Note: Descriptions are shown in the official language in which they were submitted.
- 21 ~6528
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GENEITICALLY MODIFIED CELLS FOR USE IN TRANSPLANTATION
` of ' - l
The elucidation of the molecular basis for many inherited disorders together with the
molecular isolation of the genes involved in the~ ~ disorders now offer the potential for
therapeutic treatments based upon providing a fimctional gene product to a patient having a
defect in that gene product. Gene therapy, in which a gene encoding a functional gene
product is introduced into cells of a patient to restore the activity of that gene product in the
patient, is now a realistic option for many congenital diseases. Two patients with adenosine
deaminase deficiency have already been treated for their disease by gene therapy, with
u." ~ results, amd a number of other human gene therapy protocols have received
approval for limited clinical use. Cystic fibrosis, Duchenme muscular dystrophy and
hrrnl~rhiliA are just a few of the inherited diseases which are potentially treatable by gene
therapy. Fu, Lll~l.llul~, gene therapy approaches are being applied to acquired disorders as
well, for example by illLIuduu;~lg into cells of a patient genes encoding gene products which
enhamce the l~ Ull~ ofthe patient's immune system. Novel approaches to treating
diseases such as cancer and AIDS are thus also possible by applying the principles of gene
therapy. For reviews on gene therapy approaches see Anderson, W.F. (1992) Science
256:808-S13; Miller, A.D. (1992) Nature 357:455-460; Friedmalm, T. (1989) Science
244:1275-1281; and Cournoyer, D., et al. (1990) Curr. Opin. Biotech. 1:196-208.
The general approach of gene therapy involves the illLIu~_Liull of exogenous genetic
material (e.g., DNA or RNA) into a cell such that one or more gene products encoded by the
introduced genetic material are produced in the cell, for example to restore or enhance a
fimctiûnal activity. Exogenous DNA has been successfully introduced into cells both ex vivo
(i.e., in vitro) amd in vivo. In recent years, many advances in gene therapy have been reported
that address problems relating to the types of gene delivery systems that cam be used, the
different types of genes which can be introduced into cells amd the kinds of cells which can be
modified. However, gene therapy is still limited by the need to modify autologous cells. A
patient's own cells must be modified because foreign cells, whether they are from the same
species (allogeneic) or another species (y nr,g~n~ir), are recognized as foreign by the
patient's immune system when introduced into the patient and are ~ ly rejected.
Thus, in the case of ex vivo gene therapy, cells must frrst be harvested from the patient,
modified in culture amd then l~ ..lLI, ' ' into the patient. This procedure is both time-
consuming to complete and invasive for the patient. While the ability to modify some cells in
35 vivo may overcome some of these problems, certain cell types may not be accessible for
~ "r,.l; r.. A ~ in vivo, or may not be tArgeted specifically or efficiently modified in vivo.
F~ Lll.,. ulul~ the cell .. l; r.. ; .. procedure, whether performed ex vivo or in vivo, must be
repeated for each individual patient.
, .. ,, . _, . , . . _ .... . . .. ........... ........ ............. ..... .... . ... ............ . . .. ... ... . .. ........ ..
2 1 86528
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As a means of gene therapy, it would be beneficial to provide a patient with
L~t~,lulo~uua donor cells that have been modified to express a gene product. This would
obviate fhe need to harvest cells from the patient for genetic I . ").1; ri -~ ;. ,. . thereby reducing
both the time and i~ .,Il.,aa of the procedure. In addition, a ready supply of modified
5 donor cells that CG 11'l be ~ 'UIJ~ ,J and available for ill(lUJU~,LiUII into one, or multiple,
patients could be prepared for use as needed. The ability to use modified heterûlogous dûnor
cells from either the same species as the patient ûr from a different species would also expamd
the source of cells which could be used for gene therapy.
However, ~ - ;nn of ~ ,lulO~uua cells ~i.e., allogeneic or ~, - ~.... ~ cells)
10 into a host elicits an immune response against the cells. Thus, use of modified h.,tclulOguua
cells for therapeutic purposes requires a means by which to avoid ' ~ I rejection of
the modified cells by the patient. Current approaches toward rnhibiting immune responses
against ~ J cells f~ypically involve systemic treatment of the patient, for example
with ;.. , ~ a.ve agents. This has the Jia~l~ that the patient exhibits
nnnererifir. ;.. ~ caa;UII. Additionally, ;.. ~ ,aa;vc drugâ are known to have
side effects that include am increased ,~ ;l-;l;ly to infections, renal failure, ll.y~ .,lla;u
and tumor growth. Thus, there is a need for an improved method which allows the use of
~ ,t~,lulO~ uUa donor cells for gene therapy purposes which avoids the ~IPfnmP~AfAI effects of
systemic il~ .J~ Caaall~a
S of '-l
This invention provides a means by which allogeneic cells or ~..~.... i.~ cells are
used to deliver a gene product to an individual without the need for systemic
;" ~ " " `"` '`"1'1" caa;ull of the individual~ The invention features cells which are modified to
25 express a gene product amd which have an antigen on the surface of the cell altered to inhibit
rejection of the cell when the cell is ~ into a subject. Prior to alteration, the
amtigen on the cell surface stimulates an immune response against the cell in the subject.
However, the antigen on the cell surface is altered to irlhibit ' ~, ' rejection of the
cells by the subject. Specifically, the antigen is altered to modify an interaction between the
30 antigenanda1 .., ~-,,u~). ;i~ cell,preferablyaTI~ ,UIIU~ ,inthesubject. Sincetheantigen
on the cell surface is altered prior to l. A 1~ -', the recipient subject does not require
systemictreatmentwithan;~.. ~.. l.l.,~aa;~,agenttopreventrejectionofthecell. Thus,
this mvention permits the use of allogeneic or xenogeneic cells as vehicles for delivery of a
gene product to a subject. Moreover, this invention allows for the preparation of genetically
35 modified cells which can be ~,lyu,ulcac~ ~,J and a~ ul..~ ~cJ to a subject when necessary,
thereby ~dl~UlllV.~ ;llg the need to isolate and modify autolûgous cells from the subject.
According to the invention, a cell iâ modified to express a gene product by, forexample, ilU,lUllUCUlg into the cell a nucleic acid having a nucleotide sequence which encodes
the gene product in a form suitable for expression of the gene product in the cell. In a
21 86528
W<l 95127042 ~ / O~ ~ S?
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preferred ~ l" " ,l the nucleic acid encoding the gene product is introduced into the cell
usmg a ~ viral vector, such as an adenoviral vector, an adeno-associated viral
vector or a retroviral vector. The gene product can be, for example, a secreted protein, a
membrane-bound protein or an infrAA~ r protein. Other gene products include active RNA
S molecules.
In one . . . ,1.~ of the invention, an anfigen on the surface of the cell is altered by
contacting the cell prior to ~ t ;- ~ (i.e., in vilro) with a molecule which bmds to the
antigen. A preferred molecule for altering an antigen on the cell is an antibody, or fragment
or derivative thereof, such as an F(ab')2 fragment. Alternatively, the molecule is a peptide or
10 derivative thereof k.g., a peptide mimetic) which binds the antigen and interferes with an
interaction with a I r " ~ cell. In a preferred ~ .. ,l .u.l ., ... ,1, the antigen on the cell
surface which is altered is an MHC class I antigen. Other cell surface antigens which can be
altered include a&esion molecules such as LFA-I, ICAM-I and ICAM-2
In one .. ,l ,o.l; .. l of the invention, a llu.. ~ cell is modified to express a human
15 gene product. A preferred non-human cell for use in providing a human gene product to a
subject is a porcine cell. Cell types which are modified according to the invention include
muscle cells, liver cells, neural cells, pancreatic islet cells and 1 J ' " cells.
F~ L~ IUIC;, the cell which is modified can be within a tissue or organ.
The modified cells of the invention are A-i~ -d to subjects to deliver a gene
20 product expressed by the cell to the subject. Prior to L ' ~ the cell to the subject, one
or more antigens on the cell surface are altered, e.g. by contacting the cell in vitro with a
molecule which binds to the antigen. Although a cell can be modified to express a gene
product in vivo, it is preferred that the cell is modified ex vivo, prior to A.li.. l ~ 1.0 the cell
to the subject.
2~ The invention further provides kits for use in delivering a gene product to a subject
which include a cell modified to express the gene product and a molecule (e.g., an antibody,
or fragment or derivative thereof) which bmds to am amtigen on the cell. Alternatively, the kit
includes a vector encoding a gene product with which to modify a cell and a molecule (e.g.,
am antibody, or fragment or derivative thereof) which binds to an antigen on the cell surface.
Bri~f 1~ " of ' D`
Figure l depicts the plasmid map of pCMVGH, which contains the gene encoding
humam growth hormone.
Figure 2 depicts a ,3 ~ r~ stain of cultured humam myotubes transfected with
35 plasmids pCMV~, which contains a gene encoding ~ -- l. ,ci.l ~. and pJK2Neo, which
displays neomycin resistance.
Figure 3 depicts a ~ stain of human myoblasts transfected with
plasmids pCMV13, which contains a gene encoding ~ ~ ' ' , and pJK2Neo, which
displays neûmycin resisf~ance, which were i . ' ' inih rat muscle.
2 1 86~28
WO 95/27042 P~ A~ D
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Figure 4 depicts a rabbit anti-desmin stain of human myoblasts modified with F(ab')2
fragments of the, .~ t~ antibody W6/32 and ~ 1 into u~t~ u.;ll-treated mice.
of" T
S This invention provides modified heteroloy3~s cells amd methods for delivering a
gene product to an allogeneic or xenogeneic subject without eliciting an immune response
against the cell in the subject. The invention features a ll~,tt~.ul~vu~ cell which is modified
to express a gene product and which is treated such that an amtigen on the cell surface which
stimulates an immune response against the cell in an allogeneic or ~ eic subject is
altered to inhibit rejection of the cell when 1, . .~ l into the subject. Preferably, the
antigen which is altered is am antigen which mteracts with a T Iylll,,Jllu.,y .~, in an allogeneic or
Y~"~ subject. Typically, the heterologous cell is treated to alter the amtigen on its
surface prior to ~ the cell to the subject. Thus, it is not necessary to treat the
subject ty~t~,llucally with an ;., ~ ;ve agent to prevent rejection of the
L~,tt,.ulo~;uu~ cell. Rather, following r ' ' ' ' ûf the h~,t~,.ulo~;uu~ cell (which has been
altered as described herein), the subject exhibits ;. "....~ non-l~,al~UIl ~ specific
for the cell. Preferably, the heterologous cell is also modified to express a gene product prior
to ~ "~ the cell to a subject (i.e., ex vivo). However, the cell can be modified to
express a gene product in vivo, following ~- 1 ..;, .: ..~...~ ;.... of the cell to the subject.
This invention enables gene therapy approaches to be extended to the use allogeneic
and Y,A ~,..1- ' cells as donor cells to deliver a gene product to a subject. Since both the
procedure to modify a ll~,t~ulo~;uu~ cell to express a gene product and the procedure to alter
an antigen on the surface of the cell cam be perfomed ex vivo, prior to A,l.";": . . . ;,~ the cell to
a subject, the invention allows for the preparation of genetically modified allogeneic or
25 Y~ ,.,: . cells which can be Cl,yuul-..,cil ~,d and stored until use. When the eells are needed
by a subject, the already modified cells cam be thawed, treated to alter an antigen on their
surface amd ~ '~, A l ,~ ,1 to the subject. Thus, a subject in need of gene therapy
can receive immediate treatment, Mther than havmg to wait umtil his or her own cells can be
isolated, ~u~c~ rUlly modified and IL;..~ud~ccd. Additionally, if . ~ ., ... " is necessary, a
30 second aliquot of already modified cells can easily be thawed, altered amd 1~ .1,1,;": t, .cd.
Moreover, the ability to use h.,tt~.ulo~;uu~ cells for gene therapy purposes gready extends the
supply of donor cells which can be used in this type of treatment.
