Note: Descriptions are shown in the official language in which they were submitted.
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TRRCiETED CYTOLYSIS OF CANCER CELLS
Technical Field
The field of the invention is the use of chimeric surface
membrane proteins for signal transduction. The cells expressing
such proteins are configured to recognize and act on cells
expressing TAG-72. The proteins can contain an antigen-binding
moiety which recognizes TAG-72.
Background
Regulation of cell activities is frequently achieved by
the binding of the ligand to a surface membrane receptor. The
formation of the complex with the extracellular portion of the
receptor results in a change in conformation with the cytoplasmic
portion of the receptor undergoing a change which results in a
signal being transduced in the cell. In some instances, the change
in the cytoplasmic portion results in binding to other proteins,
where the other proteins are activated and may carry out various
functions. In some situations, the cytoplasmic portion is
autophosphorylated or phosphorylated, resulting in a change in its
activity. These events are frequently coupled with secondary
messengers, such as calcium, cyclic adenosine monophosphate,
inositol phosphate, diacylglycerol, and the like. The binding of
the ligand results in a particular signal being induced.
There are a number of instances, where one might wish to
have a signal induced by virtue of employing a different ligand.
For example, one might wish to activate particular T cells, where
the T cells will then be effective as cytotoxic agents, or
activating agents by secretion of interleukins, colony stimulating
( factors or other cytokines, which results in the stimulation of
another cell. The ability of the T cell receptor to recognize
antigen is restricted by the nature of Major Histocompatibility
Complex (MHC) antigens on the surface of the host cell. Thus, the
use of a chimeric T cell receptor in which a non-MHC restricted
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ligand binding domain is linked directly to the signal traneducing
domain of the T cell receptor would permit the use of the
resulting engineered effector T cell in any individual, regardless
of their MHC genetic background. In this manner, one may change
the ligand which initiates the desired response, where for some
reason, the natural agent may not be as useful.
There is, therefore, interest in finding ways to modulate
cellular responses in providing for the use of ligands other than
the normal ligand to transduce a desired signal.
Rwlevaat Literature
The T cell antigen receptor (TCR) has a non-covalent
association between a heterodimer, the antigen/MHC binding subunit
Ti variable component and the five invariant chains: zeta (Z), eta
(t~) and the three CD3 chains: gamma (y), delta (b) and epsilon (e)
(Weisa & Imboden (1987) Adv. Immunol. 41:1-38; Cleavers et al.
(1988) Ann. Rev. Immunol. 6:629-662; Frank et al. (1990) Sem.
Immunol. 2:89-97). In contrast to the T, alpha/beta heterodimer
which is solely responsible for antigen binding, the physically
associated CD3-zeta/eta complex does not bind ligand, but is
thought to undergo structural alterations as a consequence of
T; antigen interaction which results in activation of intracellular
signal transduction mechanisms.
A description of the zeta chain may be found in Ashwell
& Klausner (1990) Ann. Rev. Immunol. 8:139-167. The nature of the
zeta chain in the TCR complex is described by Baniyash et al.
(1988} J. Biol. Chem. 263:9874-9878 and Orloff et al. (1989) ibid.
264:14812-14817. The heterodimeric zeta and eta protein is
described by Jin et al. (l990) Proc. Natl. Acad. Sci. USA
87:3319-3323. Discussion of the homodimers and heterodimera may
be found in Mercep et al. (1988) Science 242:57l-574; and Mercep
et al. (l989) ibid. 246:1162-1165. See also Susaman et al. (1988)
Cell 52:85-95. For studies of the role of the zeta protein, see
Weissman et al. (19B9) EMBO J. 8:3651-3b56; Frank et al. (1990)
Science 249:l74-177; and Zanier et al. (1989y Nature 342:803-805.
For discussion of the gamma subunit of the Fc,RI receptor,
expressed on mast cells and basophils and its homology to the zeta
chain, see Bevan & Cunha-Melo (1988) Prog. Allergy 42:123-l84;
Kinet (1989) Ceil 57:351-354; Benhamou et al., Proc. Natl. Acad.
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Sci. USA 87:5327-5330; and Orloff et al. _ (1990} Nature
347:189-191.
The zeta(() chain is structurally unrelated to the three
- CD3 chains, and exists primarily as a disulfide-linked homodimer,
or as a heterodimer with an alternatively spliced product of the
same gene, eta (~}. The zeta chain is also expressed on natural
killer cells as part of the FcyRIII receptor. The gamma chain of
the Fce receptor is closely related to zeta, and is associated
with the FceRI receptor of mast cells and baeophils and the C16
receptor expressed by macrophages and natural killer cells. The
role in signal transduction played by the cytoplasmic domains of
the zeta and eta chains, and the gamma eubunit of the FcRI
receptor has been described by Irving & Weiss (1991) Cell
64:S91-901; Romeo & Seed (1991) Cell 64:l037-1046 and Letourneur
& Klausner (1991) Proc. Natl. Acad. Sci. USA 88:B905-8909. More
recent studies have identified an 18 amino acid motif in the zeta
cytoplasmic domain that, upon addition to the cytoplasmic domain
of unrelated transmembrane proteins, endows them with the capacity
to initiate signal transduction (Romeo et al. (1992) Cell
6B:889-897). These data suggest a T cell activation mechanism in
which this region of zeta interacts with one or more intracellular
proteins.
The three CD3 chains, gamma (y), delta (d} and epsilon
(e), are structurally related polypeptides and were originally
implicated in signal transduction of T cells by studies in which
anti-CD3 monoclonal antibodies were shown to mimic the function
of antigen in activating T cells (Goldsmith & Weiss (19B7) Proc.
Natl. Acad. Sci. USA 84:6879-6B83), and from experiments employing
somatic cell mutants bearing defects in TCR-mediated signal
transduction function (Sussman et al. (1988) Cell 52:85-95).
Sequences similar to the active motif found in zeta are also
present in the cytoplasmic domains of the CD3 chains gamma and
delta. Chimeric receptors in which the cytoplasmic domain of an
unrelated receptor has been replaced by that of CD3 epsilon have
been shown to be proficient in signal transduction (Letourneur &
Klausner (1992) Science 255:79-82), and a 22 amino acid sequence
in the cytoplasmic tail of CD3 epsilon was identified as the
sequence responsible. Although the cytoplasmic domains of both
zeta and CD3 epsilon have been shown to be sufficient for signal
transduction, quantitatively distinct patterns of tyrosine
phosphorylation were observed with these two chains, suggesting
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that they may be involved in similar but distinct biochemical
pathways in the cell.
The phosphatidylinositol-specific .phoepholipase C
initiated activation by the T cell receptor ("TCR") is described
by Weise et al. (1984) Proc. Natl. Acad. Sci. USA 81:4l6-4173; and
Imboden & Stobo (l985) J. Exp. Med. 16l:446-456. TCR also
activates a tyrosine kinase (Samelson et al. (l986) Cell
46:1083-1090; Patel et al. (1987y J. Biol. Chem. 262:5831-5838;
Chai et al. (1989) J. Biol. Chem. 264:10836-10842, where the zeta
chain is one of the substrates of the kinase pathway (Baniyash
et al. (1988) J. Biol. Chem. 263:l8225-18230; Samelson et al.
(1986) supra). Fyn, a member of the src family of tyrosine
kinases, is reported to coprecipitate with the CD3 complex, making
it an excellent candidate for a TCR-activated kinase (Samelson
et al. (1990) Proc. Natl. Acad. Sci. USA 87:4358-4362). In
addition, a tyrosine kinase unrelated to fyn has been shown to
interact with the cytoplasmic domain of zeta (Chan et al. (1991)
Proc. Natl. Acad. Sci. USA 88:9166-9170).
Letourner & Klausner ((1991) Proc. Natl. Acad. Sci. USA
88:8905-8909) describe activation of T cells using a chimeric
receptor consisting of the extracellular domains of the a chain
of the human interieukin 2 receptor (Tac) and the cytoplasmic
domain of either r or y. Gross et al. ((1989) Proc. Natl. Acad.
Sci. USA 86:10024-l0028) describe activation of T cells using
chimeric receptors in which the MHC-restricted antigen-binding
domains of the T cell receptor a and ~ chains were replaced by the
antigen-binding domain of an antibody. Romeo & Seed ((1991) Cell
64:l037-l046) describe activation of T cells via chimeric
receptors in which the extracellular portion of CD4 is fused to
the tranamembrane and intracellular portions of Y, r, and r~
subunits. Letourner E Klausner (1992) describe activation of
T cells by a chimeric receptor consisting of the extracellular
domain of the IL-2 receptor and the cytoplasmic tail of CD3
epsilon (Science 255:79-82).
Based on the structural similarities between the
immunoglobulin (Ig) chains of antibodies and the alpha (a) and
beta (~) T cell receptor chains (T;), chimeric Ig-T; molecules in
which the V domains of the Ig heavy (V") and light (V~) chains are
combined with the C regions of T; a and T; ~ chains have been
described (Gross et al. (1989) Proc. Natl. Acad. Sci. USA
86:10024-10028). The role of the T; chains is to bind antigen
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presented in the context of MHC. The T, heterodimer does not
possess innate signalling capacity, but transmits the antigen
binding event to the CD3/zeta chains present in the TCR complex.
Expression of a functional antigen binding domain required
- 5 co-introduction of both VH-T; and V~ T; chimeric molecules. The
chimeras have been demonstrated to act as functional receptors by
their ability to activate T cell effector function in response to
cross-linking by the appropriate hapten or anti-idiotypic antibody
(Becker et al. (19S9) Cell 58:9I1 and Gross et al. (1989) Proc.
Natl. Acad. Sci. USA 86:l0024). However, like the native T;
chains, the V"-T; and VL T; chains do not possess innate signalling
capacity, but act via the CD3/zeta complex.
It has been speculated that antigen-specific cytolytic
immune cells might have a significant role in the modulation of
human diseases in vivo, including cancer. More recently, adoptive
T cell immunotherapy for cancer has shown promise in the clinics.
Autologous tumor-infiltrating lymphocytes (TIL's) from melanoma
patients were expanded ex vivo and reinfused into the patients.
Nine of 41 patients showed partial or complete remission
(Schwartzentruber et al. (1994) J. Clin. Oncol. 12:1475-83). A
statistically significant correlation between greater autologous
tumor lysis by the reinfused TIL's and patient responsiveness was
demonstrated in the study. In further clinical studies,
autologous TIL's were re-infused into patients at doses of up to
3x10" cells, twice weekly for 3 weeks, without significant
toxicity but with limited efficacy, moat likely due to the low
number of tumor specific T cells. The responses with TIL
therapies have been limited, however, to a few tumor types.
A significant drawback of a11 of those T cell adoptive
immunotherapies is the prolonged culture time necessary to
generate antigen-specific therapeutically relevant numbers of
cells. An alternative approach is the genetic modification of
patient T cells to express a chimeric receptor conferring the
ability of MHC independent lysis of the target cell.
HLA-unrestricted chimeric T cell receptors can redirect the
antigenic-specificity of T cell populations to recognize antigens
of choice. On binding to tumor antigen, the chimeric receptors
can initiate T cell activation, resulting in induction of effector
functions including cytolysis of the tumor cell.
TAG-72 is a human oncofetal, pancarcinoma sialylated
mucin antigen originally described by Schlom et al. at the NCI.
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TAG-72 is expressed on a variety of tumors including colon,
breast, prostate, non-small cell lung and ovarian carcinomas (Thor
et al. (l986) Cancer Rea. 46:3118). Normal tissue reactivity is
limited to low level expression in transitional.colonic epithelium
and secretory endometrium (Thor et al. (1986) supra). While
tumor TAG-72 expression is heterogeneous, data from clinical
trials conducted at several centers demonstrated that TAG-72
expression on colon, ovarian and breast carcinomas is up-regulated
with intraperitoneal (ip) administration of Y-interferon (IFN-Y)
(Greiner et al. (l992} J. Clin. Oncol.) or intravenous (iv)
administration of a-interferon (IFN-a) (Roselli et al. (1996) J.
Clin. Oncol.).
% of
cc49 Reactive patient
Tumors samples
positive
colon:
adenocarcinoma 93%
breast:
invasive ductile 94%
carcinoma
lung:
adenocarcinoma 86%
lung:
squamous cell 86%
carcinoma
ovary:
carcinoma 100%
stomach:
adenocarcinoma 95%
pancreas:
adenocarcinoma 100%
prostate:
adenocarcinoma 100%
from: Molinolo et al. (l990} Cancer Rea. 50:1291-1298
The results of hiatological data using the murine cc49
mAb which recognizes TAG-72, demonstrated a particular tumor
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reactivity as summarized in the table hereinabove (Molinolo et al.
l990, Canc. Rea. 50:129l-98).
The murine monoclonal antibody cc49 .is one of several
generated against human TAG-72 that recognize bound protein
lysatee of human carcinomas but not normal tissues (reviewed in:
Schlom et al. in Serological Cancer Markers, Sell, ed., Humans
Press, 1992, Totowa, NJ). Over 400 patients with colorectal,
breast, prostate or ovarian carcinomas have been infused with
radiolabelled murine cc49 mAb in radioimaging or
radioimmunotherapy protocols without reported significant adverse
side effects.
Several derivative molecules of cc49 have been created in
an attempt to decrease the immunogenicity of the native murine
antibody. The derivatives include a cc49 single chain antibody
(scFv or scAb) (Millenic et al. (1991) Cancer Res. 51:6363-6371),
a mouse-human chimeric cc49, a humanized cc49 antibody (Kashmiri
et al. (1995) Hybridoma 14:461-473), and a humanized cc49 scFV.
