advantageous for- immunosuppressed patients.
In still- other aspects, the invention features
a method for identifylng a cçll fusion partner that can be
fused to a tumor cell to generate a hybrid cell with
greater immunogenicity tha~ the tumor cçll. This method
involves fusing ~a tumor cell with a candidate cell and
determining the immunogenicity of the resulting cell
fusion product. Such methods can be based in vivg or ex
vivo. Clinicians and others skilled in the art are able
W0 95116775 2 1 7 8 9 5 0 PCTIUS94/14297
to determine the immunogenicity of a cell using procedures
that are well known and routine in the art.
In other aspects, the invention f eatures a
method for fusing a tumor cell with another cell. This
method involve6 expressing an artificial adhesin on either
the tumor cell, the other cell, or both. The method also
involvee combining the cells with a fusogenic agent. By
"fusogenic agent" is meant any compound that increases the
ability of a membrane from a tumor cell to fuse with a
membrane from another cell that has greater immunogenic
potential than said tumor cell.
The present invention discloses that selectivity
can be conferred to a cell fu~ion process by inducing
relevant paired cells to adhere to each other prior to
addition of a fusogenic agent. By combining such a "pre-
adhesion" step with subsequent fusion at low cellular
densities (generally below lOs cells/ml), more efficient
fusion can be achieved. Xowever, use of low cell density
is not a required parameter in this invention. As
described, a preferred method for achieving such pre-
adhesion is through the use of an artificial adhesin that
has been delivered to a relevant cell surface by any one
of a number of gene and/or protein transfer methods.
However, alternative methods that do not involve
artificial adhesins can be used to achieve pre-adhesion.
For example, a heterobifunctional antibody can be used to
adhere a tumor cell and an APC . The adherence - inducing
- method should not perturb the antigenicity and
immunogenicity of the hybrid cell to be used as a membrane
source for immunization.
In still other aspects, the invention features
an immunogenic c~ll fusion product. In this product a
membrane from a tumor cell is fused to a membrane from a
another cell with greater immunogenic potential than the
tumor cell.
.
Wo 95ll6775 2 1 7 ~ 9 5 o PCTrU594/l4297
The immunogenic cell fusion products of the
present invention are distinct from hybridomas used for
monoclonal antibody production where a cultured myeloma
cell is fused with a splenocyte. The immunogenic cell
fusion product of the present lnvention is also distinct
f rom non- immunogenic hybrid cells used in routine
laboratory experiments. However, the methods described
above for inducing immunity against a tumor cell can
utilize these hybridoma and hybrid cells. In the present
invention, the membrane of a tumor cell isolated from a
patient is fused with a membrane from another cell, such
as an APC, with greater ~ immunogenic potential than the
tumor cell. Thus, unlike monoclonal antibody production,
a primary tumor cell is involved in the fusion process.
Furthermore, unlike myelomas which have the ability to
produce antibodies, the tumor cell oi~ the present
invention need not be able to make antibodies. At any
rate, the cell ~1~sion products of the present invention
generally exclude the use of a myeloma cell fused: to a
splenocyte
One or laore of the cells that generate the cell
fusion product, or alternatively the cell fusion product
itself, may be molecularly modified prior to
administration to a patient. Molecular modifications can
be directed toward any one of a number of functional
endpoints, including ~nh~nr;nrj the fusion process,
promoting selective ~ ;rn between the parental cells,
altering the in ~ rQ tissue targeting properties of the
cell fusion products, and further ~nl~nr;ni the
immunogenicity of ~the cell fusion product above and beyond
the F~nl~nrF~ immunogenicity that would otherwise be
conferred to the hybrid cell by the parental cell.
A cell fusion partner may be modi~ied prior to
fusion with a tumor cell in order to increase the
immunotherapeutic ef icacy of the resulting hybrid cell .
This modification can be efiected by alternative methods,
Wo 9~/16775 ~ 1 7 8 9 5 0 PCTIUS94/14297
11
- including gene or protein transfer. Examples of proteins
that can be expressed or inhibited in an APC include, a
cell surface costimulator, a soluble cytokine, a selectin,
an adhesin, a major histocompatibility complex protein,
and a coinhibitor (e.g., CD8). By "foreign protein" is
meant a protein that is not normally expressed in a
particular cell. By "natural cell surface molecule" is
meant any molecule ~hat is naturally found on the surface
of a particular ell.
One molecular modification entails the coating
of one or more of the parental cells with artif icial
adhesins that promote selective adhesion between the two
cell types prior to the fusion event. A tumor cell or
another cell may be modified to enhance its fusion
potential. ~he tumor cell can be modified in vivo or in
vitro prior to cell fusion. When another cell is directly
inj ected into a tumor mass, prior to inj ection the tumor
mass may be modiiied in vivo, or the other cell may be
modified in vitro, or both.
As will be readily apparent to one skilled in
the art, the useful n vivo dosage to be administered and
the particular mode of administration will vary depending
upon the age, weight and liAn species treated, the
particular cellular compositions employed, and the
specific use for which these cellular compositions are
employed . The determination of ef f ective dosage levels,
that is the dosage levels ne--~A~Ary to achieve the desired
result, will be within the ambit of one skilled in the
art .
The present invention also provides kits
including materials used in cell fusion.
As can be seen from the above description, the
invention generally features generation of a fusion
product, e.g., through cell fusion, to provide a reagent
suitable for ; ; 7Atiorl against a tumor, either in a
prophylactic or treatment procedure, and features methods
-
. . !
'~'
WO 95/16775 2 1 7 ~ PCT/US94/14297
12
for ~identifying the optimal cell fusion partner~ to be
fused with a tumor cell derived from such a tumor.
Prior to this invention, it is believed that no
art described how tumor cell immunogenicity may be
enhanced by fusion with another cell with greater
immunogenic potential than the tumor cell. The ~use of
cell fusion provides GubGtantial advantages for practicing
tumor cell engineering and ~nh~nc;ng tumor cell
immunogenicity. Advantages of cell fusion over gene
transfer include, but are not limited to, the following:
First, the pre~ent invention obviates the need
for decoding the precise molecular signaling systems for
individual tumor~ cell:T-cell combinations. Thus, unlike
current methods which iocus upon individual defined
molecules, the present invention bypasses the general lack
of understanding of the composite set of antigenic
peptides and CoGt;~ t~ry molecules required for
effective anti-tumor T-cell responses.
Second, cell fuGion is applicable to diverse
tumor cell types. Unlike~ gene transfer, cell fusion is
not dependent upon the proliferative potential of the
tumor cell, and hence, can be applied to a variety of
tumor types which grow poorly in primary culture.
Third, cell fusion is a relatively rapid process
and does not impose a burden of excessive cell culturing,
shortening the interval between biopsy and treatment.