Accordingly, this invention provides a heterologous cell which is modified to express
a gene product and which has an amtigen on the cell surface altered to modify am interaction
35 between the amtigen and a I )i "~ cell (e.g., a T IyllllJllùt~yle). The invention further
provides methods for delivering a gene product to a subject by A.l~ .t- ;1 1~ cells of the
invention. The following ,~ . describe in detail: 1) the .. ~ 1;1;. .-1;. .I. of a llt,t~,lult;
cell to express a gene product; and 2) the alteration of arl amtigen on the cell to modify an
interaction between the antigen and a 1- t ~ cell.
woss/z7042 2 1 ~ 6528 .~
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1. M~ ;..,.ofa (~rll to F~r~ a GfnrPro.~ t
A cell of the invention is "modified to express a gene product". As used herein, the
term "modified to express a gene product" is intended to mean that the cell is treated in a
5 matmer that results ir ~he production of a gene product by the cell. Preferably, the cell does
not express the gene product prior to ~ .. Alternatively, n~ lifi~tif~n of the cell
may result in an increased production of a gene product already expressed by the cell or result
in production of a gene product (e.g., an amtisense RNA molecule) which decreases
production of another, .. l. ~;. ,.l Ir gene product normally expressed by the cell.
In a preferred ~ .l,~-l;. . -: a cell is modified to express a gene product by
mtroducing genetic material, such as a nucleic acid molecule (e.g., RNA or, more preferably,
DNA) into the cell. The nucleic acid molecule introduced into the cell encodes a gene
product to be expressed by the cell. The term "gene product" as used herein is intended to
include proteins, peptides amd functional RNA molecules. Generally, the gene product
15 encoded by the nucleic acid molecule is the desired gene product to be supplied to a subject.
Alt~.l,dti~ , the encoded gene product is one which induces the expression of the desired
gene product by the cell (e.g., the introduced genetic material encodes a . . factor
which mduces the ~ .. . of the gene product to be supplied to the subject).
A nucleic æid molecule introduced into a cell is in a form suitable for expression in
20 the cell of the gene product encoded by the nucleic acid. Accordingly, the nucleic acid
molecule includes coding and regulatory sequences required for I ..- -- - . ;I~l ;.... of a gene (or
portion thereof) and, when the gene product is a protein or peptide, translation of the gene
product encoded by the gene. Regulatory sequences which can be included in the nucleic
æid molecule include promoters, enhancers and pOl~ad~,lly;aiiol~ signals, as well as
25 sequences necessary for transport of am encoded protein or peptide, for example N-terminal
signal sequences for transport of proteins or peptides to the surfæe of the cell or for secretion.
Nucleotide sequences which regulate expression of a gene product (e.g., promoter and
erlhancer sequences) are selected based upon the type of cell in which tbe gene product is to
be expressed and the desired level of expression of the gene product. For example, a
30 promoter known to confer cell-type specific expression of a gene lirked to the promoter can
be used. A promoter specific for myoblast gene expression can be linked to a gene of interest
to confer muscle-specific expression of that gene product. Muscle-specific regulatory
elements which are known in the att include upstream regions from the dystrophin gene
(Klamut et al., (1989) MoL CeL BioL 9:2396), the creatine kinase gene (Buskin and
Hauschka, (1989) MoL Cell BioL 9:2627) and the troponin gene (Mar and Ordabl, (1988?
Proc. Na~L Aca~ Sci USA. 85:6404). Regulatory elements specific for other cell types are
known in the art (e.g., the albumin enhancer for liver-specific expression; insulin regulatory
elements for pancreatic islet cell-specific expression; vatious netlral cell-specific regulatory
elements, including neural dystrophin, neural enolase attd A4 amyloid promoters).
.. . . . .. . . .. .. .. . _ . . . ..
21 ~6528
WO95/27042 P~ 9~10 ~0 o
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A I ~. ly, a regulatory element which can direct ~u~ iLu;i v~ expression of a gene in a
variety of different cell types, such as a Yiral regulatory element, can be used. Examples of
viral promoters commonly used to drive gene expression include those derived from polyoma
virus, Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
5 Alternatively, a regulatory element which provides inducible expression of a gene l;.i~s~d
thereto can be used. The use of an inducible regulatory element (e.g., an inducible promoter)
allows for ."r~ ;. . of the production of the gene product in the cell. Examples of
potentially useful inducible regulatory systems for use in eukaryotic cells include hormone-
regulated elements (e.g., see Mader, S. and White, J.H. (1993) Proc Natl. Acad. Sci. USA
90:5603-5607), synthetic ligand-regulated elements (see, e.g. Spencer, D.M. et al. (1993)
&ience 262:1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et
al. (1993) Bioc~ernist~y 32:10607-10613; Datta, R. et al. (1992) Proc. NatL Acad. Sci. USA
89:10149-10153). Additional tissue-specific or inducible regulatory systems which may be
developed cam also be used in accordance with the invention.
There are a number of techniques known in the art for illLludu~i~ genetic material
into a cell that can be applied to modify a cell of the invention. In one ~ 1 ~ t, the
nucleic acid is in the form of a naked nucleic acid molecule. In this situation, the nucleic acid
molecule introduced into a cell to be modified consists only of the nucleic acid encoding the
gene product and the necessary regulatory elements. Alternatively, the nucleic acid encoding
20 the gene product (including the necessary regulatory elements) is containad within a plasmid
vector. Examples of plasmid expression vectors include CDM8 (Seed, B., Nature 329:840
(1987))andpMT2PC(Kaufman,etal.,EMBOJ. 6:187-195(1987)). Inanother
t, the nucleic acid molecule to be introduced into a cell is contained within a viral
vector. In this situation, the nucleic acid encoding the gene product is inserted into the viral
25 genome (or a paTtial viral genome). The regulatory elements directing the expression of the
gene product can be included with the nucleic acid inserted into the viral genome (i.e, linked
to the gene inserted into the viral genome) or can be provided by the viral genome itself.
Examples of methods which can be used to introduce naked nucleic acid into cells and viral-
mediated transfer of nucleic acid into cells are described separately in the ~ below.
A. Tntr ~ rtir,n of ~ tl N~rlrir Ari.1 intr, (~1lc
1. Transfection mediated by CaP04: Naked DNA can be introduced into cells by forming a
35 precipitate containing the DNA and calcium phosphate. For example, a HEPES-buffered
saline solution can be mixed with a solution containing calcium chloride and DNA to form a
precipitate and the precipitate is then incubated with cells. A glycerol or dimethyl sulfoxide
shock step can be added to increase the amount of DNA taken up by certain cells. CaPO4-
mediated ~ can be used to stably (or transiently) transfect cells and is only
2~86528
WO 9S127042 P~ F~
1 _ 7 _
applicable to in vifro llln.l;l; -:;..,~ of cells.' Protocols for CaPO4- mediated i.,... r ~ .. can
be found in C~rrPnt prntnrnle jn Mn~ -lDr Bioloov, Ausubel, F.M. et al. (eds.) Greene
Publishing Associates, (1989), Section 9.1 and in MolPr~llDr Cl A T ~ M~n-
~2n(1 F~lifinn Sambrook et al. Cold Spring Harbor Laboratory Press, (1 989), Sections 16.32-
5 16.40 or other standard laboratory mamuals.
2. Transfecf ion mediated b~ DEAE-dey fran: Naked DNA can be introduced into cells by
forming a mixture of the DNA and DEAE-dextran and incubating the mixture with the cells.
A d;~ yLIllrul-id~, or chloroquine shock step can be added to increase the amount of DNA
10 uptake. DEAE-dextrim 1"-"~ .... is only applicable to in vitro ",...1; f;..~;.... of cells and
can be used to introduce DNA transiently into cells but is not preferred for creating stably
transfected cells. 'rhus, this method can be used for short term production of a gene product
but is not a method of choice for long-term production of a gene product. Protocols for
DEAE-dextr m-mediated ~ l,, can be found in Cllrr~Dnt Protnrnle in Mcl
~ Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.2 and in
MnlDrlllDr Clnnin~ A L ' r~ ~ 2n~1 F~ ion Sambrook et al. Cold Spring Harbor
Laboratory Press, (1989), Sections 16.41-16.46 or other standard laboratory manuals.
3. Ele~,~ ul~ul u(~(".. Naked DNA can also be introduced mto cells by incubating the cells and
20 the DNA together in an appropriate buffer and subjecting the cells to a high-voltage electric
pulse. rhe efficiency with which DNA is introduced into cells by cl.,.,L.u~lul~iull is
inf~uenced by the strength of the applied field, the length of the electric pulse, the
~ l,u~ , the ~ l ~ f~ and ~ .. ,., of the DNA amd the ionic ~ . l ;. ., . of
the media. El~ ul~ul~iull cam be used to stably (or transiently) transfect a wide variety of
25 cell types and is only applicable to in vitro .. ,.~.l, r;..;1,.... of cells. Protocols for
u~Jul~l~illg cells can be foumd in Cllr~ent Protornle in MolDrlllor l~:nln~ov. Ausubel, F.M.
et al. (eds.) Greene Publishing Associates, (1989), Section 9.3 and m Ml ' ' Clonir~o A
T :lhnr~tnr,v M~ 2n-1 E~itinn Sambrook et al. Cold Spring Harbor Laboratory Press,
(1989), Sections 16.54-16.55 or other stimdard laboratory manuals.
4. Li,uos~ ' transfection ("liuvf~ iu,.'~: Naked DNA cam be introduced into cells
by mixing the DNA with a liposome suspension containing cationic lipids. The
DNAAiposome complex is then incubated with cells. Liposûme mediated tr?rl~fDctlnn can be
used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in
35 CllrrDnt Protocols in MnlDrlllDr Biolo_v. Ausubel, F.M. et al. (eds.) Greene Publishing
Associates, (1989), Section 9.4 and other standard laboratory manuals. Additionally, gene
delivery in vivo has been ~ 1 using liposomes. See for example Nicolau et al.
(1987) Mefh~ En2. 149:157-176; Wang and Huang (1987) Proc NatL A.cad &i. USA
Wo 9~/27042 - 8 - r~l" . c ~ '
84:7851-78~5; Brigham et al. (1989) Am. J. Med Sci. 298:278; and &ould-Fogerite et al.
(1989) Gene 84:429-438.
5. Direct Injection: Naked DNA can be introduced mto cells by directly injecting the DNA
5 mto the cells. For an in ~ ro culture of cells, DNA can be irltroduced by Illi.,I. ;
Since each cell is ~ lvill;~h~ ndividually~ this approach is very labor intensive when
modifyirlg large numbers of cells. However, a situation wherein ., - ., .; ,~1;.... is a method
of choice is in the production of transgenic animals (discussed in greater detail below). In
this situation, the DNA is stably mtroduced into a fertilized oocyte which is then allowed to
10 develop into an animal. The resultant animal contains cells ca~rying the DNA introduced into
the oocyte. Direct mjection has also been used to introduce naked DNA into cells in vivo (see
e.g., Acsadi et al. (1991) Nature 332: 815-818; Wolffet al. (1990) Science 247:1465-1468).
A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo can be used.
Such an apparatus is ,..""". .,:~lly available (e.g., from BioRad).
6. Receptor-Mediated DNA Uptake: Naked DNA can also be mtroduced into cells by
the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-
surfacereceptor(seeforexampleWu,G.andWu,C.H.(1988)J.Biol.Chem.263:14621;
Wilson et al. (1992) J. BioL Chem. 267:963-967; arld U.S. Patent No. 5,166,320). Binding of
20 the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated
~.IJu~"~ tv~;~. Receptors to which a DNA-ligand complex have targeted include the transferrin
receptor and tbe ' ~,~y~v}nui~,I.. receptor. A DNA-ligand complex linked to adenovirus
capsids which naturally disrupt en~ eo~ e, thereby releasmg material mto the cytoplasm can
be used to avoid ~ '; of the complex by ;. ' . .~ 1, Iysosomes (see for example
Curiel et al. (1991) Proc. Nat~. Acad Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.