Compared to the native cc49 antibody, mouse-human
chimeric cc49 has a similar TAG-72 binding affinity, whereas the
cc49 scFv has an 8-fold lower and the humanized cc49 antibody a
2-fold to 3-fold lower binding affinity for TAG-72. In vivo tumor
targeting in a mouse xenograft model, however, was equivalent for
humanized and chimeric cc49 antibodies (Kashmiri et al. (1995)
Hybridoma l4:461-473).
2 5 St7l~ll~tARY OF THE INVENTION
The triggering of signal transduction leading to
cytotoxic function by different cells of the immune system can be
initiated by chimeric receptors with antibody-type specificity.
The chimeric receptors by-pass the requirement for matching at the
MHC locus between target cell (i.e. virally-infected, tumor cell,
etc.) and effector cell (i.e., T cell, granulocyte, mast cell
etc.). Intracellular signal transduction or cellular activation
is achieved by employing chimeric proteins having a cytoplaemic
region associated with transduction of a signal and activation of
a secondary messenger system, frequently involving a kinase, and
a non-MHC restricted extracellular region capable of binding to
a specific ligand and transmitting to the cytoplasmic region the
formation of a binding complex. Particularly, cytoplasmic
sequences of the zeta, eta, delta, gamma and epsilon chains of TCR
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and the gamma chain of Fc,RI, or a tyrosine kinase are employed
joined to other than the natural extracellular region by a
transmembrane domain. In such a manner) cells capable of
expressing the chimeric protein can be activated by contact with
the ligand, as contrasted with the normal mode of activation for
the cytoplasmic portion. For example, the extracellular domain
can comprise a portion or derivative of an antibody which binds
TAG-72 and retains the TAG-72 specificity. The antibody can be
polyclonal or monoclonal. A preferred derivative is a single
chain antibody which binds TAG-72.
DESCRIPTION OF THE DRAWINtiB
Figure 1 is a diagrammatic depiction of the structure of
single-chain antibodies used in the chimeric receptors of the
invention as compared to the structure of native monoclonal
antibodies.
Figure 2 depicts a map of a chimera comprising a
single-chain cc49 extracellular portion and zeta as the
intracellular portion of the molecule.
Figure 3 depicts results of chromium release assays using
cells armed with a cc49-zeta chimera.
Figure 9 depicts the results of chromium release assays
using various cancer cell lines as targets.
Figure 5 depicts the result of a chromium release assay
using a mixed population of target cells comprising TAG-72' and
TAG-72' cells.
Figure 6 depicts the results of a chromium release assay
using CD4 cells ae effectors.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Novel DNA sequences, such as DNA sequences as parts of
expression cassettes and vectors, as well as their presence in
cells are provided, where the novel sequences comprise three
domains which do not naturally exist together: (1) a cytoplasmic
domain, which normally transducer a signal resulting in activation
of a messenger system, (2) a transmembrane domain, which crosses
the outer cellular membrane, and (3) a non-MHC restricted
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extracellular receptor domain which serves to bind-to a ligand and
transmit a signal to the cytoplasmic domain) resulting in
activation of the messenger system. A preferred extracellular
domain is an antibody or antigen-binding .portion thereof,
~ 5 particularly one that binds TAG-72.
The cytoplasmic domain may be derived from a protein
which is known to activate various messenger systems, normally
excluding the G proteins. The protein from which the cytoplasmic
domain is derived need not have ligand binding capability by
itself, it being sufficient that such protein may associate with
another protein providing such capability. Cytoplasmic regions
of interest include the zeta chain of the T cell receptor, the eta
chain, which differs from the zeta chain only in its most
C-terminal exon as a result of alternative splicing of the zeta
mRNA, the delta, gamma and epsilon chains of the T cell receptor
(CD3 chains) and the gamma eubunit of the Fc,RI receptor, and such
other cytoplasmic regions which are capable of transmitting a
signal as a result of interacting with other proteins capable of
binding to a ligand.
A number of cytoplasmic regions or functional fragments
or mutants thereof may be employed) generally ranging from about
10 to 500 amino acids, where the entire naturally occurring
cytoplasmic region may be employed or only an active portion
thereof. The cytoplaemic regions of particular interest are those
which may be involved with one or more secondary messenger
pathways, particular pathways involved with a protein kinase, more
particularly, protein kinase C (PKC).
Pathways of interest include the
phoaphatidylinositol-specific phoapholipase involved pathway,
which is ncrmally involved with hydrolysis of
phosphatidylinositoi-4,5-biaphosphate, which results in production
of the secondary messengers inoaitol-1,4,S-trisphosphate and
diacylglycerol. Another pathway is the calcium mediated pathway,
which may be as a result of direct or indirect activation by the
cytoplasmic portion of the chimeric protein. Also, by itself or
in combination with another pathway, the kinase pathway may be
involved, which may involve phosphorylation of the cytoplaemic
portion of the chimeric protein. One or more amino acid aide
chains, particularly tyrosines, may be phoaphorylated. There is
some evidence that fyn, a member of the erc family of tyrosine
kinaaes, may be involved with the zeta chain.
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While usually the entire cytoplasmic region will be
employed, in many cases, it will not be necessary to use the
entire chain. To the extent that a truncated portion may find
use, such truncated portion may be used in place of the intact
chain.
Suitable cytoplasmic domains arise also from other
molecules that have a signalling role in eliciting a response by
the host cell. For example, tyrosine kinaees, such ae ZAP-70 and
members of the 3anus kinase family, and ancillary molecules that
have less than a direct role in signaling, such as CD2 and CD28,
or functional portions thereof, can be found as the cytoplasmic
domain of a receptor of interest. See W096/23814.
Generally a desirable response by the host cell is
proliferation or differentiation. Manifestation of a desirable
phenotype, such as cytotoxicity, ie obtained and which can be
directed to a specific target, such as a cancer cell, by use of
a chimeric receptor of interest.
The transmembrane domain may be the domain of the protein
contributing the cytoplasmic portion, the domain of the protein
contributing the extracellular portion, or a domain associated
with a totally different protein. Chimeric receptors of the
invention, in which the tranemembrane domain is replaced with that
of a related receptor, or, replaced with that of an unrelated
receptor, may exhibit qualitative and/or quantitative differences
in signal transduction function from receptors in which the
transmembrane domain is retained. Thus, functional differences
in signal transduction may be dependent not only upon the origin
of the cytoplasmic domain employed, but also on the derivation of
the transmembrane domain.
Therefore, for the most part) it will be convenient to
have the transmembrane domain naturally associated with one or the
other of the other domains, particularly the extracellular domain.
In some cases it will be desirable to employ the
tranemembrane domain of the zeta, eta, or Fc,RI gamma chains which
contain a cysteine residue capable of disulphide bonding, so that
the resulting chimeric protein will be able to form
disulfide-linked dimers with itself, or with unmodified versions
of the zeta, eta, or FceRI gamma chains or related proteins. In
some instances, the tranemembrane domain will be selected to avoid
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binding of such domain to the transmembrane domain_of the same or
different surface membrane protein to minimize interactions with
other members of the receptor complex. In other cases it will be
desirable to employ the tranamembrane domain of zeta, eta, FceRI
~ 5 gamma, or CD3-gamma, CD3-delta or CD3-epsilon, to retain physical
association with other members of the receptor complex.
The extracellular domain may be obtained from any of the
wide variety of extracellular domains or secreted proteins
associated with ligand binding and/or signal transduction. The
extracellular domain may be part of a protein which is monomeric,
homodimeric, heterodimeric, or associated with a larger number of
proteins in a non-covalent complex.
Of particular interest are antibodies and antigen-binding
portions thereof. In particular, the extracellular domain may
consist of an Ig heavy chain which may in turn be covalently
associated with Ig light chain by virtue of the presence of CH1
and hinge regions, or may become covalently associated with other
Ig heavy/light chain complexes by virtue of the presence of hinge,
CH2 and CH3 domains. In the latter case, the heavy/light chain
complex that becomes joined to the chimeric construct may
constitute an antibody with a specificity distinct from the
antibody specificity of the chimeric construct. Depending on the
function of the antibody, the desired structure and the signal
transduction) the entire chain may be used or a truncated chain
may be used, where all or a part of the CH1, CH2, or CH3 domains
may be removed or alI or part of the hinge region may be removed.
Single-chain antibodies (scAb's) are desirable for ease
of manipulation. As depicted in Figure 1, the moat widely known
ecAb is one where the variable regions of the heavy and light
chain are tethered by a molecular linker so that the tripartite
molecule folds spontaneously to form the relevant antigen-binding
domain. Other forma of single-chain antibodies are contemplated
to fall within the scope of the invention.
scAb's are desirable because a gene thereof can be
subcloned in the proper operative relationship with the signal
sequence, tranamembrane domain and cytoplasmic domains to yield
a chimeric molecule of interest.
Antibodies to TAG-72 are preferred, with monoclonal
antibodiee being more preferred. A scAb directed to TAG-72 is a
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desired antibody derivative in the practice of the instant
invention.
Various naturally occurring cell surface receptors which
bind to TAG-72 also may be employed. For example human Heregulin
(Hrg) a protein similar in structure to Epidermal Growth Factor
(EGF), has been identified as a ligand for the receptor Herz which
ie expressed on the surface of breast carcinoma cells and ovarian
carcinoma calls (Holmea et al., Science (1992) 256:l205-1210). The
murine equivalent is the "Neu" protein (Bargman et al., Nature
3l9:226-230 (1986)). The extracellular domain of Hrg could be
joined to the zeta tranemembrane and cytoplaemic domains to form
a chimeric construct of the invention to direct T cells to kill
breast carcinoma cells.
In addition, "hybrid" extracellular domains can be used.
For example, the extracellular domain may consist of a CD
receptor, such as CD4, joined to a portion of an immunoglobulin
molecule, for example the heavy chain of Ig. See W096/24671.
Where a receptor is a molecular complex of proteins,
where only one chain has the major role of binding to the ligand,
2D it will usually be desirable to use solely the extracellular
portion of the ligand binding protein. Where the extracellular
portion may complex with other extracellular portions of other
proteins or form covalent bonding through disulfide linkages, one
may also provide for the formation of such dimeric extracellular
region. Also, where the entire extracellular region is not
required, truncated portions thereof may be employed, where such
truncated portion is functional. In particular, when the
extracellular region of CD4 is employed, one may use only those
sequences required for binding of gp120, the HIV envelope
glycoprotein. In the case in which Ig is used as the
extracellular region, one may simply use the antigen binding
regions of the antibody molecule and dispense with the constant
regions of the molecule (for example, the F~ region consisting of
the CH2 and CH3 domains).
In some instances, a few amino acids at the joining
region of the natural protein may be deleted, usually not more
than 10, more usually not more than 5. Also, one may wish to
introduce a small number of amino acids at the borders, usually
not more than 10, more usually not more than 5. The deletion or
insertion of amino acids will usually be as a result of the needs
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of the construction, providing for convenient restriction sites,
ease of manipulation, improvement in levels of expression, or the
like. In addition, one may wish to substitute one or more amino
acids with a different amino acid for similar reasons, usually not
substituting more than about five amino acids in any one domain.
The cytoplasmic domain as already indicated will generally be from
about 10 to 500 amino acids, depending upon the particular domain
employed. The transmembrane domain will generally have from about
25 to 50 amino acids, while the extracellular domain will
generally have from about 10 to 500 amino acids.
Normally, the signal sequence at the 5~ terminus of the
open reading frame (ORF) which directs the chimeric protein to the
surface membrane will be the signal sequence of the extracellular
domain. However, in some instances, one may wish to exchange this
sequence for a different signal sequence. However, since the
signal sequence will be removed from the protein, being processed
while being directed to the surface membrane, the particular
signal sequence will normally not be critical to the subject
invention. Similarly, associated with the signal sequence will
be a naturally occurring cleavage site, which will also normally
be the naturally occurring cleavage site associated with the
signal sequence or the extracellular domain.
In the embodiments provided herein various following
chimeric constructs containing as the extracellular domain an
antibody portion that binds TAG-72 were produced.
The instant invention is particularly directed to
single-chain antibody (scAb) chimeric receptors in which a ecAb
functions as the extracellular domain of the chimeric receptor
although other antibody portions can be found at the extracellular
domain of a chimera of interest. In contrast to previously
described Ig-T;chimeras (Becker et al., Gross et al., supra), the
ecAb chimeric receptors function by bypassing the normal antigen
recognition component of the T cell receptor complex, and
transducing the signal generated on antigen-receptor binding
directly via the cytoplasmic domain of the molecule.
A range of scAb chimeric receptors, for example,
anti-TAG-72 immunoglobulin-zeta (Ig-Z) chimeric receptors can be
configured.
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For example, the full-length IgG heavy chain comprising
the VH, CH1, hinge, CH2 and CH3 (Fc) Ig domains is fused to the
cytoplasmic domain of the zeta chain via the appropriate
tranamembrane domain. If the VH domain alone is sufficient to
confer antigen-specificity (so-called"single-domain antibodies"),
homodimer formation of the Ig-r chimera is expected to be
functionally bivalent with regard to antigen binding sites.
Because it is likely that both the VH domain and the VI, domain are
necessary to generate a fully active antigen binding site, both
the IgH-t molecule and the full-length IgL chain are introduced
into cells to generate an active antigen-binding site. Dimer
formation resulting from the intermolecular Fc/hinge disulfide
bonds results in the assembly of Ig-r receptors with extracellular
domains resembling those of IgG antibodies. Derivatives of this
Ig-Z chimeric receptor include those in which only portions of the
heavy chain are employed in the fusion. For example, the VH
domain (and the CH1 domain) of the heavy chain can be retained in
the extraceliular domain of the Ig-~ chimera (VH-r).