Certain gene transfer-based immunotherapeutic strategies
re~uire selection for stable transfectants. This can be
a time consuming process and complicates the clinical
practice of such methods and imposes a delay period
between biopsy and treatment.
Fourth, no biosafety hazards are known to be
associated with the cell~ fusion method of the present
invention. Gene transfer is dependent in most instances
upon genetic vectors comprising viral components which
carry with them some degree~ of biosafety hazard.
~178950
95/16775 Pl~rlUS94/14297
13
It is believed that fusing a tumor cell with
another cell with greater immunogenic potential than the
tumor cell effectively r~ ~ ;nF~ the antigenic repertoire
of such a tumor cell with the multiple molecular factors,
including cell surface costimulators, soluble cytokines,
and major histocompatibility complex (MHC) molecules, of
such other cell Different potential other cells
(including different well known APCs such as an activated
B-cell, a macrophage, and an activated endothelial cell)
differ in their molecular factor composition. Thus, by
pairing a particular APC with a particular tumor cell, one
can confer to such a tumor cell desired properties.
Cell fusion, as a means of ~nh~n~-;n~ tumor
immunogenicity according to the present invention, may be
combined with other known methods for ~nh~nf-; ng tumor
immunogenicity, including those cited herein, for example,
expreæsion of an exogenous gene in a tumor cell or
inhibition of an endogenous gene in a tumor cell. Indeed,
it is believed appropriate to combine the therapies or
methods described herein with other methods to enhance the
immunogenicity of a tumor cell.
The summary of the invention described in detail
above is not 1ntF~n~ in any way to limit the scope of the
present invention, which 18 defined in the appended
claims. Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
sRIEF DESCRIPTION OF T~iE DRaWINGS
Fig. 1 shows schematic diagrams of the pM-CSF-
GPI/R~P4~ and pM-CSFR/REP7~ expression constructs which
encode members of an artif icial adhesion pair . For the
pM-CSF-GPI/REP4c~ expression construct (panel a), the M-
CSF-DAF chimeric sequences (thicker box) are depicted in
the multiple cloning site of pREP4Om For the pM-
35 CSFR/REP7~ expression construct (panel b), the M-CSF
WO 9~/1677~ ~ ~ 7 ~ ~ ~Q PCTnJSs4/14297
14
receptor coding ser~uence ~thicker box) is depicted in the
pREP7~ vector. Abbreviations: EB~7 oriP, Epstein-Barr
virus origin of replication; EB~A-l, EBV nuclear antigen-
l; RSV 3 ' LTR, Rous sarcoma virus 3 ' long terminal repeat
promotor; PA, SV40 polyadenylation/termination sequences;
hph, 11yyl~ y~in-B resistance gene; Amp, ampicillin-
resistance gene.
DESCRIPTIO~ OF TIIE ~R~ v ENBODIMENTS
Preferred embodiments of the present invention
are described in detail below. However, the following
description of the preferred embodiments i8 not intended
to limit, in any way, the scope of the present invention
which is def ined in the appended claim~ s .
The present invention addreeses the need for
conferring a complex phenotype, immunogenicity, to diverse
types of tumor cells. Methods are provided for conferring
this complex phenotype to tumor cells ex vivo and in vivo.
In addition, methods are provided for using modified tumor
cells to generate ~T-cells to~ be used for cell transfer.
Yet other methods are provided for ~nh~nr;ng adhesion
between a tumor cell and another cell, as a prelude to
fusion of the two cells.
The preaent invention entails the fusion ~ of a
tumor cell to another cell Methods ~1~F; rn~ to promote
interc~ 1^ adhesion can be combined with conventional
non-selective cell fusion methodæ in order to selectively
target and enhance the cell iusion process.
More specifically, when tumor cells and APCs are
combined, nonselective fusion agents that are routinely
used to fuse cells will not only induce tumor cell:APC
fusions, but also tumor cell:tumor cell and APC:APC.
fusions as undesired byproducts. Herein disclosed is a
cell fusion method for minimizing these undesired fusion
events and simultaneously m~;m; 7lng the desired tumor
35 cell:APC fusion events. This is accompanied by
Wo 9511677~ ~ 4/
~ l 7 8 9 5 ~ I Cr/Uss 14297
artificially promoting adhesion between a tumor cell and
an APC, without influencing self-adhesiveness, and adding
fusing agents or performing electrofusion when adherent
tumor cell :APC conjugates are at low cell densities . The
lower cell densities would be less 'avorable to fusion
between n~ rent cells of the same cell type.
According to the present invention, a combined
adhesion/fusion method for generating tumor cell :APC
hybrids can comprise any one of a number adhesion and
fusion component methods. A method ~or altering the
adhesive propertie6 of cells that is particularly well-
suited for the adhesion/fusion method has been developed.
This method is based upon the uE:e of a class of molecules
that can be designated by the term ~artificial adhesins. "
Specifically, in one example, a glycosyl-
phosphatidylinositol (GPI)-modified variant oi the
cytokine macrophage colony stimulating factor (M-CSF),
designated M-CSF-GPI, was expressed on the surface o~
human bone marrow stromal cells. A chimeric M-CSF:decay-
2 0 accelerating f actor expression construct was used ~or M-
CSF-GPI expression . Cell: cell binding assays established
that this artificially membrane-tethered cytokine
functioned as a potent cellular adhesin, allowing for
enhanced binding to M-CSF receptor-expreeeing cellular
transfectants. Antibody blocking analyses confirmed the
M-CSF :M-CSF-receptor dependence ~ of the enhanced
intercellular binding. This capacity to direct the
cellular interactive repertoire of selected cells can in
principle be applied to other cell types and other
3 0 molecular pairs to be used in cell-based therapies .
Intercellular adhesion is mediated by homotypic
and heterotypic molecular interactions at membrane
inter~aces. There is a growing compendium of cell sur~ace
molecules that have been assigned functions as natural
adhesins in regulatory interactions between cells. Since
natural adhesins have frequently been found to be
W095/1677~ 2 1 ~8 95~ PCTIUS94/14297
16
multifunctional, they often cannot be used as neutral
molecules for altering cellular adhesive properties.
Moreover, most natural adhesins, by virtue of being
tr~n! ' d,~e hydrophobic peptide-anchored molecules, can
only be expressed on cell membranes by gene trangfer.
Therefore, methods are needed to artificially modify
adhesiveness between cells in a neutral way and without
necessarily using gene transfer.
A known method for accomplishing this is through
the use of a palmitate-conjugated antibody as an
artificial adhesin for cross-linking cells (Colsky et al.,
124 ~ l. Methods 179, 1989). However, this
approach is limited by a number of factors including the
re~uirement f or working with multichain antibody u~its .
Soluble antibodies with bifunctional specificities offer:
another potential approach ~or cross-linking cells, but
there is still the potential of signal transduction
mediated by the antibodies.