Acad. Sci. USA 90:2122-2126). Receptor-mediated DNA uptake can be used to introduce
DNA into cells eitber in vitro or in vivo amd, a~id;tiv~ has tbe added feature tb~t DNA can
be selectively targeted to a particular cell type by use of a ligand which binds to a receptor
selectively expressed on a target cell of interest.
Generally, when naked DNA is introduced into cells in culture (e.g., by one of the
F ~ techniques described above) only a small fraction of cells (about I out of 105)
typically integrate the transfected DNA into their genomes (i.e., the DNA is maintained in the
cell episomal~y). Thus, in order to identify cells which have taken up exogenous DNA, it is
a lv " to transfect nucleic acid encoding a selectable marker into the cell along with
the nucleic acid(s) of interest. Preferred selectable markers include those which confer
resistance to drugs such as G418, l~b.vIlly~,hl and methotrexate. Selectable markers may be
mtroduced on the same plasmid as the gene(s) of mterest or may be introduced on a separate
plasmid.
2 1 8~52~
_W095127042 r~"~ Cr
An alternative method for generating a cell that is modified to express a gene product
involving ;..~lUdU~,lllg naked DNA into cells is to create a transgenic arlimal which contains
cells modified to express the gene product of interest. A transgenic animal is an animal
having cells that contain a transgene, wherein the transgene was introduced into the animal or
an amcestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DN.
molecule which is integrated into the genome of a cell from which a transgenic animal
develops and which remains in the genome of the mature animal, thereby directing the
expression of an cncoded gene product in one or more cell types or tissues of the transgenic
animal. Thus, a tTansgenic animal expressing a gene product of interest in one or more cell
10 types within the animal can be created, for example, by illlludu~ lg a nucleic acid encoding
the gene product (typically linked to ~,u~.ul regulatory elements, such as a tissue-specific
enhancer) into the male pronuclei of a fertilized oocgte~ e.g., by lll;.,l~ ; and allowing
the oocyte to develop in a lu~cudu~ ~lt female foster animal. Methods for generating
transgenic animals, Iu_ ~icul_lg animals such as mice, have become cu..v~,llliullal in the art
amd are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 arld Hogan, B.
et al., (1986) A Laboratory Manual, Cold Spring Harbor, New York, Cold Spring Harbor
Laboratory. A transgenic founder animal can be used to breed more animals carrying the
tramsgene. Cells of the transgenic animal which express a gene product of interest can then
be used to deliver the gene product to a subject in accordance with the invention.
Alh,.~ .,ly, an animal containing a gene which has been modified by l"""~
,~ .. , .1.; .. ~: .. can be cull~.~ u~,t~,~ to express a gene product of interest. For example, an
....~.L, ,.,...,~genecarTiedinthegenomeoftheanimalc.nbealteredbyh~.m.^,l~O
1. ' (for inst~nce, all or a portion of a gene could be replaced by the human
h~lm.^~ gll~ of the gene to "' " the gene product encoded by the gene) or an
25 ~ gene can be "knocked out" (i.e., inactivated by mutation). For example, an
nO, ....-- - gene in a cell can be knocked out to prevent production of tThat gene product and
then nucleic acid encoding a different (preferTed) gene product is intToduced into the cell. To
create an animal with h.^lm.^~l.^.~.^~llely ,~ .. 1.:.. <I nucleic acid, a vector is prepared which
contains the DNA which is to replace or interrupt the I .. 1.^1,.~.. ~ DNA flanked by DNA
30 1 ~ y~ to the ...,.1~,~"..,.. ~ DNA (see for example Thomas, K.R. and Capecchi, M. R.
(1987) Cell ~ 503). The vector is introduced into an embryonal stem cell line (e.g., by
cl~,~,L.u,uul~tiull) and cells which have 1~ y~ 'y ICI~Vll.~ ,d the DNA are selected (see
for example Li, E. et al. (1992) cen ~9 915). The selected cells are then injected into a
blastocyst of an animal (e.g., a mouse) to form ~ co~tiull chimeras (see for example
35 Bradley, A. in T~, , and ~mbryonic Stem Cells: .4 Practical ~pproac~, E.J.
Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable ~ ,u-lu,ulcyll_l~ female foster animal and the embryo brought to term.
Progeny harbouring the 1 ~ , IC~,Ulllb;ll~,d DNA in their germ cells can be used to
breed animals in which all cells of lhe animal contain the I ~ ly Ic~,ulllll;,l.,d DNA.
2 f
wo gs/270~2 r~
- 10 -
Cells of the animal containing the l~ lvg~ y ~ci ' ' DNA which express a gene
product of interest can then be used to deliver the gene product to a subject inwith the invention
5 B. Virr' ~ " 'GrneTrmcfrr
A preferred approach for illhvdu.,;l-g nucleic acid encoding a gene produet into a cell
is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product.
Infection of cells with a viral vector has the advantage that a large proportion of cells receive
the nucleic acid, which can obviate the need for selection of cells which have received the
10 nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA
contained in the viral vector, are expressed efficiently in cells which have taken ~ viral
vector nucleic acid a~d viral vector systems can be used either in vitro or in vivo.
1. R~lr(,~ . Defective ~C;I1UV;IU~ D are well ~ ;, A for use in gene ~ansfer forgene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). A .~
retrovirus can be constructed having a nucleic acid encoding a gene product of interest
inserted into the retroviral genome. Additionally, portions of the retroviral genome cam be
removed to render the retrovirus replication defective. The replication defective retrovirus is
then packaged into virions which can be used to infect a target cell through the use of a helper
20 virus by standard tecbniques. Protocols for producing .~ ' . h vv U U1~D arld for
infecting cells in vitro or in vivo with such viruses can be found in C..rrent Prr~t~r~ ;n
M~lrrlllr ~ V. Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989),
Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable l~U~VilUD~D
include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ~Crip, ~IrCre, ~2 and IyAm. Retroviruses
have been used to introduce a variety of genes into many different cell types, including
epithelial cells, endothelial cells, I~IIIJJIIV~ D, myoblasts, I I r ~D~ bone marrow cells, in
vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and
Mulligan (1988) Proc. NatL Acad Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Na~l.
Acad Sci. USA 85:3014-3018;Armentanoetal.(1990) ProcNatl.Acad Sci. U~4 87:6141-
6145; Huber et al. (1991) Proc. NatL Acad Sci. USA 88:8039-8043; Ferry et al. (1991) Proc.
Natl. Acad Sci. USA 88:8377-8381; Chow&ury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc Natl. Acad Sc1 USA 89:7640-7644; Kay et al. (1992) ~uman
~;ene 17~erapy 3:641-647; Dai et al. (1992) Proc NatL Acad Sci. USA 89:10892-10895; Hwu
et al. (1993) J. ImmunoL 150:41044115; U.S. Patent No. 4,868,116; U.S. Patent No.
4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT
Application WO 89/05345; and PCT Application WO 92/07573). Retroviral vectors require
target cell division in order for the retroviral genome (and foreign nucleic acid inserted into
2l 8&~28
~ WO 9~il27042 P. l/- S
11
it) to be integrated into the host genome to stably introduce nucleic acid into tbe cell. Thus, it
may be necessary to stimulate replication of the target cell.
2. A~,.v . ;, . .~. . The genome of an adenovirus can be . ' ' such that it encodes and
5 expresses a gene product cr !lterest but is inactivated in telms of its ability to replicate in a
normal Iytic viral life cycle. See for example Berkner et al. (1988) BioTechni~ues 6:616;
Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dB24 or other
strr1ins of adenovirus (e.g., Ad2, AdB, Ad7 etc.) are well known to those skilled in the art.
10 R~ ...,l.;, -' ad~ uv;.u.warea~ U~..inthattheydonotrequiredividingcellstobe
effective gene delivery vehicles and can be used to infect a wide variety of cell types,
including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells
(Lemarchand et al. (1992) Proc NatL Acad Sci. USA 89:6482-6486), l I .~ (Herz and
Gerard (1993) Proc NatL Acad Sci. USA 90:2812-2816) and muscle cells (Quantin et al.
(1992) Proc NatL Acad. Sci. USA 89:2581-2584). Additionally, introduced adenoviral DNA
(amd foreign DNA contained tberein) is not inteBted into the genome of a host cell but
remains episomal, thereby avoiding potential problems that can occur as a result of
insertional ,., ~ in situations where introduced DNA becomes mtegrated imto the host
genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for
foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al.
cited supra; Haj-Ahmand and Graham (1986) ~ ViroL 57:267). Most l~ rl- defectiveadenoviral vecto}s culrently in use are deleted for all or parts of the viral El and E3 genes but
retain as much as 80 % of the adenoviral genetic material.
3. Adeno-Associated Viruses: Adeno :. ' virus (AAV) is a naturally occurring
defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper
viros for efficient replication and a productive life cycle. (For a review see Muzyczka et al.
Curr. TopicsinMicro. andlmmunoL (1992)158:97-129). Itisalsooneofthefewviruses
that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable
integration (see for example Flotte et al. (1992) ~m. J. Respir. CelL MoL BioL 7:349-356;
Samulski et al. (1989) J. ViroL 63:38æ-3828; and M~T ~ n et al. (1989) J. ViroL
62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and
can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as
tbat described in Tratschin et al. (1985) MoL Cell. Biol. 5:3251-3260 can be used to introduce
DNA into cells. A variety of nucleic acids have been introduced into different cell types
using AAV vectors (see for example Hermonat et al. (1984) Proc NatL Acad. Sci. USA
81:6466-6470; Tratschin et al. (1985) MoL CelL BioL 4:2072-2081; Wondisford et al. (1988)
MoLEndocrinoL2:32-39;Tratschinetal.(1984)J. ViroL51:611-619;andFlotteetal.
(1993) J. Biol. Chem. 268:3781-3790).
~ 1 ~6wo gs/27042 r~ c l~ ~Q
- 12 -
The efficacy of a particular expression vector system and method of ;~ udA~ lgnucleic acid into a cell can be assessed by stimdard approaches routinely used in the art. For
example, DNA introduced into a cell can be detected by a filter lI.~bI;dI~ UII technique (e.g.,
Southem blotting) and RNA produced by ~ ;-, of introduced DNA can be detected,
5 for example, by Northem blotting, RNase protection or reverse ~ -' IJUI,~'III~dS.,
chain reaction (RT-PCR). The gene product cam be detected by an appropriate assay, for
example by ;., . . " . .. ,r l~,~;;. -I detection of a produced protein, such as with a specific antibody,
or by a fimctional assay to detect a fimctional activity of the gene product, such as an
en~ymatic assay. If the gene product of interest to be expressed by a cell is not readily
10 assayable, an expression system can first be optimi~ed using a reporter gene linked to the
regulatory elements and vectûr to be used. The reporter gene encodes a gene product which
is easily detectable and, thus, can be used to evaluate tbe efficacy of the system. Standard
reporter genes used in the art include genes encoding ~ gl~ acetyl
transferase, luciferase and human growth hommone.
When the method used to introduce nucleic acid into a population of cells results in
" ,.~. I; i~. -:;.... of a large proportion of the cells and efficient expression of the gene product by
the cells (e.g., as is often the case when usin6 a viral expression vector), the modified
population of cells may be used without further isolation or subcloning of individual cells
within the population. That is, there may be sufficient production of the gene product by the
20 population of cells such that no further cell isolation is needed. Altematively, it may be
desirable to grow a 1-.. ~,. ., ~ population of identically modified cells from a single
modified cell to isolate cells which efficiently express the gene product. Such a population of
unifomm cells can be prepared by isolating a single modified cell by limiting dilution cloning
followed by expanding the single cell in culture into a clonal population of cells by standard
25 techrliques.