Co-introduction of a similar chimera in which the V and C domains
of the corresponding light chain replace those of the Ig heavy
chain (VL-r) can then reconstitute a functional antigen binding
site.
Because association of both the heavy and light V domains
are required to generate a functional antigen binding site of high
affinity, in order to generate a Ig chimeric receptor with the
potential to bind antigen, a total of two molecules will typically
need to be introduced into the host cell. Therefore, an
alternative and preferred strategy is to introduce a single
molecule bearing a functional antigen binding site. This avoids
the technical difficulties that may attend the introduction of
more than one gene construct into host cells. The "single-chain
antibody" (scAb) is created by fusing together the variable
domains of the heavy and light chains using a short peptide
linker, thereby reconstituting an antigen binding site on a single
molecule.
Single-chain antibody variable fragments (Fv's) in which
the C-terminus of one variable domain (VH or VL) is tethered to
the N-terminus of the other (VL or VH, respectively, (see
Figure 1) via a 15 to 25 amino acid peptide or linker, have been
developed without significantly disrupting antigen binding or
specificity of the binding (Bedzyk et al. (1990) J. Biol. Chem.
265:18615; Chaudhary et al. (1990) Proc. Natl. Acad. Sci.
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87:9491). The Fv's lack the constant regions (Fc)- present in the
heavy and light chains of the native antibody. In the methods of
the instant invention, the extracellular domain of the
single-chain Ig chimeras consists of the Fv fragment which may be
fused to a11 or a portion of the constant domains of the heavy
chain, and the resulting extracellular domain is joined to the
cytoplaemic domain of, for example, zeta, via an appropriate
transmembrane domain that will permit expression in the host cell,
e.g., zeta, CD4.
The resulting chimeric molecules differ from the Fv's in
that on binding of TAG-72 the receptors initiate signal
transduction via the cytoplaemic domain. In contrast, free
antibodies and Fv's are not cell-associated and do not tranaduce
a signal on TAG-72 binding. The ligand binding domain of the acAb
chimeric receptor may be of two types depending on the relative
order of the VH and VL domains: VH-1-VL or VL-1-VH (where "1"
represents the linker) (See Figure 1).
The chimeric construct, which encodes the chimeric
protein according to the instant invention will be prepared in
conventional ways. Since, for the most part, natural sequences
may be employed, the natural genes may be isolated and
manipulated, ae appropriate, so as to allow for the proper joining
of the various domains. Thus, one rnay prepare the truncated
portion of the sequence by employing the polymerise chain reaction
(PCR), using appropriate primers which result in deletion of the
undesired portions of the gene. Alternatively, one may use primer
repair, where the sequence of interest may be cloned in an
appropriate host. In either case) primers may be employed which
result in termini, which allow for annealing of the sequences to
result in the desired open reading frame encoding the chimeric
protein. Thus, the sequences may be selected to provide for
restriction sites which are blunt-ended, or have complementary
overlaps. During ligation) it is desirable that hybridization and
ligation does not recreate either of the original restriction
sites.
If desired, the extracellular domain may also include the
transcriptional initiation region, which will allow for expression
in the target host. Alternatively, one may wish to provide for
a different transcriptional initiation region, which may allow for
constitutive or inducible expression, depending upon the target
host, the purpose for the introduction of the subject chimeric
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protein into such host, the level of expression desired, the
nature of the target host, and the like. Thus, one may provide
for expression upon differentiation or maturation of the target
host, activation of the target host, or the like.
A wide variety of promoters have been described in the
literature, which are constitutive or inducible, where induction
may be associated with a specific cell type or a specific level
of maturation. Alternatively, a number of viral promoters are
known which may also find use. Promoters of interest include the
~-actin promoter, SV40 early and late promoters, immunoglobulin
promoter, human cytomegalovirus promoter, and the Friend spleen
focus-forming virus promoter. The promoters may or may not be
associated with enhancers, where the enhancers may be naturally
associated with the particular promoter or associated with a
different promoter.
The sequence of the open reading frame may be obtained
from genomic DNA, cDNA, or be synthesized, or combinations
thereof. Depending upon the size of the genomic DNA and the
number of introns, one may wish to use cDNA or a combination
thereof. In many instances, it is found that introns stabilize
the mRNA. Also, one may provide for non-coding regions which
stabilize the mRNA.
A termination region will be provided 3' to the
cytoplasmic domain, where the termination region may be naturally
associated with the cytoplasmic domain or may be derived from a
different source. For the most part, the termination regions are
not critical and a wide variety of termination regions may be
employed without adversely affecting expression.
The various manipulations may be carried out in vitro or
may be introduced into vectors for cloning in an appropriate bast,
e.g., E. coli. Thus, after each manipulation, the resulting
construct from joining of the DNA sequences may be cloned, the
vector isolated, and the sequence screened to insure that the
sequence encodes the desired chimeric protein. The sequence may
be screened by restriction analysis, sequencing, or the like.
Prior to cloning, the sequence may be amplified using PCR and
appropriate primers, so as to provide for an ample supply of the
desired open reading frame, while reducing the amount of
contaminating DNA fragments which may have substantial homology
to the portions of the entire open reading frame.
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The target cell may be transformed with the chimeric
construct in any convenient manner. Techniques include calcium
phosphate-precipitated DNA transformation, electroporation,
protoplast fusion, biolistice, using DNA-coated particles,
- 5 transfection, and infection, where the chimeric construct is
introduced into an appropriate virus, particularly a
non-replicative form of the virus, or the like.
Once the target host has been transformed, usually,
integration, will result. However, by appropriate choice of
vectors, one may provide for episomai maintenance. A large number
of vectors are known which are based on viruses, where the copy
number of the virus maintained in the cell is low enough to
maintain the viability of the cell. Illustrative vectors include
SV40, EBV and BPV.
The constructs will be designed so as to avoid their
interaction with other surface membrane proteins native to the
target host. Thus, for the most part, one will avoid the chimeric
protein binding to other proteins present in the surface membrane.
In order to achieve this, one may select for a transmembrane
domain which ie known not to bind to other transmembrane domains,
one may modify specific amino acids, e.g. substitute for a
cysteine, or the like.
Once one has established that the transformed host is
capable of expressing the chimeric protein as a surface membrane
protein in accordance with the desired regulation and at a desired
level) one may then determine whether the transmembrane protein
is functional in the host to provide for the desired signal
induction. Since the effect of signal induction of the particular
cytoplasmic domain will be known, one may use established
methodology for determining induction to verify the functional
capability of the chimeric protein.
For example, TCR binding results in the induction of CD69
expression. Thus, one would expect with a chimeric protein having
a zeta cytoplasmic domain, where the host cell is known to express
CD69 upon activation, one could contact the transformed cell with
the prescribed ligand and then assay for expression of CD69. Of
course, it is important to know that ancillary signals are not
required from other proteins in conjunction with the particular
cytoplasmic domain, so that the failure to provide transduction
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of the signal may be attributed solely to the inoperability of the
chimeric protein in the particular target host.
A wide variety of target hosts may be, employed, normally
cells from vertebrates, more particularly, mammals, desirably
domestic animals or primates, particularly humans. The subject
chimeric constructs may be used for the investigation of
particular pathways controlled by signal transduction, for
initiating cellular responses employing different ligande, for
example, for inducing activation of a particular subset of
lymphocytes, where the lymphocytes may be activated by particular
surface markers of cells, such as neoplastic cells, virally
infected cells, or other diseased cells, which provide for
specific surface membrane proteins which may be distinguished from
the surface membrane proteins on normal cells.
The cells may be further modified so that expression
cassettes may be introduced, where activation of the transformed
cell will result in secretion of a particular product. In this
manner, one may provide for directed delivery of specific agents,
such as interferons, TNF's, perforane, naturally occurring
cytotoxic agents, or the like, where the level of secretion can
be greatly enhanced over the natural occurring secretion.
Furthermore, the cells may be specifically directed to the site
using injection, catheters, or the like, so as to provide for
localization of the response.
The subject invention may find application with cytotoxic
lymphocytes (CTL), Natural killer cells (NK), TIL's or other
cells Which are capable of killing target cells when activated.
Thus, diseased cells, such as cells infected with HIV, HTLV-I or
II, cytomegalovirus, hepatitis B or C virus, mycobacterium avium,
etc., or neoplastic cells, where the diseased cells have a surface
marker associated with the diseased state may be made specific
targets of the cytotoxic cells.
By providing a receptor extracellular domain, e.g., CD4,
Which binds to a surface marker of the pathogen or neoplastic
condition, e.g., gp120 for HIV, the cells may serve as therapeutic
agents. By modifying the cells further to prevent the expression
or translocation of functional Class I and/or II MHC antigens, the
cells will be able to avoid recognition by the host immune system
as foreign and can therefore be therapeutically employed in any
individual regardless of genetic background. Alternatively, one
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may isolate and transfect host cells with the subject constructs
and then return the transfected host cells to the host.
Other appiicatione include transformation of host cells
- from a given individual with retroviral vector constructs
directing the synthesis of the chimeric construct. By
transformation of ouch cells and reintroduction into the patient
one may achieve sutologous gene therapy applications.
In addition, suitable host cells include hematopoietic
stem cells, which develop into cytotoxic effector cells with both
myeloid and lymphoid phenotype including granulocytes, mast cells,
basophils, macrophages, natural killer (NK) cells and T and B
lymphocytes. Introduction of the chimeric constructs of the
invention into hematopoietic stem cells thus permits the induction
of cytotoxicity in the various cell types derived from
hematopoietic stem cells providing a continued source of cytotoxic
effector cells to fight various diseases.
The zeta subunit of the T cell receptor is associated not
only with T cells, but is present in other cytotoxic cells derived
from hematopoietic stem cells. Three subunits, zeta, eta and the
gamma chain of the Fce receptor, associate to form homodimera as
well as heterodimers in different cell types derived from stem
cells. The high level of homology between zeta, eta and the gamma
chain of the Fce receptor, and their association together in
different cell types suggests that a chimeric receptor consisting
of an extracellular binding domain coupled to a zeta, eta or gamma
homodimer, would be able to activate cytotoxicity in various cell
types derived from hematopoietic stem cells.
For example, zeta and eta form both homodimers and
heterodimera in T cells (Clayton et al. (1991) Proc. Natl. Acad.
Sci. USA 88:5202) and are activated by engagement of the cell
receptor complex; zeta and the gamma chain of the Fce receptor
form homodimera and heterodimers in NK cells and function to
activate cytotoxic pathways initiated by engagement of F
receptors (Lanier et al. (1991) J. Immunol. 146:1571; the gamma
chain forms homodimers expressed in monocytes and macrophages
(Phillips et al. (1991) Eur. J. Immunol. 21:895), however because
zeta will form heterodimers with gamma, it is able to couple to
the intracellular machinery in the monocytic lineage; and zeta and
the gamma chain are used by IgE receptors (FcRI) in mast cells and
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basophils (Letourneur et al. (1991) J. Immunol. 147:2652) for
signalling cells to initiate cytotoxic function.
Therefore, because stem cells transplanted into a subject
via a method such as bone marrow transplantation exist for a
lifetime, a continued source of cytotoxic effector cells is
produced by introduction of the chimeric receptors of the
invention into hematopoietic stem cells to fight virally infected
cells, cells expressing tumor antigens, or effector cells
responsible for sutoimmune disorders.
Additionally, introduction of the chimeric receptors into
stem cells with subsequent expression by both myeloid and lymphoid
cytotoxic cells may have certain advantages in patients with
multiple or congenital carcinoma expressing TAG-72.
The chimeric receptor constructs of the invention can be
introduced into hematopoietic stem cells followed by bone marrow
transplantation to permit expression of the chimeric receptors in
a11 lineages derived from the hematopoietic system. High titer
retroviral producer lines are used to transduce the chimeric
receptor constructs, for example a-TAG-72/(, into both murine and
human T cells and human hematopoietic stem cells through the
process of retroviral-mediated gene transfer as described by Lusky
et al. in {l992) Blood 80:396.
For transduction of hematopoietic stem cells, the bone
marrow is harvested using standard medical procedures and then
processed by enriching for hematopoietic stem cells expressing the
CD34 antigen as described by Andrews et al. in (1989) J. Exp. Med.
169:1721. The cells then are incubated with the retroviral
supernatants in the presence of hematopoietic growth factors such
as stem cell factor and IL-6.
The bone marrow transplant can be autologous or
allogeneic, and depending on the disease to be treated, different
types of conditioning regimens are used {see, Surgical Clinics of
North America (1986) 66:589).
The recipient of the genetically modified stem cells can
be treated with total body irradiation, chemotherapy using
cyclophosphamide, or both to prevent the rejection of the
transplanted bone marrow. In the case of immunocompromised
patients, no pretransplant therapy may be required because there
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is no malignant cell population to eradicate and the patients
cannot reject the infused marrow.
In addition to the gene encoding the chimeric receptor,
additional genes may be included in the retroviral construct. The
included genes can encompass the thymidine kinase gene (Borrelli
et al. (1988) Proc. Natl. Aced. Sci. USA 85:7572) which acts as
a suicide gene for the marked cells if the patient is exposed to
gancyclovir. Thus, if the percentage of marked cells is too high,
gancyclovir may be administered to reduce the percentage of cells
expressing the chimeric receptors.
In addition, if the percentage of marked cells needs to
be increased, the multi-drug resistance gene can be included
(Sorrentino et al. (1992) Science 257:99) which functions as a
preferential survival gene for the marked cells in the patients
if the patient is administered a dose of a chemotherapeutic agent
such as taxol. Therefore, the percentage of marked cells in the
patients can be titrated to obtain the maximum therapeutic benefit
from the expression of the universal receptor molecules by
different cytotoxic cells of the patient's immune system.