Ther~ is herei~ disclosed an alternative
approach for art;f;fi~lly e~hancing adhesiveness between
cells. Genetically engineered variants of known ligand
receptor pairs can be used as art;~;r;;3l adhesins. The
strategy is to genetically alter a soluble polypeptide
ligand so that it incorporates into the surface of one
cell via a carboxy-terminal anchoring domain and yet still
retains its capacity to bind its receptor on another~cell.
The results described below document the feasibility of
such an approach.
The use o ~ a GPI moiety f or membrane anchorage
offers special potential advantages in the context of
cellular engineering. Notably, it builds upon the
demonstration (Tyk ~inqki et al., 85 ~roc. ~atl. ~ Acad.
Sci. ~SA 3555, 1988) that any polypeptide can be readily
produced as a GPI -modif ied variant through the use of
chimeric coding sequences ~n~ sing both the coding
se~uence for the protein of interest and the GPI signal
_ _ _
~Wo 95/1677~ 2 1 7 8 ~ 5 0 PCT/US94/1.1297
sequence from a naturally GPI-modified protein such as
decay accelerating factor (DAF) .
Another potential advantage of the use of GPI
anchors for adhesins stems from the fact that purified
GPI-modified proteins, by virtue of their amphophilic
properties, can be readily reincorporated into cell
membranes in the presence of low, non-lytic concentrations
of non-ionic detergents such as NP-40 ~Medof et al., 160
J. Ex~. Med. 1558, 1984) or even in the absence of
detergent. Hence, cells can be coated with purified GPI-
modified polypeptides, representing a form of "protein
paint, " bypassing the rerluirement for gene transfection
into the cell whose surface is being molecularly
engineered. Delivery of exogenous polypeptides to cells
by such a protein transfer approach circumvents problems
associated with gene transfer, particular in the case of
primary, nontransformed cells which in general cannot be
easily transfected.
Numerous applications for this artificial
adhesin technology can be envisioned, some with
therapeutic implications. For example, the
immunostimulatory and/or effector properties of cells used
in cell-based therapies, such as tumor cell:APC hybrids,
APCs, immunogenic tumor cells, or T-cells, could be
selectively .-nl~nre~l by increasing their adhesive
properties in a selective way.
A preferred protein transfer method for coating
- cells with artif icial adhesins involves the use of GPI-
modif ied proteins . Methods f or perf orming protein
transfer using GPI-~odified proteins have been described.
In all instances, this has entailed the use of dilute,
concentrations of non-ionic detergent, for example, . oo496
NP-40, in the solution containing the GPI-modified
proteins. It has been discovered that protein trans~er
can be accomplished even more effectively in the absence
of any detergent. By leaving out the detergent, higher
W095/16775 7~7895a PCr~ss4/1~297 ~
18
concentration6 of a GPI-modif ied protein can be used and
problems a6sociated with cell ly6i6 by detergent are
avoided. The optimal time for co-incubation of a cell
with a GPI-modified protein, such a3 an adhe6in-GPI
chimeric polypeptide, i3 two hour3 at either room
temperature or 37C. 25 microgram6/ml of the GPI-mQdified
polypeptide in the coating reaction with the cell6 i6
generally 6ufficient for aderiuate adhe6in coating,
although higher rrn~ ~ntrati-on6 can be u6ed with increa6ed
ef f icacy .
Another preferred protein tran6fer method for
coating cell3 with artif icial adhe6in6 entail6 the u6e of
a chimeric polypeptide in= which an adhe6in polypeptide
6equence, for example, M-CSF, i6 linked to a 6treptavidin
6equence. Method6 for~ u6ing prokaryotic expre66ion
6y6tem6 to quantitatively produce 6uch polypeptide-
6treptavidin chimera6 are~ own to tho6e familiar with the
art. In u6ing such an adhe6in-6treptavidin chimera, the
cell of intere6t i6 pre-coated with biotin. A u6eful
method for pre-coating cell6 with biotin i6 through the
use of biotin-lipid conjugates which can be used to pre-
coat cells to high bioti~ densitie6 (up to 107 biotin
molecules/cell). Alternatively, the cell6 can be
chemically biotinylated u6ing 6tandard cellular
biotinylation prQcedureE. A chimeric adhesin-6treptavidin
polypeptide i6 added to a pre-biotinylated cell, in order
to generate a cell expre6sing the artificial adhe6in at
it6 6urface. It is generally not avidin on one cell and
biotin on a 6econd cell that are being u6ed to bring two
cell6 together, but instead avidin and biotin are usually
6imply being u6ed on the 6ame cell 6urface to deliver an
;rn to that cell~6 3urface. Then, the 6ame proce66
may be applied to a 6e~con~ cell again, potentially u6ing
an avidin-biotin comple~ at that cell 3urface. Advantage6
of thi6 method; include the high 6urface den6itie6 of
art;f;~;~l adhesin that can be achieved, the feasibility
Wo 95/1677s 2 1 7 8 q 5 a PCTiUS94/l4297
19
- of producing large ~uantities of the chimeric polypeptide
using a prokaryotic expression system, and the
biocompatibility aesociated with biotin-lipid conjugates
in vivo.
According to the present invention, a tumor cell
can be coated with one member of an artificial adhesin
pair, and a conventional APC can be coated with the other
member of the pair. When cell populations of each of the
cell types are combined, intercellular conjugates form,
pairing a tumor cell with an APC. ~ When an electric
current is applied, or a chemical fusing agent is added,
to the mixed cell population, adherent cells within
conjugates preferentially fuse. Preferential fusion can
be further promoted by keeping the mixed cell population
at a low cell density. This will minimize the formation
of extraneous tumor cell:tumor cell and APC:APC hybride
with no immunotherapeutic potential.
In the example3 provided below, a preferred
method for fusing cells comprises the use of polyethylene
glycol as a fusing agent. Other methods of fusing cells
can be used, including electrofusion or use of viruses or
viral components, for example, Sendai virus, that promote
cell fusion.
The invention will be more fully understood with
reference to the examples which follow. The following
examples are intended to illustrate the invention, but not
to limit its scope which is defined in the claims appended
hereto . The f ollowing examples are presented to
illustrate the advantages of the present invention and to
- 30 assist one of ordinary skill in the art in making and
using the same, but are not intended, in any way, to
otherwise limit the scope of the disclosure or the
protection granted by letters patent hereon.
;Bxam~le l: He~atocarcinoma fused to ~Tl activated B-cell
lose their t~ ri~enicity
W0 95/16775 2 1 7 ~ PCT/US9~/14297
~ctivated B-cells are effective APCs
(hanzavechia, 140 ~ature 1985, 1985; Ron, 138 ~ rLmunol.
2848, 1987; and Kurt-Jones, 140 ~. Immunol. 3773, 1988).
Con3equently, this cell type provides an excellent APC
fusion partner for the present invention. BERH-2 is a
chemical carcinogen-induced rat hepatocellular car~cinoma
cell line from the Wistar rat. BERH-2 grows rapidly and
forma tumors in the liver of syngeneic animals.