C. Oth~r ~ fho~1c for Mo~ irl~ a C~ll to Fyrrr~ a G~-.r prn~ t
Altemative to ;llUUdU~ .tj a nucleic acid molecule into a cell to modify the cell to
express a gene product, a cell can be modified by inducing or mcreasing the level of
30 expression of the gene product by a cell. For example, a cell may be capable of expressmg a
particular gene product but fails to do so without additional treatment of the cell. Similarly,
the cell may express;-- .~r:- .1 amounts of the gene product for the desired purpose. Thus,
am agent which stimulates expression of a gene product can be used to induce or increase
expression of a gene product by the cell. For example, cells can be contacted with an agent in
35 vifro in a culture medium. The agent which stimulates expression of a gene product may
function, for instance, by increasing ~ .... of the gene encoding the product, by
increasing the rate of tr.mslation or stability (e.g., a post l. ~ -l ,.... such as a
poly A tail) of an mRNA encoding the product or by increasing stability, transport or
21 8~528
wo ssn7042 ~ r
- 13 -
l,~, .l i, I i. ., . of the gene product. Examples of agents which can be used to induce expression
of a gene product mclude cytokines and growth factors.
Another type of agent which can be used to induce or increase expression of a gene
product by a cell is a ~ ,l;.... factor which ~ ;.... 0f the gene
5 encoding the product. A i I factor which Ll~lliYUL~i. the expression of a geneencoding a gene product of interest can be provide~ to a cell, for example, by illU~ v into
the cell a nucleic acid molecule encoding the i . - facto}. Thus, this approachrepresents an alternative type of nucleic aeid molecule which ean be introduced mto the eell
(for example by one of the previously discussed methods). In this ease, the introduced
10 nucleic acid does not directly encode the gene product of interest but ratber eauses produetion
of the gene produet by the eell indirectly by inducing expression of the gene produet.
In yet another method, a cell is modified to express a gene produet by eoupling the
gene product to the cell, preferably to the surface of the cell. For example, a protein ean be
obtained by purifying tbe cell from a biological source or expressing the protein
15 I~ yusmgstandard.~ .lDNAtechnology. Theisolatedproteincarlthen
be coupled to the cell. The terms "coupled" or "coupling" refer to a chemical, enzymatic or
otber means (e.g., by binding to an antibody on the surface of the cell or genetic ~
of linkages) by which a gene product can be linked to a eell sueh that tbe gene product is in a
form suitable for delivering the gene product to a subject. For example, a protein can be
20 ehemieally vlU ~ ~lillkt d to a eell surface using ~ .,;dll~ available l,lua llu~hlv reagents
(Pierce, Rockford IL). Other approaches to coupling a gene product to a cell indude the use
of a bispecific antibody which binds both the gene product and a eell-surface moleeule on the
eell or .. l; ri. ;.. of the gene produet to mclude a lipophilic tail (e.g., by inositol phosphate
linkage) which ean insert into a cell membrane.
TT Alf~ r-ti.-n of -n Ai-ti*en on the ('ell
In addition to . . ,.-..l; I . ~1;. ..~ of a cell to express a gene produet, this invention imvolves
altering an amtigen on the eell surfaee to reduce the v ~J of the eell and thereby
inhibit rejeetion of the eell when i , ' ' into an allogeneie or ~ v i subject. In an
30 umaltered state, the amtigen on the cell surface stimulates an immune response against the eell
(also referred to herein as the donor cell) when the eell is ' ~i~ to a subjeet (also
referred to herein as the recipient ûr host). By altering the antigen, the normal ~ l 'v
~vuvlf~iull of the donor cell by the immune system cells of the recipient is disrupted and
additionally, "abnormal" imm~ . gi. al reeognition of this altered form of the antigen ean
35 lead to donor eell-speeific long term l I vv.._~ in the recipient. Thus, alteration of am
antigen on the donor eell prior to - ' v the cell to a reeipient interferes with the
initial phase of 1 v of the donor eell by the eells of the host's immume system
..... l to ~ ;.. of the cell. FL.. III.,Illlul~i, alteration of the antigen may mduce
""" ~ IlVlU~IUVll~;v~ or toleranee, thereby preventing the induetion of 1he
21 865~8
wo 9sr27042 I'~I/L~ 4~ i. ~
-14- _
effector phases of an immune response (e.g., cytotoxic T cell generation, antibody production
etc.) which are ultimately IC:.,UUlDibl~ for rejection of foreign cells in a normal imr~une
response. As used herein, the term "altered" . ~ changes that are made to a donor
cell antigen which reduce the i, . " . ,.., .., . -: ly of the antigen to thereby interfere with
~; ;"",...."~lr.~ I If ~ '' of ~.h~ antigen by the recipient's immune system. Preferably,
llu~llcaluulla~ to the donor cells m the recipient subject is generated as a
result of alteration of the antigen. The term altered is not intended to include complete
of the antigen on the donor cell since delivery of an ~ ul or
signal to the host's immune cel~s (e.g., T Iy~ O~,yt~,~) may be necessary to achieYe
10 ;"---,----i-l~;- I nu~uc~,uu..~
Antigens to be altered according to this invention include antigens on a donor cell
which can interact with a 1 p~ cell in an allogeneic or ~4.1U~ recipient and
thereby stimulate a specific immune }esponse against the donor cell in the recipient. The
interaction between the antigen and the ~ .lJU;~Lic cell may be an indirect interaction
15 (e.g., mediated by soluble factors which induce a response m the I , cell) or, more
preferably, is a dnrect mteraction between the antigen and a molecule present on the surface of
the 1 r "~ cell. As used herein, the term ~ , - cell is nntended to include T
lylll~ul~v~ t~, B IyulullOcy ~;., monocytes and other arltigen presenting cells. Preferably, the
antigen to be altered is one which interacts with a T IyulyllO~c in the recipient (e.g., the
20 antigen nor~nally bnnds to a receptor on the surface of a T Iylll,ull~ .~.).
In a preferred c~l ~ l: .l the antigen on the donor cell to be altered is arl MHC
class I antigen. MHC class I antigens are present on almost all cell types. In a normal
immune response, self MHC molecules funcùon to present antigenic peptides to a T cell
receptor (TCR) on the surface of self T Iylll~Lv~ ,a. In immune " of allogeneic or
25 ~ i cells, foreign MHC anùgens (most likely together with a pepiide bound thereto~
on donor cells are recognized by the T cell receptor on host T cells to thereby elicit arl
immune response MHC class I antigens on a donor cell are altered to nnterfere with their
IC~,O~$1..~;UII by T cells in an allogeneic or xenogeneic host (e.g., a portion of the MHC class I
antigen which is normally recogl~ized by the T cell receptor is blocked or "masked" such that
30 normal lu ~f..i(Jll of the MHC class I antigen fails to occur). Additionally, an altered form
of an MHC class I antigen which is exposed to host T cells (i.e., available for l,.c;..,u~liu., to
the host T cell receptor) may deliver an iu~.v~ , or inellffiriPnt signal to the host T cell
such that, rather than cti~"l~'rin~ arl immune response against the allogeneic or ~ g. ~i~
cel~, donor cell-specific T cell non-.~iauu.L,;~....,.,~ is mduced. For example, it is known that
35 T cells which receive an ill~y~u, or in~--ffiriPnt signal through their T cell receptor (e.g.,
by binding to an MHC antigen in the absence of a c. .~ .. y signal, such as that provided
by B7) become anergic rather than activated and remain refractoly to .c~ ;.... for long
periods of ùme (see for example Da~nle et al ~1981) Proc. Na~L ~ca~ Sci USA 78:509
510û~ Lesslauer et al. (lg86) ~ur. J: mmunol. 16:1289-1295; Gimmi, et al. (1991) Proc.
.
2~ 86528
~wo 95127042 r~/,. J~o~i~
- 15-
NatL Acad. Sci USA 88: 6575-6579; Linslèy et al. (1991 ) ~ Exp. Med 173:721-730;Koulova et al. (1991) J. ~xp. Med. 173:759-762; Razi-Wolf, et al. (1992) Proc. NatL Acad
Sci. USA 89:4210-4214).
Altemative to MHC class I arltigens, the antigen to be altered on a donor cell can be
5 .~r. MHC class Il antigen. Similar to MHC class I antigens, MHC class 11 antigens function .3
present antigenic peptides to a T cell receptor on T Iy~ u~ jt~ ~. However, MHC class 11
antigens are present on a limited number of cell types (primarily B cells, I~ IL~ o~7
dendritic cells, r.qn~rrhqnc cells and thymic epithelial cells). In addition to or alternative to
MHC antigens, other antigens on a donor cell which interact with molecules on host T cells
10 and which are known to be involved in ' O ' rejection of allogeneic or ~ .. ",. , i~
cells can be altered. Other donor cell antigens known to interact with host T cells and
contribute to rejection of a donor cell include molecules which function to increase the
avidity of an interaction between a donor cell and a host T cell. Due to this property, these
molecules are typically referred to as adhesion molecules (although they may serve other
15 functions in addition to increasing the adhesion between a donor cell and a host T cell).
Examples of preferred adhesion molecules which can be altered according to the invention
include LFA-3 and ICAM-I . These molecules are ligands for the CD2 and LFA-I receptors,
Li ~ ly, on T cells. By altering an adhesion molecule on the donor cell, (such as LFA-3,
ICAM-I or a similarly 1`~ " ,;-,~ molecule), the ability of the host's T cells to bind to and
20 interact with the donor cell is reduced. Both LFA-3 and ICAM-I are found on endothelial
cells within blood vessels in ~ ,t J organs such as kidney and healt. Altering these
antigens may facilitate I , ' of any ~ implant, by altering l~ ~,Uoll;Liu,l of
those antigens by CD2+ and LFA-I+ ho$ T-ly A Y ~.
The presence of MHC molecules or adhesion molecules such as LFA-3, ICAM-I etc.
25 on a particular donor cell can be assessed by st~ndard procedures known in the art. For
example, the donor cell can be reacted with a labeled antibody directed against the molecule
to be detected (e.g., MHC molecule, ICAM-I, LFA-I etc.) and the association of the labeled
antibody with the cell can be measured by a suitable technique (e.g.,;, . " "... ,..l ,: s~ ,. l ,.., .: ~l,
flow cytometry etc.).
A preferred method for altering an antigen on a donor cell to inhibit an immune
response against the cell is to contact the cell with a molecule which binds to the antigen on
the cell surface. It is preferred that the cell be contacted with the molecule which binds to the
antigen to be altered prior to ~ `t` ;1~ the cell to a recipient (i.e., the cell is contacted
with the molecule in v;tro). For example, the cell can be incubated with the molecule which
binds the antigen under conditions which allow binding of the molecule to the antigen and
then any unbound molecule can be removed (such as described in the Examples below).
Following a,l" ,; ~ nn of the modified cell to a recipient, the molecule remains bound to
the amtigen on the cell for a suff1cient time to interfere with ' ~ recognition by
host cells and imduce non-.~u--~ OO in the recipient.
21 865WO95/27042 r. l,- `4
- 16 -
Preferably, the molecule for binding to an antigen on a donor cell is an antibody, orfragment or derivative thereof which retains the ability to bind to the antigen. For use in
therapeutic ~ c it is necessary that the antibody which binds the antigen to be altered
be umable to fix ~ thus preventdng donor cell Iysis. Antibody ~ l ` fixation
5 can be prevented by deletion of an Fc portion of an amtibody, ~ sing an antibody isotype
which is not capable of fixing ~.. ,,l,l.. ,.. ,l or, less preferably, by using a ,;.. l l.. 1 fixing
antibody in ~ .",; .- l ,,.., with a drug which inhibits ~ r ' fixation. Al.~ ldti~.,l~,
amino acid residues within the Fc region of an antibody which are important for activating
r.. ,l,l.. 1,~ ~1 (see e.g., Tan et al. (1990) Proc. NatL Acad Sci. USA 87:162-166; Duncan and
Winter (1988) Nature 332: 738-740) can be mutated to reduce or eliminate the, ~ '
activating ability of an intact antibody. Likewise, amino acids residues within the Fc region
of an antibody which are necessary for binding of the Fc region to Fc receptors (see e.g.
Camfield,S.M.andS.L.Morrison(1991)~ p.Med.173:1483-1491;andLund,J.etal.
(1991) J. Immunol. 147:Z657-2662) cam also be mutated to reduce or eliminate Fc receptor
15 binding if an intact amtibody is to be used.
A preferred antibody fragment for altering an antigen is am F(ab')2 fragment.