The following examples are by way of illustration and not
by way of limitation.
EIAI~LE 1
CD8/Z chimera coastructioa
The polymeraee chain reaction, PCR (Mullis et al. (1986y
"Cold Spring Harbor Symposium on Quantitative Biology", NY,
263-273) was used to amplify the extracellular and transmembrane
portion of CDBa (residues 1-l87) from pSV7d-CD8a and the
cytoplasmic portion of the human t chain (residues 31-142 from
pGEM3t. Some DNA's were obtained from (Littman et al. (l985) Cell
40:237-246; CD8) and (Weiseman et al. (1988) Proc. Natl. Aced.
Sci. 85:9709-9?13; Z). Plasmids pSV7d-CDBa and pGEM3z( were
kindly provided by Drs. Dan Littman and Julie Turner (Univ. of CA,
S.F.) and Drs. R.D. Klausner and A.M. Weissman (N.I.H.),
respectively. Primers encoding the 3' sequences of the CD8
fragment and the 5' sequences of the zeta fragment (~) were
designed to overlap such that annealing of the two products
yielded a hybrid template. From this template the chimera was
amplified using external primers containing XbaI and BamHI cloning
sites. The CD8/r chimera was eubcloned into pTfneo (Ohashi et al.
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(l985) Nature 3l6:606-609) and sequenced via the Sanger
dideoxynucleotide technique (Sanger et al. (l977) Proc. Natl.
Acad. Sci. USA 74:5463-5467y.
Antibodies
C305 and Leu4 mAb's recognize the Jurkat Ti f3 chain and
an extracellular determinant of CD3 e, respectively. OKT8,
acquired from the ATCC, recognizes an extracellular epitope of
CDB. The anti-r rabbit antiserum, Q387, raised against a peptide
comprising amino acids 132-l44 of the murine r sequence (Orloff
et al. (1989} J. Biol. Chem. 264:148l2-l4817), was kindly provided
by Drs. R.D. Klausner, A.M. Weiseman and L.E. Samelson. The
anti-phosphotyrosine mAb, 4G10, was a generous gift of
Drs. D. Morrieon, B. Druker) and T. Raberts. W6/32 recognizes an
invariant determinant expressed on human HLA Class 1 antigens.
Leu23, reactive with CD69, was obtained from Becton-Dickinson
Monoclonal Center (Milpitas, CA). MOPC 195, an IgG2a, (Litton
Hionetics, Kensington, MD) was used as a control mAb in FACS
analysis. Ascitic fluids of mAb were used at a final dilution of
1:1000 (a saturating concentrationy in a11 experiments unless
otherwise stated.
Cell lines and Transfections
The human leukemic T cell line Jurkat and its derivative
J.RT3-T3.5 were maintained in RPMI 1640 supplemented with 10$
fetal bovine serum (FBS) glutamine, penicillin and streptomycin
(Irvin Scientific). Chimera-transfected clones were passaged in
the above medium with the addition of Geneticin (GIBCO, Grand
Island, NY) at 2 mg/ml. Electroporation of pTfneo-CD8/r into
Jurkat and J.RT3-T3.5 was performed in a Bio-Rad Gene Pulser using
a voltage of 250V and a capacitance of 960 pF with 20pg of plaemid
per 10' cells. After transfection, cells were grown for two days
in RPMI 5efore plating out in Geneticin-containing medium. Clones
were obtained by limiting dilutions and screened for TCR and CD8/~
expression by Flow Cytometry (see below). The Jurkat CD8 clone,
transfected with the wild-type CD8 protein, was kindly provided
by Drs. Julia Turner and Dan Littman.
Flow Cytosetry
Approximately 1 x 106 cells/condition were stained with
saturating concentrations of antibody, then incubated with
fluorescein-conjugated goat anti-mouse Ab prior to analysis in a
FACScan (Beckton Dickinson) as previously described (Weiss and
Stobo l984). Cells analyzed for CD69 expression were stained
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directly with fluorescein-conjugated Leu 23 (anti-CD69 mAb) or
MOPC l95 (control mAb).
[Ca+ij, Measureaent by Fluorimstry
Calcium sensitive fluorescence was monitored as
previously described (Goldsmith & Weiss (1987) Proc. Natl. Acad.
Sci. USA 84:6879-6883). Cells were stimulated with soluble mAb
C305 and ORTB at saturating concentrations (1:1000 dilution of
aecites). Maximal fluorescence was determined after lysis of the
cells with Triton X-100; minimum fluorescence was obtained after
chelation of Ca+Z with EGTA. Ca+~ was determined using the
equation [ Ca'Z].,--ICs ( F~",~ FM,,) / ( Fm"~-F~",~) , with I~=250 nM as
described (Grynkiewica et al. (1985) J. Biol. Chem.
260:3440-3448).
Inositol Phosphate Measurement
Cells were loaded with ['H)myo-inoeitol (Amersham) at
40 NCi/ml for 3 hr. in phosphate buffered saline, then cultured
overnight in RPMI l640 supplemented with 10% fetal bovine eerum.
Cells were stimulated for 15 min. with the indicated antibodies
at 1:l000 dilution of ascites in the presence of 10 mM LiCl to
inhibit dephosphorylation of IP,. The extraction and
quantification of soluble inositol phosphates were as described
(Imboden & Stobo (1985) J. Exp. Med. 161:446-456).
Surface Iodinations
Cells were labeled with '~I using the
lactoperoxidase/glucose oxidase (Sigma) procedure as described
(Weiss & Stobo (1984) J. Exp. Med. 160:1284-1299).
Iaaunoprecipitations
Cells were lysed at 2 x 10? cells/200 ml in 1% NP40
(Nonidet P40), 150 mM NaCl, and 10 mM Tris pH 7.8 in the presence
of protease inhibitors, 1 mM PMSF, aprotinin, and leupeptin.
Lysis buffer for lysates to be analyzed for phosphotyrosine
content was supplemented with phosphatase inhibitors as described
(Desai et al. (1990) Nature 348:66-69). Iodinated lysatea were
supplemented with 10 mM iodoacetamide to prevent post-lysis
disulfide bond formation. Digitonin lysis was performed in 1%
Digitonin, 150 mM NaCl, 10 mM Tris pH 7.8, 0.12$ Triton X-100.
After 30 min. at 4~C, lysates were centrifuged for 10 min. at
14,000 rpm.) then precleared with fixed Staphylococcus aureus
(Staph A; Calbiochem-Behring). Alternatively, lysates of cells
stimulated with antibody prior to lysis were precleared with
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aepharose beads. The precleared lysatea were _incubated with
Protein A Sepharoae CL-4B beads which had been prearmed with the
immunoprecipitating antibody. Washed immunoprecipitates were
reauspended in SDS sample buffer +/- 5% fi-mercaptoethanol and
boiled prior to electrophoresis on 12% polyacrylamide gels.
Stiaulation of calls for assessreat of phosphotprosine content.
Cells were stimulated in serum-free medium at
2 x 10' cells/200 pl with antibodies at 1:250 dilution of ascitea.
After 2 min. at 37~C, the medium was aspirated, and the cells
lysed in 100 girl of NP40 lyais buffer. Lysates were precleared,
then ultracentrifuged and samples resolved by SDS PAGE.
Iaaunohlots
Gels were equilibrated in transfer buffer (20 mM Tris
base, 150 mM glycine, 20% methanol) for 30 min. and transferred
to nitrocellulose membranes in a Bio-Rad Western blotting
apparatus run at 25 volts overnight. Membranes were blocked in
TBST (10 mM Tris HC1 [pH 8), 150 mM NaCI, 0.05% Tween 20) plus
1.5% ovalbumin, then incubated with either mAb 4G10 or rabbit
anti-~ antiserum (#387). The immunoblota were washed and
incubated with a 1:7000 dilution of alkaline
phosphatase-conjugated goat anti-mouse or goat anti-rabbit
antibody. After 1-2 hours, the blots were washed and developed
with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate substrates as per manufacturer's instructions (Promega).
IL-2 Bioassap
For stimulation, cells were coated with the indicated
antibodies at saturating concentrations {1:1000 dil. of ascitea)
for 30 min. at 4~C. After removal of unbound antibody, cells were
spun onto 24-well tissue culture plates which had been precoated
with rabbit anti-mouse Ig ( Zymed Lass ) and blocked with medium
glue 10% FBS. Phorbol myriatate acetate, PMA (Sigma) and
ionomycin (Calbiochem) were added to final concentrations of
10 mg/ml and 1 mM, respectively. Cell-free supernatants were
harvested after 20 hr. of culture and assessed for IL-2 content
utilizing the IL-2-dependent CTLL-2.20 cell line in the MTT
colorimetric assay as described (Mosmann (1983) J. Immunol. Meth.
65:55-63.
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REStTLTS -
Characterization of the CD8/Z Chiaara is T Cell Receptor-positive
and Receptor-negative Jurkat Cells
The CD8/( chimeric construct described previously was
transfected via electroporation into both the Jurkat human T cell
leukemic line, yielding clone JCD8/Z2, and a Jurkat-derived
mutant, JRT3.T3.5 deficient in full length T;f3 chain transcripts
and protein, yielding Jf3-CD8/Z14. Though JRT3.T3.5 expresses
normal levels of T; a and the CD3 subunits, its deficiency in T;13
expreesion results in the absence of TCR expression on the cell
surface (Ohashi et al. (1985) Nature 316:606-609).
Transfection of the chimera into this cell enabled
assessment of r's signalling phenotype without the complication
of the additional TCR chains. Levels of surface expression of the
chimera and TCR in atably transfected clones were quantified by
flow cytometry using mAb's which recognize either CD8 (OKTB) or
the CD3 a aubunit of the TCR (Leu 4). Fluorescence histograms of
these clones which both express high levels of CD8/r was observed;
this cell Was used as a control in a11 of the experiments.
The three clones express comparable levels of CD8
epitopes and T cell receptors with the exception of Jf3-CD8/z14,
which fails to express surface TCR. Thus the CD8/( chimera can
be expressed on the cell surface in the absence of the TCR chains.
To characterize the structure of the CD8/~ chimeric
protein, cells were surface radioiodinated, lysed in 1$ NP40 and
subjected to immunoprecipitation with OKT8 or normal rabbit
antiserum raised to a cytoplasmic peptide sequence of murine Z.
Under reducing conditions, antibodies against either CD8
or Z precipitate a single protein of 34-35kD from the
chimera-tranafected cell, while OKT8 precipitates a 29kD protein
representing wild-type CDB from Jurkat CD8. Although CD8 in its
normal environment has an apparent molecular weight of 32-34kD,
(Snow & Terhorst (1983) J. Biol. Chem. 258:14675-14681)
preliminary experirnenta comparing CD8 in Jurkat and a CD8-positive
line, HPB.ALL, suggest that the reduction in size of CD8 observed
here results from a distinct pattern of glycosylation in the
Jurkat host.
Under non-reducing conditions a more complex pattern of
proteins is seen in immunoprecipitatea of both CD8 and the CD8/~
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chimera. The complexity is characteristic of CD8 precipitates
since homomultimers and heteromultimers have been previously
observed (Snow & Terhorst (1983) supra). The two prominent
species immunoprecipitated from JCDB/r2 migrating at approximately
70 and 100 kD are likely to represent homodimers and homotrimers
of the chimera. As there are no cysteine residues for the
formation of disulfide linkages with the ( portion of the chimera,
any disulfide bonds formed in the chimera must occur through CDB.
Therefore, any protein forming a heterodimer with CD8/(
is likely to form one with the wild-type CD8 and thus should not
account for any signalling events specifically attributable to the
CD8/Z chimera.
Non-covalent association of the chimera with endogenous
CD3 gamma {y), delta (b), and epsilon (e) may complicate the
interpretation of signals traneduced by the chimera. To determine
whether removal of the extracellular and tranemembrane domains of
is sufficient to result in its expression independent of the CD3
chains, cells were surface iodinated arid lysed in digitonin, a
detergent known to preserve the integrity of the TCR complex.
Immunoprecipitates of the TCR in both Jurkat CD8 and the
TCR-expressing chimera-transfectant JCD8/(2, show identical
patterns characteristic of a CD3 (Leu 4) immunoprecipitate.
Though TCR-associated Z is not well iodinated, as its
extracellular domain contains no tyrosine residues for labelling,
Z immunoblots of CD3 immunoprecipitates confirm its presence under
such lysis conditions. A small quantity of labelled CD3 a is seen
in the Leu 4 immunoprecipitate of the TCR deficient cell despite
the fact that this same mAb failed to stain this cell. The small
amount of immunoprecipitated protein seen is likely due to
radiolabelling of internal CD3 a in a small number of
permeabilized or non-viable cells during the labelling procedure.
More importantly, no CD3 chains are detectable in
precipitates of the CD8/Z chimera in either TCR-positive or
TCR-negative cells, nor is any chimera apparent in the Leu 4
precipitate of JCDB/~ 2. Intentional overexposure of the
autoradiogram also fails to reveal TCR chains coprecipitating with
the chimeras.
To further address the question of co-association of the
chimera and TCR chains, the effect of antibody-induced down
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modulation of the TCR on chimera expression was assessed. Whereas
overnight incubation of JCDB/r2 with saturating amounts of C305,
a mAb against an epitope of the Jurkat T; l3 chain, resulted in
internalization of 94% of the TCR, surface expression of the CD8/r
chimera was unaffected. By these two independent criteria, no
discernible association exists between CD8/( and the CD3 y, b, and
a chains.