In ~periments described below, BERH-2 cells
were fused with activated B-~cells in an attempt to enhance
the immunogenicity of BERH-2 cells. The data provided
herein indicates that the hybrid cells, designated BERH-2-
B, became more immunogenic ~and less tumorigenic than the
tumor cell.
ExamPle lA: Fusion and selection of BERH-2-B cells
Activated B-cells were obtained from the spleens
of rats injected 14 days earlier with bovine serum albumin
in complete Freud' 8 adjuvant. BERX-2 cells were fused
with these purified activated B-cells using polyethylene
glycol, using a standard B-cell hybridoma fusion protocol.
The fused cells, designated BERH-2-B, were selected by
panning, first with a rabbit anti-BERH-2 antiserum and
second with a rabbit anti-rat B-cell antiserum.
The rabbit anti-BERH-2 and rabbit anti-rat B-
cell antisera : were prepared by immunizing rabbits
subcutaneously with either~BERE~-2 hepatoma or purif ied B-
cells from Wistar rats in complete Freud' 8 adjuvant .
~ctivated B-cells were purified by panning with plates
coated with purified goat_ anti-rat Ig antibody. After
repeat boosting during two_months, antiserum was collected
and purified by protein G-sepharose chromatography.
Finally, the antiserum was repeatedly absorbed with either
BERH-2 hepatoma - cells or rat B-cells .
~WO 95tl6775 ~ l 7 8 9 PCTruS94rl4297
21
ExamPle lB: ExPression ~n~ characterization of antiqens
Expression of class I MHC, class II MHC, B7,
ICAM-1 and LFA-1 on BERH-2 cells, activated B-cells and
BERH-2-B hybrid cells were assessed. Cells were washed
with phosphate-buffered saline (PBS) and stained with
monoclonal antibody to rat MHC class I (OX-18), MHC class
II (OX-6), ICAM-1 (LA 29) or LFA-1 (WT.1) as primary
antibody . To stain f or rat B7, we used a chimeric
protein, CTLA-4-Ig. Cells were incubated with the
antibodies or chimeric protein for 30 minutes on ice. A
mouse anti-human CD3 monoclonal antibody (GH3, IgG2b) and
a chimeric human CD44-Ig protein were used as llegative
controls. Cells were washed three times. FITC-goat anti-
mouse Ig or FITC-labeled rabbit anti-human Ig was used as
a secondary antibody and added for another 30 minutes on
ice. After washing, samples were fixed and analyzed in a
FACScan f low cytometer .
Parental and hybrid tumor cells were phenotyped
by immunocytochemical staining and f low cytometry . The
parental BERH-2 cells expressed low levels of class I MHC
antigen and ICAM-1, but lacked class II MHC antigen, LFA-1
and the costimulator B7. All four hybrid BERH-2-B cell
lines displayed increased class I MHC expression. In
addition, BER~-2-B hybrid cell lines expressed MI~C class
II antigen, ICAM-1, LFA-1 and B7. These BERH-2-B cell
lines have stably expressed both tumor and B-cell antigens
for more than five months.
- Exam~le lC: Com~arison of tumoriq~n; city of ~arent and
hvbrid cells
- 3 0 The tumorigenicity of parental BERH- 2 and hybrid
BERH-2-B cells were compared, and the survival data shows
e3lhanced animal survival for syngeneic anlmals injected
with BERH-2-B hybrid tumor cells as compared to BERH-~
tumor cells. Two groups of female Wistar rats (ten/group)
were injected intrahepatically with 2 x 106 BERH-2 cells or
2 x 106 BERH-2-B hybrid cells.
Wo 9S/16~75 2 ~ ~ ~ 9 ~ ~ PCTIUS94/1~297 ~
22
All animals injected with BERH-2 parental~ cells
developed liver tumors - ~nd dled within 6 0 days . In
contra~t, the BERH-2-B injected rats remained tumor-free
for more than 180 days. While the four hybrid ~11 1;nl~q
lost their ability to form tumors in syngeneic rats, they
were able to grow and f orm tumors in nude mice .
In rats injected~ with hybrid BERH-2-B ~cells,
there were abundant lymphocytic infiltrates at the site of
inj ection . The tumor inf iltrating lymphocytes present at
the site of BE~-2-B injection at two weekq were a
combination of CD4~ and CD8~ T-cells. As shown by
immunocytochemistry, moqt of the infiltrating cells were
T-cells. Seventy percent were CD8' T-cells, and thirty
percent were CD4~ T-cells. There was no inflammatory
response in animals injected with parental BERH-2 tumors.
Exam~le 2: HePatocarcinoma:activated B-cell hvbrids can
be used as a cellular vaccine to ~revent and cure
he~atocarcinoma;
The experimental data cited above established
that hepatocarcinoma cells lost tumorigenicity when fused
to activated B-cells. Moreover, the findir,g that hybrid
cells elicited a prolific T-cell response was consistent
with an immunologic explanation for the loss of
tumorigenicity. To substantiate this conclusion, tumor
prevention and cure experiments were performed.
The f;nll;n~q detailed below show that rats
injected with BERE-2-B hybrid cells became resistant to
subsequent cha~lenge with parental BERH-2 cell6.
Furthermore, these experiments est~hl; ql'~d that BERH-2
hepatomas were cured by injection of BERH-2-B hybrid
cells. Both CD4~ ar,d CD8~T cells were essential ior the
induction of protective immunity. However, only CD8 T-
cells were re~uired for ~:he eradication of pre-existing
BERH- 2 tumors .
The rats immuni2ed with BERH-2-B hybrid cells
were able to prevent tumor f ormation by parental BERH- 2
~1 789~0
~WO 95/16775 PCT/US94/14297
23
- cells. Protective immunity was induced with BERH-2-B
hybrid tumor cells.
Groups of female Wistar rats (8/group) were
immunized with 2 x 106BERH-2-B or BERH-2 cells
subcutaneouæly. Two weeks later, both groups of the rats
were challenged with 5 x 106BERH-2 cells intrahepatically.
All ratæ pre-injected with BERH-2-B and
subsequently challenged with BERH-2 remained tumor-free
for more than 150 days. In contrast, all rats pre-
injected with BERH-2 cells and then challenged with the
same BERH-2 cells died within 60 days.
Exam~le 2A: Tumor cure ex~eriments
A æeries of tumor cure experiments were next
performed to show that ; i i~tion with BERH-2-B cells
could also eradicate an established hepatoma. One set of
fourteen rats were injected intrahepatically with 2 x 106
parental BERH-2 cells. Ten days later, eight of the
injected rats were immunized with a subcutaneous injection
of 5 x 106 BERH-2-B hybrid cellæ. Theæe ratæ survived for
more than 120 days.