Antibodies can be fr~mrnt~ using cullv~ll;ùila~ techniques. For example, the Fc portion of
an antibody can be removed by treating an intact antibody witb pepsin, thereby generating an
F(ab')2 fragment. In a standard procedure for generating F(ab')2 fragments, intact antibodies
20 are incubated with immnhili7~d pepsin and the digested antibody mixture is applied to an
,...., ...l .,l; ,. ~ protem A column. The free Fc portion binds to the column while the F(ab )2
fragments passes through the column. The F(ab')2 fragments can be further purified by
HPLC or FPLC. F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab'
fragments.
An anùbody, or fragment or derivative thereof, to be used to alter an antigen can be
derived from polyclonal antisera containing antibodies reactive with a number of epitopes on
an antigen. Preferably, the antibody is a -' ' antibody directed against the antigen.
Polyclonal and ,....l,n.l....,1 antibodies can be prepared by standard techniques known in the
art. For example, a mammal, (e.g., a mouse, hamster, or rabbit) can be immuni_ed with the
30 antigen or with a cell which expresses the amtigen (e.g., on the cell surface) to elicit an
antibody response against the antigen in the mammal. Alternatively, tissue or a whole organ
which expresses the antigen can be used to elicit antibodies. The progress of; " " . ., ~
can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or
other; " ,.., ,...~ can be used with the antigen to assess the levels of antibodies. Following
~5 imml~ni7~tinn antisera can be obtaiined and, if desired, polyclonal antibodies isolated from
the sera. To produce ., .. - ~ antibodies, antibody producing cells (I,Y.A.~I~G~ ) can be
harvested from am jmmllni7~11 animal and fused with myeloma cells by standard somatic cell
fusion procedures thus illllllUI ~dL~ these cells and yielding hybridoma cells. Such
techniques are well known in the att. For example, the hybridoma technique originally
2 1 86~28
~ WO 95127042 r~
- 17 -
developed by Kohler and Milstein ((1975) Nature 256:495-497) as well as other techniques
such as the human B-cell hybridoma techqique (Kozbar et al., (1983) Immunol. Today 4:72),
and the EBV-rlyl,llJu...a technique to produce humaq " ,...~ antibodies (Cûle et al.
(1985) l ' . ~' 'Antibodies in Cancer TheralJy, Allen R. Bliss, Inc., pages 77-96) cam be
used. Hybridoma cells caq be scI:,sqed i.. , .h. 1 ~.. '- All,~ for production of antibodies
specifically reactive with the antigen and -' ' antibodies isolated.
Another method of generating specific antibodies, or aqtibody fragments, reactive
with an antigen is by use of expression libraries encoding ~' ' " genes, or portions
thereof, expressed in bacteria which can be screened with the antigen (or a portion thereof).
10 For example, complete Fab fragments, VH regions, Fv regions and single cham amtibodies
caq be expressed in bacteria using phage expression libraries. See for example Ward et a'~.,
(1989) Nature 341:544-546; Huse et a'.., (1989) Science 246:1275-1281, aqd McCaffer~ et
a'~. (1990) Nature 348:552-554. Altematively, a SClD-hu mouse caq be used to produce
antibodies, or fragments thereof (available from Genpha~.~m). Antibodies of the a,uylul
15 binding specificity which are made by these techniques can be used to alter am aqtigen on a
donor cell.
Aq aqtibody, or fragment thereof, produced in a llu.. I subject can be recog uzed
to va~ying degrees as foreign wheq the antibody is _ ' J to a human subject (e.g.,
when a donor cell with aq antibody bound thereto is aJIl~ufl~t~ l"J to a humaq subject),
20 resulting in an immune response against the antibody in the subject. One approach for
...,..;,..;, g or ~1;...,., ~;,.g this problem is to produce chimeric or humanized antibody
derivatives, i.e., antibody molecules comprising portions which are derived from non-human
amtibodies aqd portions which are derived from human antibodies. Chimeric antibody
molecules can include, for example, am antigen bindiqg domain from an antibody of a mouse,
25 rat, or other species, with human const,mt regions. A variety of approaches for making
chimeric antibodies have been described. See, for example, Morrison et a'~., Proc. NatL Acad.
Sci. U.S.A. 81, 6851 (1985); Talceda et al., Natwe 314, 452 (1985), Cabilly et al., U.S. Patent
No. 4,816,567; Boss et a'.., U.S. PatentNo. 4,816,397; Tanaguchi et a'.., European Patent
Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB
2177096B. Foruseintherapeutical.l.l; -1;~.. ~ itispreferredthatamantibodyusedtoaltera
donor cell aqtigeq not contain an Fc portion. Thus, a humani_ed F(ab')2 fragment in which
parts of the variable region of the antibody, especia'lly the conserved framework regions of
the antigen-binding domain, are of humaq origin aqd only the hyy~ ;al,l~ regions are of
non-humaq origin is a preferred antibody derivative. Such altered ;IIUI_ ~,7 1 _''
35 molecules caq be produced by any of several techniques Icnown in the att, (e.g., Teng et al.,
Proc.Natl.Acad Sci. U.S.A., 80,7308-7312(1983);Kozboretal., T ' ~;yToday, 4,
7279 (1983); Olsson et al., Meth. ~inzymol., 92, 3-16 (1982)), and are preferably produced
according to the teachings of PCT Publication W092/06193 or EP 0239400. T~llmA~li7-
~
2 1 86528
wo ss,27042 r~ 6
- 18-
antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road,
T~ c~ Middlesex, Great Britam.
Each of the cell surface antigens to be altered, e.g., the MHC class I antigens, MHC
class 11 antigens, LFA-3 and ICAM- I is well-~ I . .. ~. r .; ,. .1 and antibodies reactive with these
S anti~ r r~s are f.."" ~: lly available. For example, an antibody reactive with human MHC
class I antigens (i.e., an anti-HLA class I antibody),W6132, is available from the American
Type Culture Collection (ATCC HB 95). This antibody was raised against human tonsillar
ly~ cytc ' and binds to HLA-A, HLA-B and HLA-C (Barnstable, C.J. et al.
(1978) Cell 14:9-20). Another anti-MHC class I antibody which can be used is PT85 (see
10 Davis, W.C. et al. (1984) Hybridorna Technology in Agricultural and Vetrinary Research
N.J. Stern and H.R. Gamble, eds., Rownman and Allenheld Publishers, Totowa, NJ, pl21;
CJ~ IY available from Veterinary Medicine Research D~ Y~IU~ Pullman WA).
This antibody was raised against swine leukocyte antigens (SLA) and binds to class I
antigens from several different species (e.g., pig, human, mouse, goat). An anti-ICAM-I
15 antibody can be obtained from AMAC, Inc., Maine. Hybridoma cells producing anti-LFA-3
antibûdies can be obtained from the American Type Culture Collection, Rockville, Marylamd.
A suitable antibody, or fragment or derivative thereof, for use in the invention can be
identified based upon its ability to inhibit the; ~ g;~ ~I rejection of allogeneic or
r~ ir cells using a protocol such as that described in the Examples. Briefly, an20 antibody (or antibody fragment) to be tested is incubated for a short period of time (e.g., 30
minutes at room; I ~) with cells or tissue to be . ' ' and any unbound
antibody is washed away. The cells or tissue are then 1, .~ t ~1 into a recipient animal.
The ability of the antibody ,ul~ ,ll. to inhibit or prevent rejection of the . ' ~ cells
or tissue is then determined by monitoring for rejection of the cells or tissue compared to
25 untreated controls.
It is preferred that an antibody, or fragment or derivative thereof, which is used to alter an
antigen have an affinity for binding to the antigen of at least 10-7 M. The affinity of an antibody or
other molecule for binding to an an antigen cam be determmed by CUII~ ~,I..iUIIfll techniques (see
Masan, D.W. and Williams, A.F. (1980) Biochem. d'. 187:1-10). Briefly, the antibody to be tested is
30 labeled with I125 and incubated with cells expressing the antigen at increasing c..,.. --~,.~;....~ until
f q~lilihn~n is reached. Data are plotted graphically as 'bound antibody]/[free antibody] versus
iboumd antibody] amd the slope of the line is equal to the kD (Scatchard analysis).
Other molecules which bind to arl antigen on a donor cell and produce a functionally
similar result as antibodies, or fragments or derivatives thereof, (e.g., other molecules which
35 interfere with the interaction of the antigen with a I ~~~ r '- cell and induce
;",.",~ ,lf~S~I n.,~ .,ll.,.,.,) can be used to alter the antigen on the donor cell. One
such molecule is a soluble form of a ligand for an antigen (e.g., a receptor) on the donor cell
which can be used to alter the antigen on the donor cell. For example, a soluble form of CD2
(i.e., comprising the Plf~f`PII~ domain of CD2 without the 11 r 1~ ~ or ~ Lulul~llll-,
WO9SJ27041 2 1 8 6528 ~ s ~6~
- 19 -
domain) can be used to alter LFA-3 on the donor cell by binding to LFA-3 on donor cells in a
manner analogous to an antibody. ~ .ly, a soluble form of LFA-I cam be used to alter
ICAM-I on the donor cell. A soluble form of a ligand can be made by standard, ~
DNA procedures, using a ~ .,.,.l.:., .l expression vector containing DNA encoding the
5 ligand ~ ,p. an ey~ domain (i.e., lacking DNA cn-oding the l"...~..,....1... rand cy ~ul ' domains). The ~c ' expression vector encoding the . . l, .. F 11 . .~.
domain of the ligand can be introduced mto host cells to produce a soluble ligand, which can
then be isolated. Soluble ligands of use have a binding affinity for the receptor on the donor
cell sufficient to remain bound to the receptor to interfere with ;.., , , ~lh~ C~U~liLiUII
10 and induce non-lc~u~ when the cell is a~ vt~,.cd to a recipient (e.g., preferably,
the affinity for binding of the soluble ligand to the receptor is at least about 10-7 M).
Additionally, the soluble ligamd cam be in the form of a fusion protein comprising the receptor
binding portion of the ligand fused to another protein or portion of a protein. For example, an
imr~-mrl~l-lb~lin fusion protein which includes an FY~rDII.~IDr domain, or functional portion
of CD2 or LFA-I linked to an ill =' I " heavy chain constant region (e.g., the hinge,
CH2 and CH3 regions of a humam;, . - - . ~ gl, l, ~l; . . such as lgGl ) can be used.
T.",."".~ sb"l," fusion proteins can be prepared, for example, according to the teachings of
Capon, DJ. et al. ( 1989) Nature ~:525-531 and U.S. Patent No. 5, 11 6,964 to Capon and
Lasky.
Another type of molecule which can be used to alter an MHC antigen (e.g., and MHC
class I antigen) is a peptide which binds to the MHC antigen and interferes with the
interaction of the MHC amtigen with a T IYIII~ O~ y ..,. In one ~, . ,l .o.l;. ., ~l the soluble
peptide mimics a region of the T cell receptor which contacts the MHC antigen . This
peptide can be used to mterfere with the interaction of the intact T cell receptor (on a T
I~ o-,~Lc) with the MHC amtigen. Such a peptide binds to a region of the MHC molecule
which is ~ ~,;r~ recognized by a portion of the T cell receptor (e.g., the alpha-l or alpha-
2 loop of an MHC class I antigen), thereby altering the MHC class I antigen and inhibiting
~c~ u~f~L;u~ ûf the amtigen by the T cell receptor. In amother . . . ,l .o.l; . ., . ~l the soluble peptide
mimics a region of a T cell surface molecule which contacts the MHC amtigen, such as a
region of the CD8 molecule which contacts an MHC class I antigen or a region of a CD4
molecule which contacts am MHC class Il antigen. For example, a peptide which binds to a
region of the alpha-3 loop of an MHC class I antigen can be used to inhibit bmding to CD8 to
the amtigen, thereby inhibiting .c ,, of the antigen by T cells. T cell receptor-derived
peptides have been used to inhibit MHC class l-restricted irnmune responses (see e.g.,
Clayberger, C. et al. (1993) Transplanf Proc 25:477-478) and prolong allogeneic skin graft
survival in vtvo when injected ~ ' y into the recipient (see e.g., Goss, J.A. et al.