To determine whether a covalent link exists between
endogenous ( and the CD8/t chimera, r immunoblot analysis was
performed comparing ~ and OKT 8 immunoprecipitates in Jurkat CD8
and JCDB/Z2. The anti-r antiserum immunoprecipitates both the
chimera and r from JCDB/=2, but only endogenous ( from the Jurkat
CD8 control. In contrast to the anti-t antiserum, OKTB
immunoprecipitatea the chimera but not r in JCDB/Z2, while neither
species is detected in Jurkat CDB. Collectively, the results from
these experiments and those described above, argue against an
interaction between the chimera and endogenous T cell receptor
subunits.
stimulation of CD8/~ Results in Activation of the
Phosphatidylinositol and Tyrosine Rinase Pathways
To determine whether binding of the extracellular domain
of CD8/r would result in intracellular signalling events, the
ability of OKTB to elicit an increase in cytoplasmic free calcium
((Ca+~);) in chimera-transfected cells was examined. A fluorimetry
tracing obtained with JCDB/L2 on stimulation of its TCR with the
anti-T, 13 monoclonal antibody C305 was obtained. With the addition
of soluble OKTB) a substantial increase in calcium ((Ca+2];) is
seen, suggesting that the cytoplasmic domain of r is capable of
coupling to signalling machinery which results in the activation
of phospholipase C.
The ability of the chimera to transduce a signal in cells
lacking surface expression of the TCE chains was examined next.
Stimulation of the TCR-negative Jti-CD8/(14 with C305 results in
no detectable increase in [Ca+Z];; however, OKTS is still able to
elicit a strong calcium response. The lack of significant
increase in (Ca+Z]; with OKTS stimulation in Jurkat CD8
demonstrates that the ~ portion of the chimera is required for the
elicited (Ca+2); response.
Since the increase in [Ca'~]; which occurs with TCR
stimulation is attributed to increases in inositol phosphates, the
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ability of CD8/r to induce PIPi hydrolysis was tested by assessing
changes in total soluble inositol phosphates following stimulation
with OKT8. Stimulation of CD8/~ with OKTB resulted in the
generation of inositol phosphates in both .chimera-expressing
S cells. In contrast, no inositol phosphates were noted with
stimulation of the wild-type CD8 protein in Jurkat CDB.
Stimulation of TCR in Jurkat CD8 and CD8/~2 induced increases in
inoaitol phosphates, whereas in the TCR-deficient transfectant,
J13-CD8/r14, no such increase was observed upon TCR stimulation.
These results are consistent with the calcium fluorimetry data and
confirm the chimera's ability to activate phoapholipase C even in
the absence of endogenous cell surface TCR chains.
As stimulation of the T cell receptor activates a
tyrosine kinase pathway in addition to inositol phospholipid
pathway, it was important to determine whether chimera stimulation
would result in tyrosine kinase activation. Western blots reveal
a small number of tyrosine-phosphorylated proteins existing in a11
three clones prior to stimulation. Upon stimulation of Jurkat CD8
and JCDB/r2 with C305, (anti-T;f3), the tyrosine kinase pathway is
activated as demonstrated by the induction of tyrosine
phosphorylation of a number of proteins.
As expected, C305 has no effect in the TCR-negative
transfectant, J13-CD8/t14. Stimulation of the chimera on both
JCD8/r2 and J13-CD8/r14 with OKT8 results in the appearance of a
pattern of tyrosine-phosphorylated bands indistinguishable from
that seen with TCR stimulation. In contrast, stimulation through
wild-type CD8 in Jurkat does not result in induction of tyrosine
phoaphoproteins. Thus, the CDB/r chimera, in the absence of T; and
CD3 y, 8, and e, is capable of activating the tyrosine kinase
pathway in a manner analogous to that of an intact TCR.
Since JCD8/2 expresses two discernible forma of Z on its
surface, endogenous r and the CD8/r chimera, each of which could
be stimulated independently, the specificity of receptor-induced
phoephorylation was addressed.
Immunoprecipitates of r derived from the three clones,
either unstimulated, or stimulated with C305 or OKTB, were
analyzed by western blotting with an anti-phoaphotyrosine
antibody. A small fraction of the r immunoprecipitates were
blotted with ~ antiserum to control for differences in protein
content between samples. Analysis of the lysate derived from
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TCR-stimulated Jurkat CD8 cells reveals a typical pattern of
phosphorylation with the multiplicity of bands from 16-21 kD most
likely repreeenting the varying degree of phosphorylation of the
seven cytoplasmic tyrosine residues of C.
In this experiment, a small degree of constitutive r
phosphorylation is detected in Jurkat CDB; however, this is not
augmented by stimulation of the wild-type CD8 protein. Whereas
phosphorylation of r ie seen with stimulation of the TCR in
JCDB/r2 though weaker than that seen in C305-stimulated Jurkat
CDB, no induced phosphorylation of the chimera is apparent.
Conversely, stimulation of the CD8/Z chimeric receptor on both
JCDB/(2 and J!3-CD7/~14 results in a high degree of phosphorylation
of the chimera exclusively, seen as an induced broad band from
34-39 kD. This result indicates that the receptor-activated
kinase responsible for phosphorylation of r recognizes its
substrate only in a stimulated receptor complex.
Stiaulation of CD8/r Results in Fats Events of T Cell Activation
T cell activation results from the delivery of
receptor-mediated signals to the nucleus where they act to induce
expression of specific genes. One such gene encodes the
activation antigen CD69, whose surface expression is induced
within hours of T cell receptor stimulation and appears to be
dependent on activation of protein kinase C (Testi et al., J.
Immunol. 142:l854-l860). Although the function of CD69 in T cell
activation is not well understood, it provides a marker of distal
signal traneduction events.
Flow cytometry reveals a very small degree of basal CD69
expression on unstimulated cells. Maximal levels are induced on
a11 cells with phorbol rnyristate acetate, PMA, an activator of
protein kinase. Stimulation of the TCR results in induction of
CD69 on Jurkat CD8 and JCDB/~2, but not on the TCR-negative clone,
Jfi-CD8/r14. Moreover, stimulation of cells with OKTB induces CD69
on both cells expressing the CD8/( chimera. Though a minimal
degree of CD69 induction is apparent with stimulation of wild-type
CD8 protein, this level is no higher than that observed with
stimulation of Jurkat CD8 with a Class I MHC antibody w6/32.
Perhaps the most commonly used criterion to assess late
activation events is the production of the lymphokine,
interleukin-2 (IL-2) (Smith (1986) Science 240:1269-1176). The
IL-2 gene is tightly regulated, requiring the integration of a
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number of signals for its transcription, making- it a valuable
distal market for assessing signalling through the CD8/( chimera.
Stimulation of Jurkat CD8 and JCDB/r2 cells with TCR antibodies
in the presence of PMA results in production of IL-2.
JCDB/r2 and Jurkat CD8 cells were stimulated With the
indicated mAb or inomycin (1 um) in the presence of PMA
(10 ng/ml). IL-2 secretion was determined by the ability of
culture supernatants of stimulated cells to support the growth of
the IL-2~dependent CTLL-2.20 cells. Since PMA alone induces no
IL-2 production in Jurkat, yet has a small direct effect on the
viability of the CTLL 2.20 cells, values obtained with PMA alone
were subtracted from each response value) yielding the numbers
shown above Data from two independent experiments are presented.
Table - Induction of IL-2 Production
Treatment IL-2 (Units /ml1
Jurkat CD8 JCDB/t2
Experiment Experiment
#1 #2 #1 #2
Unetimulated <0.1 <0.1 <0.1 <0.1
C305 + PMA 13.5 9.1 3.7 2.1
OKTS + PMA <0.1 <0.1 6.8 7.0
C305+OKT8+pMA -- -- -- --
W6/32 + PMA <0.1 <0.1 <0.1 <0.1
Ionomycin+pMA 30.4 4.2 24.2 24.6
Importantly, while treatment with OKTS on Jurkat CD8
induces no IL-2, similar treatment of JCDB/2 results in levels of
secreted IL-2 consistently higher than those produced in that cell
with TCR stimulation. Jf3-CD8/~14 responded more weakly to a11
experimental stimuli in this assay, but the data were
qualitatively similar in that this cell reproducibly secreted IL-2
in response to OKT8 but not to C305.
The data confirm that in addition to early signal
transduction events, later activation events occur upon
stimulation of the CD8/r chimera, thus demonstrating its ability
to couple to the relevant signal transduction pathways in a
physiologic manner.
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EXAMPLE 2
CD4-Zeta Chisieric Receptor In Signal Transduction
Construction of CD4-:eta Chiaeras
Plasmid pGEM3zeta bears the human zeta cDNA and was
provided by Dr. R.D. Klausner and Dr. S.J. Frank (NIH, Bethesda,
MD). The plasmid p8S.L3T4 bears the human CD4 cDNA, and was
provided by Dr. D. Littman and Dr. N. Landau (University of
California San Francisco, CA). A BamHI-ApaI restriction fragment
(approximately 0.64 kb) encompassing the entire human zeta chain
coding sequence from residue 7 of the extracellular (EXT) domain,
was excised from pGEM3zeta, and subcloned into the eamHI and ApaI
restriction sites of the polylinker of pBluescript II SK (+) 9pSK
is a phagemid based cloning vector from Stratagene (San Diego,
CA), generating pSK.zeta. Subsequently, a BamHI restriction
fragment encompassing the entire CD4 coding sequence
(approximately 1.8 kb) was excised from pBS.L3T4, and subcloned
into the BamFiI site of pSK. zeta, generating pSK. CD4. zeta. See
U.S. Pat. No. 5,359,046.
Single-stranded DNA was prepared from pSK.CD4.zeta
(Stratagene pBluescript II protocol), and used as a template for
oiigonucleotide-mediated directional mutagenesis (2oller & Smith
(1982) Nucleic Acids Res. 10:6487-6500) to generate CD4-zeta
chimeras with the desired junctions described below. CD4-zeta
fusion: 1, 2, and 3 were subsequently sequenced via the Sanger
dideoxynucleotide technique (Sanger et al. (1977) Proc. Natl.
Acad. Sci. 74:5463-5467), excised as EcoRI-ApaI restriction
fragments and cloned into the polylinker of expression vector
pIK.l.l or pIK.l.l.Neo at identical sites.
An EcoRI-BamHI restriction fragment (approximately
1.8 kb) encompassing the entire coding region of CD4 was excised
from pSK.CD4.zeta, and subcloned between the EcoRI and BglII sites
of the plK.l.l or pIK.l.l.Neo polylinker.
The plasmid pUCRNeoG (Hudziak et al. (1982) Cell
31:l37-l46) carries the neomycin gene under the transcriptional
control of the Rous Sarcoma virus (RSV) 3' LTR. The RSV-neo
cassette was excised from PURCNeoG as a HincII restriction
fragment (app. 2.3 kb), and subcloned between the two SspI sites
of pIK.l.l, generating plK.l.l.Neo.
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pIK.l.l is a mammalian expression vector_ constructed by
four successive cassette insertions into pMF2, which was created
by inserting the synthetic polylinker 5'-HindIZI-SphI-EcoRI-AatII-
BglI-XhoI-3' into KpnI and SacI sites of pSKII .(Stratagene), with
loss of the KpnI and SacI sites. First, a BamHI-XbaI fragment
containing the SV40 T antigen polyadenylation site (nucleotides
2770-2533 of SV40, Reddy et al. (1978) Science 200:494-502) and
an NheI-SalI fragment containing the SV40 origin of replication
(nucleotides 5725-5578 of SV40) were inserted by three-part
ligation between the BglII and XhoI sites, with the loss of the
HglII, BamHI, XbaI, NheI, SalI and XhoI sites. The BamHI-XbaI and
NheI-SalI fragments were synthesized by PCR with pSV2Neo (Southern
& Berg (1982) J. Mol. Appl. Gen. l:327-34l) ae the template using
appropriate oligonucleotide primer pairs which incorporated BamHI,
Xbal, NheI and SalI sites at their respective ends.
Second, an SphI-EcoRI fragment containing the splice
acceptor of the human al globin gene second exon (nucleotides +l43
to +251) was inserted between the Sphl and EcoRI Bites. The
SphI-EcoRI fragment was synthesized by PCR with p~rSVaHP (Treisman
et al. (1983) Proc. Natl. Acad. Sci. 80:7428-7432y as the template
using appropriate oligonucleotide primer pairs, which incorporated
SphI and EcoRI sites at the respective ends. Third, the synthetic
polylinker 5'-EcoRI-BglII-Apal-AatII-3' was inserted between the
EcoRI and the AatII sites. Fourth, a HindIII-SacI fragment
containing the CMV IE enhancer/prompter (nucleotides -674 to -19,
Boshart et al. (19B5) Cell 41:521-530) and a SacI-SphI fragment
containing the CMV IE first exon/splice donor (nucleotides -19 to
+170 ) were inserted by three-part ligation between the HindIII and
SphI sites. The HindIII-SacI fragment was prepared by PCR with
pUCH.CMV (M. Calos, Stanford University, Palo Alto, CA) as the
template using appropriate oligonucleotide primers which
incorporated HindIII and SacI sites at the respective ends. The
SacI-Sphl fragment was chemically synthesized.
RESULTS
Design of CD4-seta Chireras
Three CD4-zeta chimeric receptors (F1, F2 and F3) were
constructed from the extracellular (EC) and cytoplasmic (CYT)
domains of CD4 and zeta respectively. The transmembrane (TM)
domains of the CD4-zeta receptors were derived from zeta (F1, F2y
or CD4 (F3). F2 and F3 possess a11 four V domains.
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Fl retains only the V1 and V2 of the CD4 EC domain
(residues 1-180 of the mature CD4 protein), the TM domain of zeta
(residues 8-30 of the mature zeta chain) and the CYT domain of
zeta (residues 31-142 of the mature zeta chain).