In contrast, ratæ injected both times with
parental BERH-2 cells all died within 42 days. A second
set of rats were surgically implanted with a small
fragment of BER~1-2 hepatoma intrahepatically. Fourteen
rats were intrahepatically irnr)l~nt~tl with a small fragment
(0.3 mm x 0.5 mm) of BERH-2 tumor. Two days later, eight
of the animals were inj ected subcutaneously with 5 x 106
- BERH-2-B hybrid cells. The other six rats were injected
subcutaneously with same number of BERH-2 cells. Ten days
later, a subset of the tumor-implanted animals were
injected with BERH-2-B cells, the ~ ;n;ng control rats
were injected with parental BERH-2 cells. Whereas all 6
rats injected with BER~-2 cells died within 50 days, only
2 of 8 rats injected with BERH-2-B hybrid cells developed
tumors; the latter died at 71 and 74 days after tumor
WO 95~16775 PCTiU594/14297
21 78q53
24
implantation, respectively. Six of the animals lived for
more than 180 day8 after tumor implantation.
Example 2B: Determ;n~tion of type of T-cell mediation
It was ne~t determined whether the rejection of
BERH-2-B cells is mediated by CD4~ ar.d/or CD8~ T-cells.
Rats were depleted of CD4~ cells or CD8 ' cells by antibody
treatment prior to injection of BERH-2-B cells. BERH-2-B
cells were able to form tumors in both CD4-depleted and
CD8-depleted rats. The effects of depletion of CD4~ or
CD8+ cells on the growth of BERH-2-B and BERH-2 tumor
cells in vivo are discussed below.
~xi~m le 2C: Grou~s A-D
Female Wistar rats (Groups A, B, C, D) were
treated with purified anti-rat CD4 (OX38), or anti-CD8
(OX-8) or ;~ control mouse anti-diethyl~ m;n~ pentaacetic
acid monoclonal antibody. Each animal received 500 /lg of
the purified antibody intravenously twice per week for
three weeks.
Two days before injection of tumor cells,
peripheral blood lymphocytes were obtained from indiviaual
treated rats and stained with monoclonal antibodies to CD4
or CD8 to verify the depletion of CD4~ or CD8~ cells,
respectively. Treatment with anti-CD4 monoclonal antibody
depleted more than~ 9596 of the CD4 ' cells, and treatment
with anti-CD8 monoclonal antibody depleted close to 9596 of
the CD8 ' cells; treatment with control antibody did not
alter the number of CD4~ and CD8~ cells. Three days after
the last injection of the antibodies, all rats were
injected intrahepatically with 5 x lO6 BERH-2-B tumor
cells.
Exam~le 2D: Gro~lns E-G
Rats were first immuni_ed with BERH-2-B cell6
and then depleted of CD4* or CD8+ cells 14 days later.
Female Wistar rats (Groups E, F, G) were first; ;7f"l
with 2 x lO6 BERH-2-B cells subcutaneously. Two weeks
after; ;7i~tion, animals were treated with anti-CD4, or
~17895~
~Wo 95/16775 PCT/US9 ~/14297
anti-CD8 or control antibody, and the efectiveness of the
depletions was verified by immuno1uorescence and flow
- cytometry. Three days after the last injection of
monoclonal antibody, all animals received 5 x 106 BERH-2
cells intrahepatically. These exp~riments have been
repeated twice with comparable results.
Table 1
Effects of Depletion of CD4' or CD8+ Cells
on the Growth of BERH-2-B and ~ -2 Cells In Vivo
Treatment Protocol Number
of
Antibody Animals
Speci~icity immunize f lrst With
Ab treat f irst then treat Tumors
then immunize with Ab
None - 0/6
CD4 + 4 / 6
CD8 + 5/6
15Control Ab + 0/6
CD4 + 0 / 5
CD8 + 5/5
Control Ab + /5
These CD4-or CD8-depleted rats were then
challenged with BERH-2 cells. Tumors developed in CD8-
depleted, but not in CD4-depleted rats. This indicates
that whereas both CD4' and CD8+ cells are necessary for the
induction o~ protective anti-tumor immunity, once the
immune response has been induced, CD8+ cells are sufficient
alone to mediate tumor cell destruction alone. These
results contrast with those reported previously for murine
m.~l An~mA cells transfected with the B7 costimulator gene
where CD4+ cells were not required for inductiol~ of the
anti-tumor immune response (Chen, 71 5~1 1093, 1992;
Townsend, 259 Science 368, 1993).
WO 95/16775 PcrNS94114297
2~ 78950~ ~
26
ExamE~le 2E: ~umor-specificity of immunity
It was next determined whether the immunity
induced by BERH-2-B cells is tumor-specific. NBT-II is a
bladder carcinoma that grow6 rapidly in sYngeneic Wistar
rats. Immunization with BERH-2-B hyb~id cells prevented
the growth of the parental BERH-2 cells. However,
immunization with BERH-2-B was unable to inhibit~ the
growth of NBT- II cells .
The specificity of the anti-BERH-2 immune
response ~1 i r; tP~l ~ by BERH-2-B hybrid tumor cells was
documented. Female Wistar rats were injected with 2 x lO6
BERH- 2 -B cells subcutaneously . Two weeks af ter
l7~tjon, one -group of t=he rats were injected with 5
x lO6 BERH-2 cells intrahepatically. Another group oi rats
were injected with 5 x 106 ~;IBT-2 rat bladder carcinoma
cells (obtained frDm the American Tissue Type Collection) .
Tumor developed locally in the inj ected site in
rats ; ~ 7C.(l with BERH-2-B tumor cells and challenged
with NBT-2 tumor cells in all eight animals. All ahimals
in this group died within 45 days aiter tumor challenge.
In addition, CD8~ T-cells from BERH-2-B-immunized rats
killed BERH-2 cells but did not kill NBT-II cells
vitro .
Table 2
Specif icity of the Immune Response
Elicited bY BERH-2-B Hvbrid Tumor Cells
Number oi Animals
Immunization ~ ~h~ nge With Tumors
BERH-2-B BERH-2 0/8
BERH-2-B ~ NBT-II 8/8
3 o Exam~le 2F, NecessitY of in vitrQ 8election
It was-determined whether the ;n vitro 8election
step for hybrid cells is rer~uired for effective induction
of anti-tumor immunity.
_ _
~Wo 95/16775 2 1 7 8 9 5 ~ Pc~r/usg4/l4297
27
Tumor protective immunity inducea with BERE~-2
tumor cells fused with activated B cells does not require
in vitrQ selection. Three groups of rats ~8/group) were
injected subcutaneously with 5 x 106 BERE~-2 cells, 5 x 106
BERH-2 cells mixed with 5 x 106 activated B cells, or 5 x
106 BERH-2 cells fused with 5 x 106 activated B cells in
the presence of PEG. Fused cells were washed three times
with PBS, resuspended in PBS and inj ected subcutaneously .