(1993) Proc. Natl. Acad. Sci. USA 90:9872-9876).
An antigen on a donor cell further can be altered by using two or more moleculeswhiGh bind tû the same ûr different antigens. For example, two different antibûdies with
,,, . ,,,,, . , , .. , . , . . _ , . . . . .. ....
2 1 86528
wo 95/27042 ~ o :^ ~Q
-20 -
specificity for two different epitopes on the same antigen can be used (e.g., t~vo different anti-
MHC class I antibodies can be used in ., " "l .~ .). Alternatively, two different types of
molecules which bind to the same arltigen can be used (e.g., an anti-MHC class I antibody
and arl MHC class l-binding peptide). A preferred ~ ' of anti-MHC class I
S antibodies which can be used with k l?nan donor cells is the W6/32 antibody and the PT85
antibody or F(ab')2 fragments thereof. Another anti-MHC class I antibody which can be used
is the .. "f~ antibody 9-3 generated at Diacrin, Inc. 9-3 reacts with porcine MHC class
1. The epitope for the .~ .. " ,~ antibody 9-3 has been shown to be on the alpha-3 domam
of MHC class I (the alpha-3 domain of porcine MHC class I is known-see, e.g., Satz, M.L. et
al. (1985)J. ImmunoL 135:2167-2175)andisseparatefromtheepitopeforthe .,.
antibody PT85.
When the donor cell to be ' ' to a subject bears more than one
-- ,JU;.,lic cell-interactive antigen, two or more treatments can be used together. For
example, two antibodies, each directed agairlst a different antigen (e.g., an anti-MHC class I
15 antibody and an anti-lCAM-I antibody) can be used in ~ l .: - ~: or two different types of
molecules, each binding to a different antigen, can be used (e.g., an anti-lCAM-I antibody
and an MHC class l-binding peptide). Alternatively, a polyclonal antisera generated against
the entire donor cell or tissue containing donor cells can be used, following removal of the Fc
region, to alter multiple cell surface antigens of the donor cells.
Alternative to binding a molecule (e.g., an antibody) to an antigen on a donor cell to
inhibit ;.. .. ~ rejectioD of the cell, the antigen on the donor cell carl be altered by
other means. For example, the antigen c~m be directly altered (e.g., mutated) such that it can
no longer interact norrnally with a l r ' "~ cell ~e.g., a T l~ o~ e) in aD allogeneic
or J ' ~ recipient and induces ;. .-- - - .l~-g,~l IIOII-I~D~ IID;~ DD to the donor cell in
25 the recipient. For example, a mutated form of a class 1 MHC antigen or adhesion molecule
(e.g., LFA-3 or ICAM-I) which does not contribute to T cell activation but rather delivers aD
or ;..~ signal to a T cell upon bmding to a receptor on the T cell can be
created by ~ and selection. A nucleic acid encoding the mutated form of the
antigen can then be iDserted Dnto the genome of a non-human animal, either as a transgene or
30 by l~ ..p.. ~ lc ' ' (to replace the ~ ,...., gene encoding the wild-type
amtigen). Cells from the non-human animal which express the mutated form of the anùgen
can therl be modified to express a gene product of interest according to one of the procedures
described earlier. The modified cell expressing the gene product of interest and the mutated
(i.e., altered) form of the antigen can then be used as a donor cell to deliver a gene product to
35 an allogeneic or ~.,..~ig~ , recipient.
Alternatively, an antigen on the donor cell can be altered by .1.~ , or
altering its level of expression on the surface of the donor cell such that the interaction
bet~veen the antigen and a recipient l ~ ji. cell is modified. By decreasing the level
of surface expression of one or more antigens on the donor cell, the avidity of the irlteraction
w09sl27042 2 ~ 8 ~ 5 2 8 r~"~ - n
-21 ~
between the donor cell and the ' ' r cell (e.g., T lyl.l,ull~c~l~;) can be reduced. The
level of surface expression of an antigen on the donor cell can be duvv~ by
inhibiting the ~ , translation or transport of the antigen to the cell surface. Agents
which decrease surface expression of ~he antigen can be contacted with the donor cell. For
S exam~'o, a number of oncogenic viruses have been ~1.., ..~..~1 ... ` . l to decrease MHC class I
expression in infected cells (see e.g., Travers et al. (1980) Int'L Symp. on Aging in Cancer,
175180; Rees et al. (1988) Br. J. Cancer, S7 374-377)~ In addition, it has been found that this
effect on MHC class I expression can be achieved using fragments of viral genomes, in
addition to intact virus. For example" . ~ .... of cultured kidney cells with fragments of
l 0 adenovirus causes .l;, ., ~ . of surface MHC class l antigenic expression (Whoshi et al.
(1988)J. E~p. Med. 168 2153-2164)~ ForpurposesofdecreasingMHCclasslexpressionon
tbe surfaces of donor cells, viral fragments which are non-infectious are preferable to whole
viruses.
Alternatively, the level of an antigen on the donor cell surface can be altered by
capping the antigen. Capping is a term referring to the use of antibodies to cause a~ aLiul.
and illa~ utiull of surface antigens. To induce capping, a tissue is contacted with a first
antibody specific for an antigen to be altered, to allow formation of CUI~ . . ntiho~y immune
cnmrlPy~ ly~ the tissue is contacted with a second antibody which forms
immune complexes with the first antibody. As a result of treatment with the second antibody,
the first antibody is aggregated to form a cap at a single location on the cell surface. The
technique of capping is well known and has been described, e.g., in Taylor et al. (1971)~ Nat.
New BioL 233 225-227; and Santiso et al. (1986)~ Blood, 67 343-349~ To alter MHC class I
antigens, donor cells are incubated with a first amtibody (e.g., W6/32 antibody, PT85
antibody) reactive with MHC class I molecules, followed by incubation wjth a second
antibody reætive with the donor species, e.g., goat amti-mouse antibody, to result in
a~LI ~;a~iUII.
TTT. Gl~n~tir~ y Mo~1ifip~1 CPllq with Al~ red Sllrf~rp Ant~enc ~g D-~nnr (~Plle for ~Plivery of
~PnP protll-rte to AllQ~çnpir or XPn~ nPir Su~jects
This invention provides a means for modifying a variety of cell types to express a
gene product amd for reducing the " ~/ of such cells in an allogeneic or
host. Depending on the type of cell to be modified, a gene expression system
(e.g., vector with . . r regulatory elements) is selected to allow expression of a gene
product in that particular cell type. One or more appropriate molecule(s) is also selected to
bind to amtigen(s) on the cell surface to alter the antigen (e.g., one or more anti-class I
antibodies which bind the MHC class I antigens on that palticular cell type). Cells which can
be modified amd altered according to the invention include liver cells (e.g., I . yt~
muscle cells (e.g., myoblasts, myocytes, myotubes), neural cell, pamcreatic islet cells and
r~u;~,lic cells. The use of 1- ~.1 yt~,~ for the expression of a palticular gene product
, ,,, . ,,,,, _,, , .. , ... , . , . , .,,, .. , .... , ,,, ,, , _ , , ,
- 2 1 ~6~2~
wo 95127042 P~ 'C4
-22 -
allows for the production of proteins Lhat require a specific co- or pOSt-Llall~la iullal
" ,n.~ ,. ., such as vitamin K-dependent l~a ku~yla~iul, (e.g., many of the blood clotting
factors require this mt-tlifi~ ti- n for biologic,al activity). Myoblasts have the advantage that
injected myoblasts fuse with existing muscle fibers in a recipient (see e.g., Partridge, et al.
(1989) Nature 337:176; and Karpati et al. (1989) Am. J. Pa~hology I '.r 27). T1. ~. ,~. .;. 1;~
stem cells are adv ,, in ~hat they continue to divide and repopulate a number of cell
types (see e.g., Chang and Johnson (1989) Ins J. Cell Cloning 7:264; Williams (199û) Hum.
Gene Ther. 1:229; Karlsson et al. (1985) Proc. NatL Acad. Sci. USA 82:158; and Bodine et al.
(1989) Proc. Na~l. Acad. Sci. USA 86:8897). Al ~ly, mature I r cells, such
as l.ylllUllU~,yt~,~ or monocytes, can be used. A gene product can be CUl~ lVUaly produced by
modifying a cell which continues to divide (e.g., a stem cell, such as a I l stem
cell) or a gene product can be produced in a limited amounts by modifying a li~l~ h.'.,d
cdl which does not divide (e.g., a myotubes or a neural cell). Rc~ u. . ,l .;, ,l retroviral vectors
are suitable for modifying dividing cells but are not suitable for modifying non-dividing cells.
The modified cells can be contained within a tissue or whole organ. For example, a
tissue or organ can be modified to express a gene product by infecting the tissue or organ
with a l" . ,...1.;..,..,1 virus (e.g., retrovirus, adenovirus, adeno-associated virus etc.). One or
more antigens on tbe tissue or organ can be altered by contacting the tissue or organ with a
molecule which binds the antigen. For example, an organ can be perfused with a solution
20 containing the molecule (e.g., an antibody) using ~,vllv~,llLiolldl organ perfusion methods.
This invention further allows cells to be modified to express a variety of gene
products and thus allows many different types of gene products to be delivered to a subject.
For example, the gene product can be a secreted protein. In this situation, the modified donor
cell secretes the gene product in tbe subject, either locally or systemically. Non-limiting
25 examples of secreted gene products of therapeutic interest which a cell can be modified to
express include ~ uaiLa~ lu~.uululllua.~ ~ ccl ~LI.~uaill~ pllwl.11ala~
.ylaa-" tyrosine h.~hu~ylaa~, ornithine i ; yl~la~ r.~- -- ~ synthetase,
TJDp-~;lu~ ~llull~ayl j r apoAI, TNF, soluble TNF receptor, humam growth hormone,
insulin,cl~ IhlulJu;~Lul~ anti-~ factorsand;"t l .k;..~ Forexample,tbesecreted
30 protem can replace a missing function in a subject (e.g., insulin in a diâbetic subject) or can
stimulate a response in a subject (e.g., TNF or IL-2 can be produced m a tumor-bearing
subject to stimulate an immune response against the tumor in the subject). Alternatively, the
gene product can be a m~mhmr~^-bound protein. In this case, the gene product remains
associated with the membrane of the modified donor cell and fimctions, for example, by
35 binding a soluble subst~mce in a host (e.g., binding of LDL cholesterol by an LDL receptor)
or by binding to another I o-bound protein (e.g., a receptor) on cells of the host to
trigger a signal within the recipient cells. Non-limiting examples of ll.~,lll~l~e-boumd gene
products which a cell c~m be modified to express mclude the LDL receptor, CFTR and CD18.
Alternatively, the gene product can be an intr~ r protein. The intr~r~ r protein
21 86528
_~VO 95r27042 P~ 'C I-
- 23 -
within modified donor cells can be introduced into cells of a recipient by fusion of the donor
cells to recipient cells (e.g., fusion of modified myoblæts or myocytes with muscle cells
within the recipient, e.g., to delivemly t~l~ . ' ) An ;. .~ f 11 1 , protein can also function by
acting upon substances within a recipient that are taken up by the modified cell (e.g., to
5 detoxify substances within the recipiert~ Non-limiting examples of intr~P~ r proteins
which a cell can be modified to express include dystrophin"~-globin arld adenosine
deaminæe.
In one ..l,o.~: . ..1 the cell to be modified is a non-human cell and the gene product
is a human gene product. Humarl gene products have been expressed in IIU,A I cells (see
e.g., Dai, Y. et al. (1992) Proc. NatL Aead. Sci. USA 89:10892-10895; Armentano, et al.
(1990) Proc. Natl. Acad &i. USA 87:6141-6145; van Bc--e~rh~m V.W. et al. (1992) Proc.