F2 retains the CD4 EC domain comprising a11 four V
regions (residues 1-370 of the mature CD4 protein), the TM domain
of the zeta chain (residues 8-30 of the mature zeta chain) and the
CYT domain of zeta (residues 31-142 of the mature zeta chain).
F3 retsina the CD4 EC domain comprising a11 four V
domains (residues 1-371 of the mature CD4 protein), the TM domain
of CD4 (residues 372-395 of the mature CD4 chain), and the CYT
domain of zeta (residues 31-142 of the mature zeta chain).
Transient Expression of CDd-zeta Recegtors
Chimeric receptors F1, F2, and F3, and the native CD4
gene were introduced into an expression vector pIK.l.l which
directs transcription via the CMV promoter/enhancer. To evaluate
the structural integrity and cell surface levels of expression of
the chimeric receptors, a highly efficient transient expression
system was employed. Constructs were introduced by
electroporation into the human embryonic kidney cell line, 293
(American Type Culture Collection, ATCC, Rockville, MD), cells
were harvested 24 hours later, and subsequently analyzed by FRCS
employing a FITC-coupled mAb specific for the Vl domain of CD4,
OKT4A. Although similarly high levels of surface F2 and F3 were
detected by OKT4A, the level of Fl detected by the antibody in the
same transient assay was extremely low.
To address whether F1 was present in the membrane, and to
assess the structure of the chimeric proteins, immunoprecipitation
of radiolabelled proteins was carried out. T~renty hours after
electroporation of 293 cells with either F1, F2 or F3, cells were
pulse-labelled with 35S-methionine for four hours, lysed in 1$
NP40, and subjected to immunoprecipitation by either OKT4A (Ortho
Pharmaceuticals, NJ) or a rabbit antiserum raised against a
cytoplasmic peptide of marine zeta (obtained from R. Klausner,
NIH, MD). The level of radiolabelled Fl relative to either F2 or
F3 was significantly higher when anti-zeta antiserum instead of
OKT4A was used as the immunoprecipitation agent. The results
suggest that the F1 receptor may not present the necessary
topology for efficient binding of Vl-specific mAb's.
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F1 and F2 Form Disulfide-Linked Homodimers; F3 is a Dlonomer
Native zeta exists as a disulfide-linked homodimer or as
a heterodimer in which the zeta chain is associated with an
alternatively spliced product of the same gene, Eta. F1 and F2
both possess the TM domain of zeta, and therefore should have the
potential to form a homodimer (and possibly a heterodimer with
native zeta) via the membrane proximal cysteine residue (position
11 of the mature zeta chain). In contrast, the tranemembrane
domain of F3 is derived from CD4, and would therefore be expected
to confer the native monomeric state of the native CD4 molecule
to the F3 receptor.
To determine whether the receptors do form covalent
linkages, immunoprecipitates of radiolabelled 293 cells which have
been electroporated with each of the constructs under evaluation,
were analyzed under reducing and non-reducing conditions. Under
both reducing conditions, a single protein of approximately 70 kb
was immunoprecipitated by OKT4A from 293 cells electroporated with
F3. As expected, CD4 also gave rise to a single protein of
approximately 60 kd under both reducing and non-reducing
conditions.
In contrast, F1 and F2 gave rise to proteins of
approximately 70 kd and 150 kd, respectively under non-reducing
conditions, approximately double that seen under reducing
conditions (approximately 34 kd and 70 kd respectively). The
results demonstrate that F1 and F2, like native zeta, exist as
disulfide-linked homodimers, whereas F3 exists as a monomer, as
does native CD4. The data do not rule out the ability of F3 to
form a noncovalently associated dimer.
Introduction of CD4-zeta Receptors into a Human T Cell Line
The chimeric receptor genes F1, F2, and F3, and the
native CD4 gene, were introduced into a derivative of pIK.l.l
bearing a selective marker, pIK.l.lNeo. Each construct was stably
introduced via electroporation into the human T cell leukemia
line, Jurkat, and independent Jurkat clones obtained by limiting
dilution and selection of G418. Cell surface expression of the
chimeric receptor was assessed by FACS analysis of Jurkat clones
employing FITC-coupled OKT4A.
Although native Jurkat cells express a low level of CD4
on the cell surface, transfectants expressing high levels of F2
or F3 were readily identified due to the significantly higher
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levels of fluorescence observed relative to untransfected cells.
Similarly, stable clones expressing high levels of CD4 were also
identified. In contrast) none of the clones isolated from cells
electroporated with the F1 receptor construct revealed levels of
OKT4A-specific fluorescence higher than that seen with native
Jurkat cells.
FRCS analysis of over 100 Jurkat clones, revealed that
the F3 receptor has the potential to be stably expressed in Jurkat
cells at significantly higher levels (up to 50-fold) than the F2
receptor.
Induction of CD69 Expression Upon Stimulation of Native and
Chiaeric Receptors
CD69 (Leu-23) is an early human activation antigen
present on T, B, and HK lymphocytes. CD69 ie detected on the cell
surface of T lymphocytes within 2 hours after stimulation of
CD3/TCR, reaching a maximal level by 18 to 24 hours. CD69 is
therefore the first detectable cell surface protein induced in
response to CD3/TCR-mediated signals, and represents a reliable
marker of T cell activation. The ability of the CD4-zeta chimeric
receptors to specifically mediate CD69 induction in the Jurkat T
cell line was investigated. Representative Jurkat clones
expressing either F2, F3, or CD4 were selected for functional
analysis.
Monoclonal antibodies specific for the Ti a/S or CD3
chains can mimic the effect of antigen and serve as agonista to
stimulate signal transduction and T cell activation events. Cells
were stimulated with immobilized mAb's specific for (a) the T;
chain Jurkat, (C305), (b) the CD3 a chain (OKT3)) and (c) the V1
domain of CD4 (OKT4A). W6/32 recognizes an invariant determinant
of human HLA class 1 antigens, and was used in some experiments
as negative control.
CD69 expression was assayed by FRCS analysis
approximately 18 hours post-stimulation) employing FITC-couples
anti-Leu 23 mAb. Unstimulated cells exhibited a very low level
of basal CD69 expression but upon stimulation with a
pharmacological activator of protein kinase C, phorbol myristate
acetate (PMA), maximal expression was induced. Stimulation of
native Ti with the C305 mAb, or native CD3 with the OKT3 mAb, also
resulted in induction to the CD69 marker. However, stimulation
by OKT4A gave rise to a high level of CD69 expression only for
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those tranefectants expressing a chimeric CD4-( receptor. Indeed,
for a number of transfectants, particularly F3-derived, the level
of CD69 induction observed upon stimulation was equal to that seen
with PMA.
Stimulation of wild-type CD4 with OKT4A resulted in
little or no induction of CD69, when assayed in a number Jurkat
CD4-transfectants. Similarly, treatment of tranefectants with the
class 1 antibody, w6/32, had no significant effect in this assay.
Furthermore, secretion of IL-2 upon stimulation with OKT4A has
been observed.
The results demonstrate that CD4 chimeric receptors
possessing the cytoplasmic tail of zeta function effectively in
initiation of T cell activation events. Specifically, chimeric
CD4-zeta receptors bearing the CD4 TM domain (F3) mediate T cell
activation more efficiently (with respect to CD69 induction) than
those bearing the zeta TM domain (F2), despite the fact that the
latter retains the homodimeric form of native zeta.
F3 differs from F2 and native zeta, in that it does not
exist in the form of a covalent homodimer. The data therefore
demonstrate that covalent dimerisation of the chimeric receptor
is not essential for initiation of T cell activation as measured
by CD69 induction.
EXAMPLE 3
Siagle Chain Antibody-Zeta Chiaeric Receptor
The chimeric receptor cc49-zeta is composed of several
subunits: an scFv consisting of the humanized VH and VL regions
from the cc49 murine antibody that binds the TAG-72 antigen,
linked to the gamma 1 hinge and CH3 domains of human IgG, the
human CD4 transmembrane domain, and the human CD3-zeta
intracellular domain (Figure 2). The VH and VL regions are joined
by an synthetic (Gly4 Ser)3 linker that has been used in several
scFv antibodies and is retained from the original humanized cc49
antibody. Of the Ig constant regions, only the CH3 domain was
retained in the cc49-zeta receptor to preclude the binding of
cc49-zeta positive T cells to FcR positive cells mediated by the
Ig CH2 domain. The cc49-zeta construct is depicted in the pRT43.2
retroviral vector (Figure 2).
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pRT43.2 is a derivative of the pIK vectors discussed
hereinabove and disclosed in W094/29438. The vectors carry MMLV
gag sequences to improve packaging and the XhoI-ClaI fragment of
pZIPneoSVX is deleted. The EcoRI-ApaI polylinker from pIKl.l was
inserted downstream of the splice acceptor to enable transfer of
inserts from pIK plasmide into retroviral constructs. The
resulting plaemid is called pRTDl.2 and contains both 5' and 3'
MMLV LTR's.
The 5' LTR U3 region of pZIPneoSVX (Cepko et al. (1985)
Cell 37: l053-l062 ) was replaced with the MMSV U3, derived from the
HindIII/SacI fragment of pIKMMSV, to generate pRTD4.2.
In pRTD2.2, the U3 region of the 5' LTR of pZIPneoSVX was
replaced with the HindIII/SacI fragment of pIKl.l encoding the CMV
early immediate enhancer/promoter, which was fused to the MMLV R
region by an oligo that encodes nucleotides 19 to +1 of the HCMV
promoter linked to nucleotides +1 to +32 of MMLV (Schinnick et al.
(1980) Nature 293:543-548).
pRTD2.2SVG was constructed by replacing the 750 by
SacI/BstEII fragment of pRTD2.2 with the 736 by SacI-BstEII
fragment of LXSN (Miller & Rosman {1989) Biotechniques 7:980-990).
pRTD2.2SSA was constructed by replacement of the 1441 by
SacI-EcoRI fragment of pRTD2.2 with the 1053 by SacI-EcoRI
fragment of LXSN.
pRTD2.2SVGE- was constructed by synthesis of an oligo
encoding bases 2878-2955 of pLXSN (GenBank Accession M28248)
which had been appended by addition of an ApaI site at the 5' end.
That was used to replace the ApaI-Nhel fragment of pRTD2.2SVG,
which contains the DNA sequence 3' of the polylinker and 5' of the
NheI site in the 3' LTR.
To permit replication of the plasmid in cells which
express the SV40 T antigen, the sequences between the 5' and 3'
LTR's of pRTD2.2 were cloned between the SacI and EcoRI sites of
pIRl.l to form pIKT2.2. pIKT2.2SVG was constructed by insertion
of a SacI-EcoRI fragment, which contains part of the HCMV promoter
at the 5' end and includes an additional 750 by downstream from
the 3' LTR, between the SacI and EcoRI sites of pIKl.l.
pIKT2.2SVGE-F3 was constructed by replacing the 182 by ApaI-Nhel
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fragment of pIKT2.2SVGF3 with the 80 by ApaI-NheI fragment from
pRTD2.2SVGE-F3.
pRT43.2F3 was derived from pIKT2.2SVGE-F3 by replacing
the EcoRI-ApaI polylinker downstream from the 3' LTR with a
synthetic oligo containing an AacI site. Also, the NdeI site at
the 3' end of the viral gag sequences was converted to an XhoI
site by oligo insertion.
pRT43.3PGKF3 was derived from pRT43.2F3 first by removal
of the 3' LTR and insertion of a 3' LTR in which the sequences
from PvuII to XbaI were deleted (MMLV, GenBank accession #J02255,
nucleotide numbers 7938-8l15). In addition the MMLV splice
acceptor region was replaced with a human phoephoglycerate kinase
gene promoter (GenBank Accession #M11958, nucleotides 2-516) which
was cloned into a polylinker with an XhoI site at the 5' end and
EcoRI site at the 3' end.
Construction of Retroviral Vector with pQR Enhancer Driven
cc49-seta with IgY2 CH2 ((ilya~ Mutated to Ala)
( pRT4 3 . 3 P(iICHuCC4 9 P'vg 2 3 7 a II~1T 1 )
A PGK enhancer driven cc49-g237a-zeta retroviral vector
(pRT43.3PGKHuCC49Fvg237aINT1) containing the VH and V~ regions of
the humanized single-chain cc49 linked to the human Igyl and IgY2
CH2 (Glyz,~ mutated to Ala), IgY2 CH3 and CD3 zeta domains was
obtained. A 1212 by Ncol-SmaI fragment containing the humanized
single-chain cc49 scFv was excised from the pTAHuCC49SCLgdCHl
vector, a PCR clone of the humanized cc49 scAb (provided by J.
Schlom) and ligated in two steps to the 7401 by XhoI-PmlI fragment
and the 559 by NcoI-Xhoi fragment from pRT43.3PGKF25g237a, another
PGK enhancer driven ecFv-8237a-zeta retroviral vector encoding the
scFv of an anti-HIV gp120m" antibody of 447D.
Construction of Retroviral Vector with P6K Eahancer Drives
cc49-seta with IgYl CH3 Dovain (pRT43.3PaRCC49dCH2)
A PGK enhancer driven cc49-8237a-zeta retroviral vector
(pRT43.3PGKCC49dCH2) containing the V" and VL regions of the
humanized single-chain cc49 linked to the human Igyl hinge and
IgylCH3 and zeta domains was created. pRT43.3PGKHuCC49FVg237aINT1
was digested with RsrII and NsiI to yield an 8243 by vector
fragment. A 279 by NsiI-NspI fragment containing the Y1 hinge and
Ig yl CH3 domain from pRT43.3PGKF15dCH2, a PGK enhancer driven
scFv-8237a-zeta retroviral vector encoding the scFv of an anti-HIV
gp4lW" antibody 98.7, was cloned into the larger fragment. The
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construct was completed by an oligonucleotide linker that had
RsrII and NspI ends.