Two weeks later, all groups of rats were challenged with
5 x 106 BERH-2 tumor cells ; ntr~hf~ratically
BERX-2 tumor cells were fused with activated B-
cells. After fusion, cells were washed and injected into
syngeneic rats with in ~itro selection. The efficiency of
the fusion in such experiments ranged form 30% to 50%. As
controls, BERX-2 tumor cells mixed with activated B-cells
without PEG were injected subcutaneously. All animals
were then challenged with the parental BERX-2 cells
intrahepatically .
The data indicated that fused cells were
immunogenic in the absence of in vitro selection. Only
animals ; ; 7f"l with tumor cells fused with activated
B-cells were able to reject the parental tumor cells;
simply mixing tumor cells with activated B-cells was not
effective. This finding that protective immunity can be
induced by tumor cells fused with activated B-cells
without an in vitro selection step simplifies the
clinical therapeutic application of this method.
- These findings indicate that an effective BERX-2
hepatocarcinoma-specific vaccine can be generated by
fusing tumor cells with syngene~c, activated B-cells. It
is believed that in addition to class II MHC and B7
costimulator, hybrid BERH-2-B cells may express oth~=
molecules that are essential for the activation of anti-
tumor T-cells. This may include, but is not limited to,
soluble cytokines. Production of cytokines by hybrid
_ _ _ _ _ _ _ _ _ _ _ _ _ . . .. . . . _
r
WO 9~/16~75 ~ ~ 7 ~ 5 ~ PCTIUS9.1/14297
28
tumor cells may be important in the elicitation of host
immune responses.~
Example 3: GlYcos~l~hos~hatid~linositol ~"GPI")-modified
c~tokine can function as an- 2trtificial ~lh~cin
For purpoæes of achieviny high level stable
expression of cell surface molecules on human cellula~
transfectants, self-replicating EBV episomal expression
vectors were employed (GrQger et al, 81 Gene 285, =1989).
In the present study, two EBV vector variants designated
pREP4~ and pREP7,B were used, both of which share a
transcriptional cassette in which the RSV 3' LTR promoter,
a multiple cloning site, and the SV40 late
polyadenylation/termination signal are linked in tandem~
An expression construct for M-CSF-GPI was
generated as f ollows ~ The 1. 8 kb M- CSF coding region
fragment (XhoI - EcoRI) of p3ACSFR1 (Fig. 1) was inserted
into the corresponding sites of pBluescript (pBT,
Stratagene, Inc. ) . This generated a GPI-anchored variant
of M-CSF, pM-CSF/BT was cut with ~L, filled-in with the
pollk, and subsequently cut with BamHI. The 3 ' AvaII
(filled-in) to BamHI from the DAF subclone pDF2.1/BT was
subcloned into this vector~ to generate an in-frame M-CSF-
DAF chimeric se~uence. The KpnI-BamXI fragment of the
resultant plasmid containing the chimeric sec~uence was
subcloned into- the corresponding sites of pREP4Y to
generate pM-CSF-GPI/REP4~.
An M-CSF receptor EBV episomal expression
construct was generated as follows: A 4 ~ Okb EcoRI
fragment of pc-~f~ 102 was subcloned into the EcoRI site
of ~Bluescript (Stratagene, Inc~) to generate pM-CSFR/BT~
The 3.6 kb ~XI fragment=of this subclone, containing the
entire M-CSFR ~coding region, was subcloned in a sense
orientation into the ~HI site of pREP7~3 to generate pM-
CSFR/REP7 ~ .
The overall experi~Lental strategy of t~is study
was to use stable gene transfer to modify the ~=adhes`ive
. . .
21 7~950
o 95116775 PCTIUS94/14297
29
- properties of cells. The paired cellular targets chosen
were the human SV40 large T-immortalized bone marrow
stromal cell line KM-102 and the human myeloid leukemia
cell li~e K562. Previous work with both the KM-102 and
K562 lines have shown them to be eficient transfection
targets with EBV expression vectors.
With the goal of ~nh~nr; n,r adhesion between KM-
102 and K562 cells, we stably transfected them with
episome-based expression constructs for an artificial GPI-
modified variant of M-CSF, designated M-CSF-GPI, in which
natural M- CSF coding sequence is linked in- f rame to the
GPI signal sequence of human DAF, into KM-102 stromal
cells . Indirect immunof luorescence staining demonstrated
a high level surface expression of M-CSF epitopes on the
pM-CSF GPI/REP4o! hyg~KM-102 transfectants. No M-CSF
epitope was detectable on the surface of nontransfected
KM-102 cells or on KM-102 cells t-ansfected with the
irrelevant EBV episome pRSVCAT~/22 0 . 2 .
pM-CSFR/REP7,(~ and pM-CSF GPI/REP4~r were
introduced into K562 and U937 cells, respectively, by
lipofection. The KM-102 stromal cell line was kindly
provided by K. Harigaya and m~;n~{l;n~r~ in McCoy's 5a
medium ~Gibco, Inc. ) supplemented with 109~ heat-
inactivated fetal bovine serum (FBS) (M.A. Bioproducts) /10
mM HEPES/40 ~lg/ml gentamycin sulfate in a humid 5% CO~
atmosphere at 37C. pM-CSF-GPI/REP4(Y was introduced into
KM-102 cells by lipofection. Briefly, cells were grown to
5096 confluence in six-well plates, and washed twice with
PBS and once with opti-MEM (Gibco) . Cells were then
incubated for 5-8 hours at 37C with 1 ml opti-MEM,
cnn~;ning 10 ~g DNA and 30 /lg lipofectin, before adding
1 ml of complete medium rnnt~ining 2096 FBS.
Seventy-two hours post-transfection, selection
for stable transfectants was begun by replacing the medium
with fresh medium cr,n~in;ng 75 ~Lg/ml hygromycin B
(Calbioehem, Inc. ) Stably hygR transfeeted eolonies were
_ _ . _ _ _ . _ _ _ _ _ _
WO 95/16775 PCTr~594/14297
2178950
picked at 2-3 weeks using cloning rings (Bellco, Inc. )
n~ d and main~ained in 100 llg/ml hygromycin B.
Surface M-CSF expression was established by indirect
immunofluorescence using a polyclonal rabbit anti-M-CSF
antibody (Genzyme) and a FITC-anti~rabbit IgG (BMB)
secondary antibody. M-CSFR expre~sion was confirmed by
indirect i~munofluorescence .and flow cytometry (FACS),
using a rat monoclonal anti-M-CSFR primary antibody
(Oncogene Sciences) and FITC-conjugated rabbit anti-rat
IgG secondary antibody (Miles ICN) .
In parallel, X562 leukemic cells were
transfected with human M-CSF receptor (M-CSFR or c-fms)
episomal expression construct, designated pM-CSFR/REP7~.