NafL Acad. Sci. USA 89:7640-7644). The human gene product expressed in a non-human
cell can be the human version of a gene product typically expressed by that cell type, e.g.,
human insulin can be expressed in non-human islet cells or humarl Factor IX can be
15 expressed in non-human ' , ~ (see, e.g. Armentano, et al. (1990) Proc. Natl. Acad Sci.
USA 87:6141-6145). Alternatively, the human gene product expressed irl the non-human cell
can be a gene product which is not normally expressed by that cell type. For example, human
growth hormone can be expressed in l.u.. ' myoblæts or tyrosine lly ~u~.yl~Lt~ can be
expressed in non-human myoblasts (see e.g. Jiao, S. et al. (1993) Natwe 362:450-453 and
Dai, Y. et al. (1992) Proc. NatL Acad Sci. USA 89:10892-10895 for examples oftheexpression in a cell of a gene product not normally expressed by that cell type). A preferred
non-human cell for use m the methods of this invention is a porcine cell. Genetically inbred
strains of pigs (e.g., miniature pigs), which have organs of ~ equivalent size to
humans and have well ~ ;, i MHC antigens, are available in the art.
Alterationofoneormoreantigerlsonthesurfaceofacellpriortoll--.~l.lA.: :;---
reduces the ;~ .f~ ;y of the cell such that rejection of the cell by an allogeneic or
recipient is inhibited following ~ Accordingly, the invention
providesamethodforreducingthe;l.,....f.~,....: .;lyofacellwhichismodifiedtoexpressa
gene product in which the cell is contacted, prior to ~ ;.... (i.e., in vitro), with at
30 least one molecule which binds to at least one antigen on the cell surface. The amtigen to be
altered stimulates an immune response against the cell in an allogeneic or Y' -~... ; subject.
Thus, alteration of the antigen on the cell surface irlhibits rejection of the cell when
1, ,., .~1.l - ' ~i imto a subject. It is preferred that the cell is contacted in vitro with a molecule,
(e.g., antibody, or fragment or derivative thereof, such as an F(ab')2 fragment) which binds to
35 tbe amtigen on the cell surface but does not activate ~ or induce Iysis of the cell.
Preferably, the antigen on the cell surface which is altered is an MHC class I antigen.
A modified cell of the invention is used to deliver a gene product to a subject by
_.l.":. -. . ;..~ the cell to subject. The term "subject" is mtended to mclude marnmals,
preferably humans, in which an im~mme response is elicited against allogeneic or ~, ... L,. .. i~
- 2l~6528
wo 95/27042 ~ J
-24 -
cells. A cell can be a~Llflll;~ c ;I to a subject by any appropriate route which results im
delivery of the gene product to a desired location in the subject. For example, cells can be
allllli~l;~t"lcd i-~ ,llu....ly, I ~ lel)lally~
(e.g., under the kidney capsule) or i~ 'ly. The cells can be a Inl..u~t~.cd in a5 physiolo~ieaLlly compatible carrier, such as a buffered saline solution. When cells are within
a tissue or organ, the tissue or organ can be ~ into a suitable location in the subject
by CUII~ techniques.
It is preferable that a cell is modified to express a gene product prior to r ' ' ' ' ' g
the cell to a subject (i.e., modified ex vivo). It is also preferable that the cell is modified to
10 express the gene product prior to altering an antigen on the cell surface. However, a cell can
be modified to express a gene product ex vivo after the antigen has been altered, so long as
the ,...~ r.. -1;.... method does not disrupt an association bet~veen the antigen on tbe cell
surface amd the molecule (e.g., an antibody) which is bound to the antigen. Ful~ lc, a
cell can be modified to express a gene product in v~vo following alteration of a surface
1~ antigen(s) ex vivo and ~ -- to a subject. ~n vivo methods for genetically modifying
a cell (e.g., using retroviral or adenoviral vectors) are known in the art. These n~
are .. ~ by the invention.
The methods of the invention for delivering a gene product to a subject can further
comprise additional treatments which inhibit rejection of the , ' ' cells by the subject.
20 For example, an ;.. ..... ~ .- .l.l.. ~ ,~;v~ agent (e.g., a drug) can be ~ d to tbe subject at
a dose and for a period of time sufficient to induce tolerance to the l . .- ~ I cells in the
subject. A preferred ;.,."... ~ . agent for "-~ to a subject is ~,y~luc~uliul
A. Other i~ ,;vc agents which can be used include FK506 and RS-61443. Such
; .... - - .- -,- l ~.. - .,~,~ . agents can be used in r.~ ;- . with a steroid (e.g., ~IU~OCUI ~i. u;ds
25 such as prednisone, l~l~tll~ uluilc and fl -- - . Il.- -~) or ~ l~llu~ t;~, agent (e.g
; .P and ~ - - . l ), or both. Alternatively, an agent which depletes or
inhibits T cell activity in the subject can be a ' ' ' ' cd to the subject. For example, am
antibody which binds to a surface antigen on T cell in the subject can be used to deplete T
cells within the subject. Preferred surface antigens to which a T cell-depleting amtibody can
30 bind include CD3, CD2, CD4 and CD8. Other arltibodies which carl be used to inhibit T cell
activity in a subject include amtibodies agamst IL-2 or other T cell growth factors and
antibodies against the IL-2 receptor or other T cell growth factor receptors.
Another aspect of the invention pertains to a kit for use in delivering a gene product to
a subject. In one r~ u li . I the kit includes a cell which is modified tû express a gene
35 product; the cell having am amtigen on the surface which stimulates an immune response
against the cell in an allogeneic or xenogeneic subject. The kit fi~r~er includes a molecule
(e.g, an antibody, or fragment or derivative thereof) which binds to tbe antigen on the cell
surface. In another ~ I.o-l;..~ ,~ the kit mcludes a vector encoding a gene product in a form
suitable for expression of the gene product in a cell and a molecule (e.g., am antibody, or
.
wo 95l27042 2 1 8 6 5 2 8 P~l/l s c ,- ~
- 25 -
fragment or derivative thereof) which binds to the antigen on the cell surface. In this
ho.l ;. ". . ,1 the kit can optionally mclude a cell which has the antigen to be altered on the
cell surface. When cells are included m the kit (e.g., genetically modifled cells or cells to be
genetically modified), the cells can be ~ u~l~a~.l v~d (e.g., in liquid nitrogen or on dry ice)
and thawed before use. The molecule (e.g., an antibody, or fragment or ~ivative thereof)
which binds to an antigen on a cell can be provided in the kit in a ~)h~a;olG~ ,dlly acceptable
ca~rier, such as a buffered saline solution. A preferred molecule for binding to an antigen,
such as an MHC class I antigen on a cell is a F(ab')2 fragment. The t I ' of the kit
can be supplied in A,u,ulul containers (e.g., tubes, vials) for each component (e.g., cells,
antibodies, vectors) and each component supplied within an appropriate holder (e.g.,
container). A kit of the invention can also include illaLI ul,liulla for use of the kit to deliver a
gene product to a subject.
This invention is further illustrdted by the following Examples which should not be
construed as limiting. The contents of all references and published patents and patent
All~ cited throughout the application are hereby ill ,UllJ~ ' ' by reference.
~X ~.MPT .T~,~
EXAMPLE I: PRODUCTION OF GENETICALLY MODIFIED HUMAN
MYOBLASTS AND TRANSPLANTATION OF THE MODIFIED
MYOBLASTS INTO MICE
Satellite myoblasts were isolated from a frozen biopsy of human muscle following 10
minute digestions in trypsin/Ar~ buv;l~, serum albumm mix at 37C. Released cells
from each digestion were seeded in 100 mm plates in the following growth medium (GM):
MCDB 120(JRHp:eri~-nrPg Lenexa,KS)+epidermalgrowthfactor+ ~ +
20% fetal bovine serum. Primary cultures were re-fed once with GM before being
trypsinized for ~ u,uu~iul~ ten days after digestion.
Cell harvests from digestions 3 through 10 were combined and wdshed twice with ice
cold Hepes buffered saline pH 7.0, counted and 1~ d at a final .. . ~ . .- of 2.6 x
105 cells/ 0.8 ml of Hepes buffered salme for placement in an 0.4 cm gap width
cl~ uuuld~iull cuvette. Cells were mcubated on ice for 10 minutes with 20 ~g of ScaI
digested pCMV~ plasmid (Clontech, Palo Alto, CA), a plasmid which contains the gene
encoding ~ ,, ' ' , and 2 llg of Nsil digested pkJ2Neo (Dinsmore, J.H. and Solomon,
F.(1993)Ne~.u~,u~u.,ùl~2:19-23),whichdisplaysneomycinresist;mce,orpCMVGH,a
plasmid containing a gene encoding human growth hormone and which also displays
neomycin resist~mce. pCMVGH was constructed æ follows: plasmid p0GH (Selden, R.F. et
al. (1986) MoL CelL BioL 6(9): 3173-3179) was cut with BamHI and EcoRI and cloned into
plasmid pcDNA3 (Clontech, Palo Alto, CA) previously cut with the same enzymes. The
resultant pldsmid, pCMVGH, which is shown in Figure 1, contains the gene encoding human
2 ~ 86~8
WO 95127042
-26 -
growth hormone. The pCMVGH plasmid was then lineari~ed with ScaI in preparation for
;IlLluducLiu-l into human myoblasts via el~ Llu~uul_liul~ Electroporation was performed with
a BioRad Cl~.LlulJuld~iull device with . ~ extender at 240V, 50011F. After
ck,~,L u!,ul~Lion, cells were left to recover for 10 minutes at room ~ and seeded into
four 6-well plates at 6.5 x 104 cells per we?! (assuming no cell death during el. ~ L~ul,ul~iiùn),
and cultured in GM + 800?1gG418/m?~ for 11 days to select for stable ~.A..~rA ..~-.,t~ Cloning
efficiency was 0.01 - 0.02%. El~ ~LIu,uul~lLiu.~ was determined to be more effcient for
;, .. . than either lipQfection (B?~L) or ca?cium i ' ~, ' -mediated 1, ~ "A~ ~ ~ ;"
(Rosenthal, N. (1987) Meth EnzymoL 152:704-720).
For determing expression in v;fro, cells transfected with pCMV~ and pJK2Neo werefixed in 0.05% O' ' ' ' ~.lc, rinsed three times (5 minutes each rinse) in PBS, stained with
X-GAL for 3-24 hours (Na2HP04-80mM, NaH2PO4-20mM, MgCI2-1.3mM, X-GAL-
lmg/ml, K3Fe(CN)6-3mM, K4Fe(C?~)6-3mM in dH2O) according to st~mdard protocols.
Clones after G418 selection often showed sporadic expression of ~-gal when stained with X-
GAL substrate mix. However, expression increased with Cu~ ,utivc passages amd was at
100% upon fusion of cells (FiguAre 2). For d ~ ., ,..,.~ expression of human growth hormone
in vifro, medium from cells transfected with pCMVGH was sampled amd measured forhuman growth hormone content using a growth hommone ' y from Nichols
Labs, San Jan Capistnmo, CA. Human growth hormone was produced at 800-1800 ng/106
20 cells/hour.
For 1~ nude mice were ~;1,. ~;,. .I by; ~
, ." ,;~ of Avertin (250 mgAcg body weight) and a flank incision was made to expose
the kidney. A small incision was made on the kidney and a small fire polished glass rQd was
irlserted between the kidney epithelium and the kidney tissue tQ create a space for cells to be
25 i . ' ' Prior to ~ 106 cells were spun down in an Eppendorf pipette tip,
then were placed under the ?~idney capsule together with a piece of sponge blocking the tip.