Construction of Retroviral Vector with P(ig Enhancer Driven
cc49-zeta with Igyl CH3 Domain (pRT43.2LTRCC49dCH2)
An MMLV LTR enhancer driven cc49-8237a-zeta retroviral
vector (pRT43.2LTRCC49dCH2), containing the VH and Vi regions of
the humanized single-chain cc49 linked to the human Igyl hinge and
IgylCH3 and zeta domains was made. pRT43.3PGKCC49dCH2 was
digested with EcoRI and ApaI to yield an 1B34 by EcoRI-ApaI
fragment containing the cc49 ecFv which was ligated to a 6702 by
EcoRI-ApaI fragment containing the retroviral vector and MMLV LTR
enhancer sequences from pRT43.2F3, an MMLV LTR enhancer driven CD4
(V1,V2,V3,V4)-zeta retroviral vector.
Construction of Retroviral Vector with PGR Eahancer Driven
cc49-zets With Igy2 CH2 (Glya~ i~tutated to Ala)
(pRT43 . 3PaiCCC49g237 a )
A PGK enhancer driven cc49-8237a-zeta retroviral vector
(pRT43.3PGKg237aCH2), containing the V" and V~ regions of the
humanized single-chain cc49 linked to the human Igy2 CH2 (Glyv~
mutated to Ala), Igy2 CH3 and zeta was obtained.
PGKHuCC49Fvg237aINT1 was digested with RsrII and NsiI to yield an
8243 by vector fragment. Into that was cloned the gamma2 hinge
and the 8237a mutation as a 622 by Nail-Hinpl fragment from
pRT43.3PGKF25g237a. The construct was completed by an oligo
linker that had RsrII and HinpI ends.
Construction of Retroviral Vector with LTR Enhaacer Driven
cc49-seta with Igy2 CH2 ((~lyn., Dtutated to Ala) Igy2 CH3 and zeta
Dos~aia (pRT43.2LTRCC49g237a)
A PGK enhancer driven cc49-8237a-zeta retroviral vector
(pRT43.2LTRg237a), containing the VH and VL regions of the
humanized single-chain cc49 linked to the human Igy2 CH2 (Gly~7
mutated to Ala), Igy2, CH3 and zeta was constructed. The 6702 by
EcoRI to ApaI retroviral vector fragment from pRT43.2F3 was
ligated to the cc49 scFv containing 2l67 by EcoRI to ApaI fragment
from pRT43.3PGKCC49g237a in a two part ligation.
cc49-zeta Receptor Expression on T Lymphocytes following
Retroviral Transduction
Using the kat producer cell lines of Finer et al. (1994
Blood 83 p. 43-48) high levels of stable T cell expression of
cc49-zeta receptor were achieved; typically, greater than 30~ of
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human T cells tranaduced with retrovirua encoding. cc49-zeta in a
single exposure to virus.
T cell cc49-zeta expression was stable. Over the course
of 35 days of continuous culture (the length of the experiment was
that long), the expression of cc49-zeta did not decrease as,
determined by FRCS.
Cytolytic Activity of cc49-aata CD8+ Lymphocytes: TAG-72+''
Leuka~ia Cell Lines
Most of the cell lines examined to date are negative for
TAG-?2 expression. That result is consistent with the observation
that few gastrointestinal-derived tumor cell lines are TAG-72+ in
vitro and those that are express low heterogeneous levels of
TAG-72 (Hand et al. (1985) Canc. Res. 45:833-840). The levels of
TAG-72 expressed by cultured LS174T cells were several logs lower
than that observed when those cells were maintained in vivo in
nude mice and are lower than levels of anti-TAG-72 staining
observed with patient samples.
In a chromium release assay, cc49-zeta* CD8 T lymphocytes
(closed squares) were extremely potent killers of Jurkat cells
(Proc. Natl. Acad. Sci. (1991) 8S:2037-2041) with significant
target cell specific lysis observed at effector to target (E: T)
ratios as low as 0.3:1 (Figure 3, top left panel) . cc49-zeta+
human T lymphocytes, however, did not kill another human T cell
line CCRF-CEM (Cancer Res. (1967) 27:772-783) (Figure 3, top right
panel). Non-tranaduced donor effectora (closed triangles) were
used as a control for non-specific lysis.
Cytolytic Activity of cc49-zata CD8+ Lp~phocytes: TAO-72+
Oastrointestiaal Carcinosa Call Lines
The specific cytolytic capacity of cc49-zeta CD8+ T
lymphocytes was tested with a series of TAG-72 positive and
negative gastrointestinal tract-derived tumor cell lines. Among
the positive cell lines were NCI H508 (Cancer Res. (19B7)
47:67l0-6718) (cecum adenocarcinoma) (Figure 4, top left panel},
LS-174T (colon adenocarcinoma) (Figure 4, top center panel) and
LS-180 (Cancer Res. (1967) 45:833-840) (colon adenocarcinoma)
(Figure 4, top right panel). cc49-zeta* CD8* T lymphocytes (closed
circles) lysed a11 TAG-72 positive cell lines and the level of
cytolysis correlated with H508, LS-174T and LS-l80 target cell
TAG-72 expression levels (Table A). Non-transduced CD8+
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lymphocytes were included as a control for non-specific lysis
(closed squares).
specificity of cc49-zeta CD8+ Lysiphocyte Cytolytic Activity
Lysis of TAG-72+ LS-174T target cells was not observed
with either CD8 T lymphocytes expressing an irrelevant chimeric
scFv-zeta receptor, F25, directed against HIVgp120env or
non-transduced T cells. Furthermore, cc49-zeta CD8 T cells did
not lyre syngeneic T cells.
Also, cc49-zeta CD8' lymphocytes did not lyre any of the
TAG-72' gastrointestinal carcinoma cell lines tested. The TAG-72
negative cell lines included MIP (colon carcinoma), SNU-1 (Cant.
Res. (1987) 47:6710-6718) (gastric adenocarcinoma) and NCI H716
(cecum adenocarcinoma) (Cant. Res. (1987) 47:6710).
Cytolytic Activity of cc49-zeta CD8+ Lymphocytes: TACT-72+"
Noa-gastrointestinal Carcinoaa Cell Lines
As several of the potential clinical targets include
ovarian, breast and non-small cell carcinomas, the target
specificity of cc49-zeta CD8* T lymphocytes was tested with
several cell lines derived from such tumor types. The CTL targets
included, the TAG-72 positive KLE-B (endometrial adenocarcinoma)
(Richardson, Mass. Gen. Hosp., Boston, MA) and the TAG-72 negative
cell lines, BT20 (J. Natl. Cancer Inst. (195B) 21:1131-1147)
(breast carcinoma), A549 (J. Natl. Cancer Inst. (1973)
51:1417-1423) (lung carcinoma) and COLV-6 (breast carcinoma).
While KLE-B cells express heterogeneous levels of TAG-72
by FRCS, there was extensive lysis of KLE-B cells by cc49-zeta
CD8+ T lymphocytes. No significant lysis of TAG-72 negative
breast and lung carcinoma-derived cell lines by cc49-zeta CDS' T
lymphocytes was observed. Non-transduced ND1 donor lymphocytes
were included as a control for non-specific lysis.
In an overview of the lysis studies performed with
various target cell lines and cc49-zeta CD8+ human T cells, the
data set forth in Table A demonstrate the relative levels of
TAG-72 expression on the target cells and the level of target cell
lysis in a standard 4 hour chromium release assay with cc49-zeta
human T lymphocytes. The amount of target lysis is directly
proportional to the expression of TAG-72 by the target cell and
there was no statistically significant cc49-zeta CD8+ T cell lysis
of TAG-72 negative target cells.
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CELL LINE ORIGIN TAG-72 LYSIS BY CC49-r+
EXPRESSION HUMAN T CELLS
BY.
FRCS (@E:T OF 30:1j
JURKAT T cell line +++ 52%
KLE-B endometrial +.~+++ 42%*
adenocarcinoma
LS-174T colon +~++ 25%
adenocarcinoma
NCI H508 cecum +.r++ 22%
adenocarcinoma
LS-l80 colon -.i+ 15%
adenocarcinoma .
COLV-6 breast carcinomand 9%*
CCRF-CEM T acute -/+ 8%
lymphoblastic
leukemia
MIP colon carcinoma- 4%
SNU-1 gastric - 2%
adenocarcinoma
BT20 breast carcinoma-- 1%
A549 lung carcinoma - -1%
NCI H716 cecum nd -7%
adenocarcinoma
Table A *high non-specific lysis
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Cytolytic activity of cc49-seta CD8+ lymphocytes: Absence of
bystander target call lysis
Schlom reported that TAG-72 expression was down-regulated
following in vitro culture of TAG-72+ tumor. cell lines (Hand
et al. (1985) Canc. Res. 45:833-840). As Jurkat cells expressed
constitutive high levels of TAG-72 (similar to primary tumor
samples), Jurkat cells Were used in the subeequent studies to
assay the different of cc49-zeta human T lymphocytes activities.
While cc49-zeta CD8+ T lymphocytes mediated only minor
cytolysis of TAG-72 negative cell lines, in patients, TAG-72+
cancer cells will be adjacent to TAG-72' normal tissue. Therefore
an important measure of the specificity of cc49-zeta T cells is
the absence of lysis of TAG-72' targets when cultured with TAG-72'
targets.
A mixed culture assay of TAG-72+ and s~Cr-labeled TAG-72'
cells was set up to address whether lysis of the antigen positive
targets resulted in the non-specific lyeis of antigen negative
bystander cells. No increase in non-specific cc49-zeta T cell
mediated lysis was observed following coculture of increasing
numbers of unlabelled (cold) TAG-72+ Jurkat cells (closed circles)
in the presence of s~Cr-labeled TAG-72' Snu-1 gastric
adenocarcinoma cells (open circles) (Figure 5).
To determine whether the presence of TAG-72 negative
cells interfered with cc49-zeta T lymphocytes-mediated cytolysis
in vitro, a cell mixing experiment was performed to determine the
influence of increasing numbers of TAG-72 negative cells on the
cc49-zeta T lymphocytes mediated lysis of Crs'-labeled TAG-72
positive targets. The results of those experiments indicated that
there was no significant interference of lysis of TAG-72+ Jurkat
targets by TAG-72' Snu-1 gastric adenocarcinoma cells (compare
closed circles and closed triangles) (Figure 5).
Cytolytic Activity of cc49-seta CD4+ Lymphocytes: Against
TAG-72'"'- Cell Lines
In a 4-hour chromium release assay cc49-zeta CD4+ T
lymphocytes (with 15% contaminating CD8+ lymphocytes) lysed Jurkat
targets while nontranaduced CD4+ lymphocytes did not (Figure 6).
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EEA~~LE 4
Construction of CD4-CD3y, CD4-CD3b, and CD4-CD3e Chimeric
Receptors
Cloning of CD3 Chainss Gfass;a (y), Dalta (b), aad Epsilon (e):
cDNA sequences encompassing the tranamembrane and
cytoplaemic domains of the gamma, delta and epsilon chains were
isolated by standard PCR techniques from Jurkat cell RNA.
Coastructioa of Chimeric CD4-CD3E, -CD3b, and -CD3y Receptors
The PCR products obtained were digested with NarI and
ApaI) and the resulting NarI-ApaI restriction fragments (y=276 bp,
b=276 bp, e=305 bp) were eubcloned into the expression vector
pIKI.ICD4 (as described above) between unique NarI and ApaI sites.
Oligonucleotide-mediated deletion mutagenesis was used to generate
chimeric receptors with the following compositions:
1. CD4-CD3y
(i) CD4 extracellular and transmembrane domain (CD4
amino acids 1-395) and CD3y cytoplasmic domain (CD3Y amino acids
117-160);
(ii) CD4 extracellular domain (CD4 amino acids 1-370}
and CD3y transmembrane and cytoplasmic domains (CD3y amino acids
83-160).
2. CD4-CD3S
(i} CD4 extracellular and transmembrane domain (CD4
amino acids 1-395) and CD35 cytoplaemic domain (CD3b amino acids
107-150);
(ii) CD4 extracellular domain (CD4 amino acids 1-370)
and CD3b tranamembrane and cytoplasmic domains (CD3b amino acids
73-l50).
3. CD4-CD3e
(i) CD4 extracellular and transmembrane domain (CD4
amino acids 1-395) and CD3e cytoplasmic domain (CD3e amino acids
132-185);
(ii) CD4 extracellular domain (CD4 amino acids 1-370)
and CD3e transmembrane and cytoplasmic domains (CD3e amino acids
98-185).
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E~LE 5
Stea Cell Transductioa
By engineering, hematopoietic stem cells, a multi-lineage
cellular immune response can be mounted against the disease
target, such as, cancers expressing TAG-72. After transduction of
stem cells followed by bone marrow transplantation, the engineered
bone marrow stem cells will continually produce the effector cells
abrogating the need for ex vivo cell expansion. Because stem cells
are self-renewing, once transplanted, these cells can provide
lifetime immunologic surveillance with applications for chronic
diseases such ae malignancy.