Abundant sur~ace expression o~ natural human M- CSFR was
demonstrated by indirect immunostaining and ~low
cytometry. To control for ~ episomal transfectants
demonstrating non-specific up-regulation of ~ their
endogenous adhesion molecules, K562 cells were transfected
with the irrel evant episome pRSVCATo!/220 . 2 .
To con~Eirm that pM-CSF-GPI/REP4~Y yields a GPI
membrane anchored product, phosphatidylinositol-specific
phospholipase C (PIP~C), an enzyme which specially cleaved
GPI moieties made by certain cells f rom their surf aces,
was used. ~IPLC treatment o~ KM-102 cells transfected
with pM-CSF-GPI/REP4c~ did not result in a significant
release of either M-CSF or DAF, another GPI-anchored
protein serving as control, f rom the surf ace, as
determined indirect immunofluorescence. This suggested
that KM-102 is similar to some other cell types that are
known to express~ a GPI anchor variant that is resistant to
PIP~C cleavage.
PIPI.C cleavage was performed by incubating 1 x
106 cells with 1 unit of PIP~C ~Boehrin~er Mannheim
Biochemicals) at 37C for one hour in RPMI 1640 medium
containing 10~6FBS and 0 . 01!'s sodium azide . Cleavage was
assessed using an anti-DAF antibody and ~low cytometry.
~Wo 95/16775 3 PCr/US94/1429~
In light of this f inding, pM- CSF-GPI/RBP4~ was
additionally transfected into the U937 cell line which is
known to produce PIPLC-sensitive GPI anchors. PIPLC
susceptibility of M-CSF and endogenous DAF on these cells
was assessed. Nontransfected U937 cells did not express
M-CSF on their surface. The pM-CSF-GPI/REP40! U937
transfectants expressed high levels of cell surface GM-
CSF . The tethered M- CSF could be specif ically cleaved
with PIPIIC to an extent ~similar to that seen with
endogenous GPI-anchored DAF protein. The PIP~C cleavage
yielded removal of surface M-CSF epitope to the same
extent as DAF. This indicates that the pM-CSF-GPI/RBP4
construct generates a GPI-anchored form of M-CSF.
It was next de~rm;nf~fl whether the pM-CSF-
GPI/RBP40~ KM-102 transfectants would bind preferentially
M-CSFR' c~ r targets. Cell:cell binding between
adherent KM-102 cells and M-CSFR~ n-~n~r~h~orent K562 targets
cells was enhanced approximately three-fold when M-CSF-GPI
was presen~ on the surface of the KM-102 cells.
Intercellular adhesion between adherent KM-102
transfectants and n-~n~ rent K562 transfectants was
measured using a cell:cell binding assay. Normal KM-102
cells (nontransfected), a stable transfectant expressing
GPI-anchored M-CSF ~pM-CSF-GPI/REP40~), a control
transfectant expressing the lymphoid cell surface molecule
(pCD8/RBP2 . 1), or an irrelevant transfectant (pcYIL-
6/RBP5.1) were separately seeded into wells of a
polyvinyl, flat-bottom 96-well plate. 35S-labeled K562
target cells, eIther nontransfected (none) or expressing
the M-CSF receptor (pM-CSFR/RBP7~) or Cl~T (pRSVCAT/220.2),
were added to the wells and allowed to bind to the KM-102
cells. The number of K562 target cells which remained
adherent following an - inverted centrifugation was
calculated by measuring the speclf ic activity of the
target cells added. A signi~icant increase in adherence
between pM-CSFR-REP4~ transfected KM-102 and pM-CSFR-REP,B7
_ _ . .. . . . . .. . . ..
WO 95/16775 PCrlUS94/14297
217&950
32
transfected KM-102 was noted whereas no increase in
adhesion was noted for other combinations.
This was specific for M-CSF expression, since
control KM-102 transfectants rrnt~;n~ng either pCD8/REP2.1
encoding the irrelevant surf ace protein CD8 or
alternatively p~Y~I,-6/REP5.1~ driving antisense II.-6 RNA
expression, demonstrated no ,~nll~n~rPrl binding to M-CSFRt
targets. Moreover, no significant augmentation of
adhesion was evident when nontransfected K562 cells, or
K562 cells transected with pRSVCAT~/220.2 were used as
cellular targets. Xowever, ~K562 transfectants overall do
appear to be slightly more~ adhesive than nontransfected
cells .
To def initively establish that it is the
membrane-associated M-CSF, and not secondary expressed
surface molecules on the pM-CSF-GPI/REP4~x KM102
transfectants, that was specifically responsible for the
enhanced adhesion, antibody blocking analysis was
performed. Prior incubation of M-CSFRt K562 target cells
with antibodies directed against the M-CSFR or
alternatively, o~ surface M-CSFt KM102 cells with
polyclonal anti-M-CSF ~n~;ho~ , each partially inhibited
this specif ic cellular interaction .
The ef ~ects o~ blocking antibodies upon the
bindiny of M-CSF-GPI and M-CSFR-positive cells to each
other was assessed in cell:cell binding ~assays.
Nontransfected K562 cells (none) or K562 cells stably
expressing the M-CSFR (pM-CSFR/REP7~) or CAT
(pRSVCAT/220.2) were allowed to bind to KM-102 cells
expressing GPI-anchored M-CSF (pM-CSF-GPI/REP40~) .
Blocking antibodies were added to the appropriate cells,
as indicated, 3 0 min . prior to adding the cells to the
wells. Anti-M-CSF, polyclonal anti-M-CSF antibody; anti-
M-CSFR, monoclonal anti-M-CSF receptor (c-fm~) antibody;
an anti-TRF, monoclonal anti-transferrin receptor
. . . _ _
~17~S~
~WO 95/16775 PcrNss4/l4297
33
antibody. Normal rabbit serum (NRS) was added, as
indicated, to prevent F receptor cross-linking.
The simultaneous addition of these two blocking
antibodies, directed against both members of the
ligand:receptor pair, completely blccked the specific
binding. Normal rabbit serum, which did not inhibit
binding, was included in all eXpAr;rAntA to prevent the
cross-linking of cells through Fc receptors expressed on
the K562 cells. The antibody-mediated inhibition observed
was specific for the M-CSF :M-CSFR pair, since antibodies
against the human transferrin receptor - (TFR), known to be
expressed on K562 cells, had no blocking effect.
Cell:cell binding assays employed a modification
of a published method (McClay et al., 78 Proc. Natl. Acad.
Sci ~SA 4975, 1981). Briefly, 3 x 10~ nontransfected or
transfected KM-102 cells were placed in wells of a
polyvinyl, flat-bottom 96-well plate (Dynatech
~aboratories) with 0.1 ml complete medium per well, and
the cells were incubated at 37C for two days. Wells were
pretreated with fetal bovine serum for two hours to
promote attachment of the KM-102 ce~ l 8 . The KM-102 cells
were generally 60-809~ confluent at the time of the
cell:cell binding assay. The K562 target cells, labeled
with 35S-methionine, were washed, resuspended in complete
medium at 5 x 105 cells per ml, and 0.1 ml was added
directly to the wells.