This type of tramsplant can be easily loca?iized even with a bare eye. The skin mcision was
then closed with a wound clip and the animal transferred to a cage for recovery. Mice ~ ,' .d with myoblasts were sacrificed thirLy days after ~ " and
30 then the region of muscle which received the transplant was dissected and placed in 0.5%
O' ' ' ' ydc. After fixation, tissue was rmsed three tnmes (five minutes each rinse) in PBS
amd then frozen The frozen tissue was later thawed, sectioned and stained with X-GAL for
24-48h (X-GAL solution was the same as above except for increased K3Fe(CN)6 and
K4Fe(CN)6 ~ ~ .- . . ,1. ..1 ;l ll . to 50mM). ~-gal expression was detected 30 days after
35 I,,..,~lll~"l-l;~m
21 ~28
W0 95127042 1
-27 -
EXAMPLE Il: TRANSPLANTATION OF GENETICALLY MODIFIED
HUMAN MYOBLASTS/MYOTUBES INTO CYCLOSPORIN-
TREATED RATS
Satellite myoblasts were isolated from human muscle, cultured and transfected as5 described i~. ~xample 1. ~-gal and human growth hormone expression in these cells were
also measured as described in Example 1. ~-gal expression was measured after 34 days in
vitro (Figure 2). This was the latest time point exammed amd does not reflect a limit on the
length of time in which expression is expected.
Expression of hGH in stable ~ in vitro was maintained over S passages up
10 to 14 days in culture. Clones were never cultured till senescence to assay for GH but
expression is stable m cultures from different frozen passages. In myoblasts, hGH was
expressed at 200-500 ng/l 06/hour; in myotubes, hGH was expressed at 800-
1 500ng/1 06/hour.
The Lewis rats used for the l . ~ .., experiment were obtained from Charles
15 River, Wilmington, MA. Rat tibialis anterior muscle was damaged by injection of pivacaine
and 1~ Jllida~ three days prior to ~ ;. ." One dây prior to ~ : -:;.." and
daily thereafter, the rats were treated with cyclosporin (10-15mg/kg). Human myoblasts
stably transfected with ~-gal or hGH expression vectors as described above were i . '
as myoblasts or induced to form myotubes then i , ' ' to the damaged site in recipient
20 muscle. Two weeks after ~ ,1 - llnl ;. .l lJ the muscle was removed for l, ~. ,l. ~;;. ~l analysis
to detect ~-gal expression. Rat serum was also sampled to measure circulating hGH levels at
days 3 and 7 post ~ ;. ." Production of human growth hormone in the rats, as a
result of expression of human growth hormone by the introduced modified cells, is monitored
by detecting the presence of human growth hormone in the circulation of the raOE. Aliquots
25 of blood from the rats were collected periodically, from the tail vein, and hGH present within
the blood sample was detected using a growth hormone - - .y from Nichols Labs,
Sam Ju~m Capistrano, CA.
~ -gal expression was measured 14 days after ~ . ' in vivo (Figure 3). This
was the latest trme point examined and does not reflect a limit on the length of time in which
30 expression is expected.
In vivo detection of human growth hormone production was observed to last for tbree
days. After myoblast amd myotube injection into normal Lewis rat TA muscle, a much
reduced number of cells were detected with a hurnan specific probe 14 days post
;.." Thus, the mability to detect hGH expression past three days post-
35 ~ ;nn would appear to be due to poor cell survival rather than a loss of GHexpression from tbe transgene.
` 2186~28
WO 95/27042 r~
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EXAMPLE III: :~ TRANSPLANTATION OF MODIFIED HUMAN MYOTUBES
INTO CYCLOSPORIN TREATED MICE
Humam myotubes were modified by incubation with purified F(ab')2 fragments of &e5 ..,...,.~ amtibody W6/32 (F(ab')2 fragments were were generated usir~- .he ~
F(ab')2 ~ JalaLiull kit sold by Pierce Chemical Company, Rockford, Illinois) at a
, of l O llg of antibody fragment for ~ u~ ,ly 5 x l O6 cells in SOO ~ll of PBS
for one hour on ice wi& ;. ,t- . . ,.;li. . ,l shaking. After &e incubatiorl, the treated myotubes
were washed, spun down once and ,~ ;i at transplant ~.. ~ ,.~ ;.. , . in PBS amd &en
l0 i " '~ i (5 x I o6 nuclei per mouse) into &e kidney capsules, as described inExample I, of four ^l~ ;. ,.- -: - l Baib/c mice. Two of &ese mice were treated with
cyclosporin (20 mg/kg) beginning one day prior to i , ' amd continued daily
&ereafter. The o&er two mice were masked wi& F(ab')2 fragments of &e l l
antibody W6/32 and not subject to c.y..lu~l~ul;ll treatment. As a positive control, untreated
15 myotubes (5 x I o6 nuclei per mouse) were l, nl l~/l ~ 't ~I under &e kidney capsules of two
nude mice. As a negative control, umtreated myotubes (5 x I o6 nuclei per mouse) were
f ~1 under &e kidney capsule of two normal Balb/c mice.
The mice kidneys were removed 30 days after l ", "~l,t . .~ -, ;. ,. . fixed in 4%
I - .r... I.. l 1. ..y~ for 24 hours, and stained wi& rabbit anti-desmin from BioGenex Labs,
20 San Ramon, CA (Figure 4). E~uman myotubes were detected irl all . ^l, ;... - ' ~ kidrleys 30
days after ~ . including the kidrleys from &e negative control. The negative
control, however, showed evidence of ongoing rejection of &e myotubes, e.g., vacuolated
areas with obvious cell necrosis. Evidence of ongoing rejection was not observed in &e test
mice (mice 1 ","~,1 .t l with myotubes and subject to ..yl.,lO~lJUl;ll treatment, mice
~ l I wi& F(ab')2 modified myotubes, and nude mice l. ~ l wi& myotubes).
EXAMPLE IV: PRODUCTlON AND TRANSPLANTATION OF
GENETICALLY MODIFIED PORCINE MYOBLASTS
SUITABLE FOR T~ANSPLANTATION
An expression vector containing a gene encoding human growth hormone is
introduced into porcine myoblasts to create a modified cell which expresses human grow&
hormone. The modified porcine cell is &en treated wi& an anti-MHC class I antibody
F(ab')2 fragment, thereby altermg porcine MHC class I antigens (i.e., SLA antigens) on &e
cell. The genetically modified cells wi& altered MHC class I antigens are &en ~LIl;.l;~..,.~d
35 to a mouse to ~ &e delivery of a human gene product to a subject using a cell which is ~ g~ . to the subject.
Nucleic acid encoding &e gene for human growth hormone (hGH) is cloned into the
~;ullllll.,l~,;ally available plasmid expression vector pCDNAIII. Nucleic acid encoding &e
human grow& hormone gene can be obtained by a standard procedure based upon &e
21 8~528
~wo 9~/27042 r~ . c
-29 -
reported nucleotide sequenee of the gene (Goeddel et al. (1979) Nature ~1:544), for example
either by desiglung PCR primers based upon the gene sequence amd amplifying a fragment of
DNA ~ P. the coding region of the gene by PCR or by screening a cDNA or
genomic DNA library with primers based upon the gene sequence. Nucleic acid encoding
S human growth hormone is cloned into the pGl~ linker of pCDNAlII to create a vector (phGH)
containing the human gro~vth hormone gene umder the i . ' control of the
u ~ t~ C ~alu~ promoter. The vector also contains a bacterial selectable marker (arnpicillin
resistance) and a " selectable marker (neomycin resistance).
A~ul~ ly prepared phGH plasmid (e.g., cesium chloride purified and linearized)
10 is introduced into porcine myoblasts by c lc ~ Uy~Jla~iUll. In a general procedure,
~,Uyl~ / 1 x 107 cells are suspended in 0.5 ml of ice-cold c IC.~IIuyulaLiull buffer
(possible el~ . Llu,uula iull buffers include: PBS without Ca2+ or Mg2+, HEPES-buffered
saline and tissue culture medium without fetal calf serum. e.g., RPMI) and phGH DNA
(aU~ J 1-10 llg) is added. The DNA/cell suspension is placed in an eh ~ ClUUUla~iUll
15 cuvutte and incubated on ice for 5-10 minutes. The cuvette is then placed in am
eh,~ uyula~iull apparatus (e.g., ~ullllll~ ially available from BioRad) and pulsed at a desired
voltage and -~ setting. The voltage and ~ - c conditions for most efficient
Cl~ ~ ~luyula~iu.. are deter~nined l,~ ,ally (for example by testing a variety of conditions,
.l. ~.... -.;..~ the percentage of cell death ~vith each condition and selecting a condition that
achieves ~yylu~ 50 % cell death) but typical conditions are between 200 to 300 Vwith 500 to 1000 IlF ~ After the cells are shocked, the cells are again incubated on
ice for 10 minutes and then washed and plated in an appropriate culture media.
To select cells which have ill~Uly~ ' the introduced phGH plasmid, the antibiotic
G418 is added to the culture media three days after el~ Lluyula~iull and the cells are refed on
alternate days with G418-contairling media. The .~.. ,1".1;" of G418 used for efficient
selection of cells is determined empirically (for example, by !' ' ' ' ~ the dosage of G418
which is needed to kill mock transfected cells ) but typically is in the range of 400 ~g/ml to
800 ~g/ml. Selective cell death of cells which have not taken up the phGH plasmid occurs
begim~ing about on day 5 after the start of drug selection and by day 30 surviving cells are
30 considered to be selected. Individual clones of cells are then isolated and expamded into
specific cell lines and arlalyzed for expression of the gene product.
Expression of hurnan growth hormone by the tr.msfected cells is detected using aCullllll~ ly available two-site " ~ (RIA; from Nichols Institute
Diagnostics) with pure hGH standards for , to determine the level of hGH
35 expression by the cells. Human growth hormone produced by the cells is secreted into the
culture medium, thereby allowing assessment of hGH expression by sampling am aliquot of
the culture media for the presence of hGH usmg the RIA. Thus, the production of hGH by
the cells can be ~ '~r monitored by repeatedly sampling the culture media over time.
2 1 86528
wo ss/2~042 ~ Q4- ~Q
-30 -
A stably tranfected cell line which produces maximal expression of the introduced
hGH DNA is chosen by screening different clones that have been drug selected for hGH
production. Such a cell line can then be prepared for 1., y.~ ... into a ~ ... ir
recipient anima~ by treating the cells witb an F(ab')2 fragment of the PT85 anti-MHC class I
5 antibody (~UIIII. .~1 ~,;o,lly available from Veterinary Medicine Research De ~ Pullman
WA) which binds to SLA class I antigens. An F(ab')2 fragment is prepared from intact
antibodies as follows: Purified PT85 at 20 mg/ml is incubated with ' 1i7.od pepsin for 4
hours at 37 C in a pH 4.7 digestion buffer in a shaking water bath. The crude digest is
removed from the pepsin and i ' ~J neutrali7ed with a pH 7.0 binding buffer. The10 crude digest is applied to an ;, .. nl ,;1 ;,. ;1 protein A column and the eluate containing the
F(ab')2 fragments is collected. The F(ab')2 fragments are dialy_ed against PBS for 24 hours
using 50,000 MW cutoffdialysis tubing to remove any ~ Fc fragments. CHAPS
is added to the dialysis bag at a of 10 mM. The ~ r . - of the F(ab')2
digest is monitored by silver staining of 15 % SDS gels. Final IJUI;rl~d~iUII of the fragments
15 is achieved by FPLC using a Superose 12 column (Pharmacia, Upsala, Sweden).
Cells modified to express hGH are prepared for 1~ ;nn by incubating the cells
with the purified F(ab')2 fragments at a ~ l ;nl~ of l mg of antibody for ~ S,
I x Io6 cells for 30 mmutes at room ~ t~ . After the incubation, the treated cells are
washed once in Hank's buffer contarning 2 % fetal calf serrlm and then " 'y
20 Il, .~ r.l into Balb/c mice by syringe injection at an appropriate site (e.g., into the muscle
of the hindleg). Production of human growth hormone in the mice, as a result of expression
of human growth hormone by the introduced modified cells, is monitored by detecting the
presence of human growth hormone in the circulation of the mice. An aliquot of blood from
the mouse is collected periodically, for example from the tail vein, and hGH present within
25 the blood sample is detected using the " ~ described previously.
EQUIVAr .F,~TS
Those skilled in the art will recognize, or be able to ascertain usmg no more than
routine~-l...,.,.. --';nn mamyequivalentstothespecificc~ ' oftheinvention
30 described herein. Such equivalents are intended to be ~. ~ ' by the following claims.