The plates were incubated at 37C for 2.5 hours
to allow for maximal binding. Medium was added to each
well to produce a positive meniscus, and then plates were
- 30 carefully sealed with adhesive plate sealers (Dynatech
I-abs, Inc. ) . The plates were inverted and centrifuged for
10 minutes at room temperature using Sorvall micro-plate
carriers. A relative centrifugal force (RCF) of 900 x g
was used for most experiments. Post-centrifugation, still
inverted plates were flash-frozen at -80C, and the
bottoms of each well, containing the stromal cells and
WO g5116775 PCTIUS94114297
2 1 7gq50 34
bound targets, were cut off and placed in s~;nt;ll;3tion
vials for counting. The number of K562 cells bound per
well was calculated as follows:
CPM BOUND TO STROMAL CELLS
CELLS BOII~D/WELL = x (5 x l0~)
TOTAL CPM ADDED/WELL
Each group represents the means of at least
triplicate samples. Representative KM-102 stromal cell
wells were harvested and counted to ensure that e~[uivalent
l0 cell numbers were present in each well.
For antibody blocking experiments, antibodies
were added directly to cell suspensions or to wells (as
indicated) and incubated ~at room temperature ior 3 0
minutes just prior to thç addition of target cells to the
wells. The final concentratiors of antibodies used in the
blocking studies were: rabbit anti-human M-CSF polyclonal
antibody (Genzyme), l011g/ml ; rat anti-c-~ma/CSF-l receptor
(Oncogene Science, Inc. ), 2 ~Lg/ml; and mouse monoclonal
anti-human transferrin receptor (Hybritech, Inc.) 8 ~Lg/ml.
Xeat-inactivated normal rabbit serum (4 ~l/l x l05 cells)
was added in each case to prevent the cross-linking of
cells through Fc receptors.
Hence, in this example, a method for selectively
altering inter~ r adhesion has been demonstrated.
Spe~;f;~lly, it has shown that an artificial GPI-modified
variant of a model cytokine M-CSF, when anchored at the
cell surface, can augment c~ r binding to M-CSF
receptor-bearing~ tumor cells. High level expression of
both the M-CSF-GPI and M-CSFR molecules on their
respective cells could be efficiently obtained via gene
transfer using- episomal_ expression vectors. The
M-CSF-dependence of the effect was verified by anti-M-CSF
and anti-M-CSF receptor antibody blocking. Clearly, the
invention is nct limited to these variants since GPI-
anchored variants of multiple ligands other than M-CSF
could be used as alternative artif icial adhesins .
~WO95/16775 2 l 7 8 9 50 PCT/US9~/14297
In the above experiment, only the M-CSF
component of the M-CSF:M-CSFR pair was GPI-anchored. The
M-CSFR cl~prn~nt could also be GPI-anchored. Together,
this would permit coating one cell with a ligand:GPI
(e.g., M-CSF:GPI) chimera and a second cell with a
receptor:GPI (e.g., M-CSF~:GPI) chimera and thereby
selectively ~nl~nr;ng adhesiveness between the respective
cells via protein transfer. Such a use of a ~'disabled~
GPI-modified receptor, as part of an artificial adhesin
pair, would obviate the possibilities for unwanted
signaling through the receptor. Although there is
evidence for cis signaling through certain natural GPI-
anchored proteins, it seems likely that most cytokine
receptors, when artificially GPI anchored, will not be
functionally responsive to their corresponding cytokines
due to distortions in molecular topology.
The pre-adhesion step entails the mixing of
three components, namely, a tumor cell, a second cell with
greater immunogenic potential, and artif icial adhesin
molecules. After a co-incubation period, generally
lasting greater than 2 0 minutes, the mixture is
centrifuged at 400g for 5-l0 minutes in d~L~ ate tissue
culture medium supplemented with antibiotic and
resuspended in the same medium. The cell suspension is
kept at room temperature for 30 minutes with gentle
stirring. Subsequently the mixture i8 spun through a
sterilized isotonic sucrose ~320 mOsm) solution r~-nti:lining
2 mM sodium phosphate buffer, pH 7.2 (400g for 5-l0
minutes). The cell pellet comprising tumor cell:second
cell conjugates is gently suspended in l ml of isotonic
sucrose buffer.
Following pre-adhesion using artifical adhesins
and subsequent enrichment f or heterologous cell
conjugates, fusion is carried out. A preferred method for
fusing the heterologous cells in conjugates is
electrofusion. Methods for performing electrofusion are
Wo 95/16775 PCTIUS94/14297
21 78q50 ~ -
36
well-known to those f;~ r with the art. Of note, most
electrofusion protocols comprise two-steps: the induction
of membrane-membrane contact followed by application o~
the fusogenic pulse. The dielectrophoresis method is u3ed
in most experiments to achieve t~e f irst step of
congregating cells jbefore fusion-;n~ f ;n~ electric pulses
are applied. Xowever, it is known that the two steps can
be dissociated, that is, one need not congregate cells by
dielectrophoresis in order to achieve fusion (Sowers, 220
Methods in Enzvmoloqv 196, 1993). A preferred method
according to the present invention entails omitting the
dielectrophoresis step for congregating cells non-
selectively, and instead applying the fusogenic pulse to
heterologous cell l conjugates that have been pre-adhered
with artificial i~hF~C;nR The ~usogenic pulse can be
applied with a high voltage generator, and altenlative
instruments are coTnmercially available. Though it is
preferable to use con~ugates at low conjugate densities
(less than 10~ per ml), one can readily go up to 107 per ml
or higher in practicing this invention. Typical
electrofusion coIlditions use five square wave pulses of
2 . 5 kV/cm and 5 microsecond duration, at a controlled
temperature of 35C and with intervals of 15 sec between
pulses to permit dissipation of Joule heat. Optimization
of fusion conditions icr particular cell types can be
readily performea. (~ess optimal ~ ;nc are~those
described by Tsong and Tomita, 220 Methods in EnzYmoloq~
238, 1993).
~hile the present invention has been described
in conjunction with the preferred embodiments and
examples, one of ordinary skill, af ter reading the
foregqing specification, will be able to effect various
changes, substitutions ~or e~uivalents and = other
alterations to the invention provided herein. It is
therefore intended that the protection granted by letters
patent hereon be limited only by the definitions c-~nt~;n,o-l
.
1 78950
WO 95/16775 PCl'NS94/14297
37
in the appended claims, and equivalents thereof. It will
be understood that changes may be made and the details o~
~ormulation without departing :~rom the spirit o~ the
invention as de~ined in the ~ollowing claims.