Effector cells including T cells, neutrophils, natural
killer cells, mast cells, baeophils and macrophages are derived
from hematopoietic stem cells and utilize different molecular
mechanisms to recognize the targets. T cells recognize targets by
binding of the T cell receptor to a peptide in the groove of a MHC
molecule on an antigen presenting cell. In the previous examples,
it was shown that the chimeric receptors of the invention can
bypass the MHC-restricted T cell receptor in T cells. Other
cytotoxic cells of the immune system recognize targets through F
receptors. F~ receptors bind to the F~ portion of antibody
molecules which coat virally infected, fungally infected, or
paraaite infected cells. In addition, antibodies against tumor
antigens induce antibody dependent cellular cytotoxicity (ADCC)
against the tumor cell by cytotoxic cells harboring F~ receptors.
It was demonstrated that in addition to the capability of chimeric
receptors of the invention to by-pass the MHC-restricted T cell
receptor, they are also able to by-pass the Fc receptor and
redirect the cytoxicity of neutrophils derived from transduced
stem cells.
The transduction method used for introducing the chimeric
receptors into stem cells was essentially the same as described
in Finer et. al. (1994) Blood 83:43-50. On the day prior to the
transduction, 293 cells transfected with the thymidine kinase gene
were plated at 103 cells/well in a Corning 6-well glate. The
cells serve as transient viral producers. On the day of
transfection, CD34+ cells were isolated from low density
mononuclear human bone marrow cells using a CellPro LC34 affinity
column (CellPro, Bothell, WA). Recovered cells were plated out
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in Myelocult H5100 media (stem Cell Technologies Inc., Vancouver,
B.C.) containing 100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu
IL-6 and 10'i M hydrocortisone for a period of 48 hours for
"pre-stimulation".
The next day, the 293/TK cells were transfected as
described by Finer et. al., supra. The following day, the CD34+
cells were collected and reauapended in infection media consisting
of IMDM, 10% FBS, Glutamine, 100 ng/ml hu SCF, 50 ng/ml hu IL-3,
ng/ml hu IL-6 and 8 ~g/ml polybrene. 3-5 x 10s cells were
10 added in 2 ml total to each well of the tranafected 293 cells to
initiate the co-culture.
Forty-eight hours later the CD34+ cells were collected.
Briefly, the 2 mls of cell supernatant were removed and additional
adherent CD34+ cells were dislodged using an enzyme free/PBS based
cell dissociation buffer. Cells were then expanded and
differentiated in vitro in Myelocult medium with addition of
100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu IL-6, and 10 NM
Gancyclovir to inhibit 293 proliferation. The cells will not
survive under gancyclovir selection, due to carrying the thymidine
kinase gene.
At approximately day 10 after transfection, cells were
cultured in 10 ng/ml hu SCF and 2 ng/ml hu G-CSF. From day 14
onward, the cells were driven toward becoming neutrophila by
culture in 10 ng/ml G-CSF alone.
Cells were monitored via cytoapina and differentials to
ascertain the degree of differentiation and maturity of the
neutrophils. Between days 16-24, the cells can be used for testing
effector functions such as cytotoxicity, and ascertaining the
degree of transduction by FACS and PCR analysis.
The differentiated neutrophila express the CD15 antigen,
and the neutrophils derived from transduced stem cells also
express the human CD4 extracellular domain (derived from
CD4-zeta). In one experiment, approximately 18% of the neutrophila
were expressing CD4-zeta, and the correction was factored in the
calculation of effector:target ratio. The cytotoxicity of the
neutrophils was tested according to the following methods.
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Cptotoxicitp Assail
Raji target cells, expressing the envelope protein of HIV
(gp160)) were labeled with sodium "Cr chromate (Amersham,
Arlington Heights, IL), generally 50 ~eCi/106 cells for 2 hours.
The targets were then washed 3 times to remove loosely bound S'Cr,
and resuspended at 10' cells/ml in RPMI1640, 10% FHS, and
glutamine.
Modified CD34-derived neutrophils, expressing the
CD4-zeta chimeric receptor, were plated in triplicate and titrated
1:2 in a final volume of l00 ill. The E:T ratio is dependent on
the cell number available, but usually was in the range of
l00-200:1. A 100 gel portion (l0,000 cells) of the target cell
solution was added to each well. Plates were then spun for
2 minutes at 500 RPM and then allowed to incubate for 5 hours at
37~C and 5% COz. s'Cr released in the supernatant was counted using
a Y counter.
The percentage of cytotoxicity was calculated as: 100%
x EXP-SR/MR-SR, where EXP are the counts released in the presence
of effector cells, SR = those spontaneously released, and MR = the
maximal release achieved when targets are incubated and lysed with
a 1% Triton-X solution (Sigma, St. Louis, MO).
Cytotoxicity against Raji cells expressing the envelope
protein of HIV was observed. Eliciting no response are the same
transduced neutrophils against the parental Raji line not
expressing HIV envelope, and nontransduced neutrophils against the
envelope expressing Raji cells. The chimeric receptor-bearing
neutrophils specifically recognized and killed cells expressing
HIV envelope protein. The transduced cells do not recognize the
parental Raji cells not expressing HIV envelope, and nontransduced
neutrophils do not kill Raji cells expressing envelope. The data
demonstrate the feasibility of redirecting other cytotoxic cell
types derived from stem cells beeides T cells.
It is evident from the above results that one can provide
for activation of various signalling pathways in a host cell by
providing for expression of a chimeric protein, which may serve
as a surface membrane protein, where the extracellular domain is
associated with a ligand of interest, while the cytoplasmic
domain, which is not naturally associated with the extracellular
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domain) can provide for activation of a desired pathway. In that
manner, cells can be transformed so as to be used for specific
purposes, Where cells will be activated to a particular pathway
by an unnatural ligand. That can be exemplified by using CD4 as
the extracellular domain, where binding of an HIV protein can
result in activation of a T cell which can stimulate cytotoxic
activity to destroy infected cells. Similarly, other cells may
be modified, so as to be more effective in disease treatment, or
to immune effects and the like.
EZAMPLE 6
Human natural killer (NK) cells can be genetically
modified to express high levels of CD4( using retroviral
transduction. In addition, the CD4r chimeric receptor is
biochemically active, as cross-linking of CD4( on NK cells results
in tyrosine phosphorylation of CD4r and multiple cellular
proteins. The CD4t chimeric receptor is functionally active, and
can direct NK cells to specifically and efficiently lyre either
natural killer-resistant tumor cells expressing the relevant
ligand, gp120, or CD4+ T cells infected with HIV.
HK Cells
The human NK3.3 clone has been previously described in
Kornbluth et al. (1982) J. Immunol. 129:2831. Cells were
maintained in NK media: RPMI 1640 supplemented with 15% fetal
bovine serum, glutamine, penicillin, streptomycin and 15%
Lymphocult-T (Biotest, Denville, NJ). Cell density was maintained
at less than 1 x 106 cells/ml, and media were replaced every two
days.
Retroviral Transduction of HR cells with CD4r
Retroviral transduction of NK3.3 cells was carried out
employing the kat retroviral producer system previously described
for transduction of CD8+ T lymphocytes (Roberts et al. (1994)
Blood 84:2878 and Finer et al. {1994) Blood S3:43) with the
following modifications. 293 cells were plated at 1 x 106 cells
per plate in a 6-well plate with 2 ml of media per well {293-1),
and 48 hours later were transiently tranefected with 10 ug of
retroviral vector encoding CD4(, pRTD2.2F3 and 10 ug of packaging
plasmid. 24 hre post transfection, media were replaced with NK
media. 4 hrs later, 3 x 106 NK cells were added per transfected
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293-1 plate and co-cultivated in the presence of polybrene
(2 ug/ml). After a 24 hour cocultivation period, NK3.3 cells were
removed from the 293-1 plate, and subjected to a second round of
co-cultivation with freshly transfected 293 cells for an
additional 24 hrs. Transduced NK3.3 cells were then harvested and
allowed to recover for 24 to 48 hrs in NK media. Stable
expression of the CD4( chimeric receptor in transduced NK3.3 was
analyzed 15 days poet transduction by flow cytometry with
FITC-conjugated anti-CD4 mAb'a ae described below. CD4(+ NK cells
were subsequently purified by immunoaffinity anti-CD4 mAb-coated
flasks (Applied immune Sciences).
Antibodies
Anti-FcyRIII mAb 3G8 was from Medarex (West Lebanon, NH);
anti-CD4 mAb OKT4A was from Ortho Diagnostic Systems (Raritan,
NJ); sheep affinity purified F(ab')2 fragments to mouse IgG;
biotin-conjugated F(ab')z fragment goat anti-mouse IgG were from
Cappel (Durham, NC); anti-phoaphotyroeine antibody 4G10 was from
Upstate Biotechnology (Lake Placid, NY); anti-Z rabbit anti-serum,
g387, raised against a peptide comprising amino acids 132-144 of
the human ( sequence, was kindly provided by Dr. L. E. Samelson
(NIH); FITC conjugated-antibodies, Gammal, anti-CD16 (-FcyRIII),
and anti-CD4 OKT4A mAb'a were obtained from Becton-Dickinson (San
Jose, CA). Rabbit anti-human lymphocyte serum Was from Accurate
Chemical and Scientific Corp. (Westbury, NY). Anti-gp120 mAb was
from Dupont/NEN Research Products (Wilmington, DE);
allophycocyanin streptavidin was from Molecular Probes, (Eugene,
OR). MOPC 21 (IgG,), used as a control mAb in three colored FRCS
analysis, and goat serum were from Sigma (St. Louis, Mo).
Anti-human class II (HLA-DP) mAb was from Becton Dickinson (San
Jose, CAy. Sheep anti-mouse Ig peroxidase, donkey anti-rabbit Ig
peroxidase, and the ECL Western blotting system were from Amersham
(Arlington Heights, IL).
HR Cell Stisulation and Immunoprecipitation
NK3.3 and CD4~+ NK3.3 cells were fasted in RPMI 1640
containing 1 mg/ml BSA for 2-3 hra prior to stimulation. Cells
were then spun down and resuapended in the same medium at a
density of 2 x 10' cells/ml. The cell suspensions were incubated
with mAb to FcyRIIIA (3G8) or CD4 (OKT4A) for 15 minutes at 4~C,
and then washed to remove unbound antibody. Sheep affinity
purified F(ab')2 fragments to mouse IgG were then added at 37~C for
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3 minutes in order to cross-link FcYRIIIA or CD4~. For
immunaprecipitationa, cells were lysed at 2 x 10' cells/200 ml of
1% NP-40, 150 mM NaCl, and 10 mM Tris (pH 7.8) in the presence of
protease inhibitors (1 mM PMSF, aprotinin, leupeptin), and
phosphatase inhibitors (0.4 mM EDTA, NaHC03, 10 mM Na4PzOT~lOHzO) .
After 30 minutes at 4~C) lyeates were centrifuged for 10 minutes
at 14,000 rpm, and pre-cleared with protein A Sepharoae beads. The
pre-cleared lysatea were then incubated with the
immunoprecipitating anti-r serum at 4~C for 30 minutes, followed
by protein A Sepharose beads at 4~C overnight. Washed
immunoprecipitatea were then subjected to SDS-PAGE under reducing
conditions.
Immunoblot Analysis
Separated proteins were transferred to nitrocellulose
membranes. Membranes were subsequently incubated with the
primary antibody (anti-phosphotyroaine or anti-~ antiserum).
Bound antibody was detected With horseradish peroxidase-conjugated
sheep antibody to mouse or rabbit IgG , followed by a non-isotopic
enhanced chemiluminescence ECL assay (Amersham).
Flow Cytometry
Approximately 1 x 106 cells per condition were washed once
with PBS plus 2% FCS, then incubated with saturating
concentrations of fluoreacein isothiocyanate (FITC)-conjugated
OKT4A for detection of CD4Z expression, or anti-CD16 for detection
of FcyRIIIA expression. FITC-conjugated isotype-matched
antibodies served as negative controls. Cells were then analyzed
in a FACScan cytometer (Becton Dickinson, CA). HIV-gp120
expression was analyzed by staining with mouse anti-gp120 mAb or
isotype negative control, followed by incubation with goat
ant i-mouse biotin F ( ab~ ) Z, followed by al lophycocyanin-streptavidin
prior to analysis. Allophycocyanin-stained cells were then
analyzed using a Becton Dickinson Facstar Plus.
Cytotoxic assays
Cytotoxicity was determined using a standard 4 hr
chromium-51 ( s~Cr ) release assay ( Matzinger ( 1991 ) Immunol . Methods
145:185) with the following modifications. 1x106 target cells
(Raji or Raji-gp120} were incubated with 50 pCi of S~Cr in 50 girl
of media for 2 hrs at 37~C. Labeled target cells were then plated
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into 96-well plates (1 x 10~ cells per well) together with
unmodified or CD4(+ NK3.3 cells at the target:effector ratios
indicated, and incubated at 37~C for 4 hrs. For control
experiments demonstrating CD16-mediated ADCC, effector cells were
pre-incubated with a saturating concentration (l/16 dilution) of
rabbit anti-human lymphocyte serum for 30 minutes at 4~C prior to
addition of target cells. At the end of the 4 hour incubation
period, plates were spun at 600 rpm for 2 min. About 100 ul of
supernatant were removed from each well and counted in a gamma
counter for the assessment of "Cr release. Percentage specific
lysis was calculated from triplicate samples using the following
formula: [(CPM-SR)/(MR-SR)] x 100. CPM = cpm released by targets
incubated with effector cells, MR = cpm released by targets lyeed
with 100 N1 of 1% triton x-l00 (i.e., maximum release), SR = cpm
released by targets incubated with medium only (i.e. spontaneous).
The CEM.NXR human T cell line is described in Byrn et al.
(l990) Nature 344:667. When uninfected or HIV-1 IIIB infected
CEM.NKR T cells were employed as target cells, the JAM teat was
employed for measuring cell lysis (Matzinger 1991), and is based
on the amount of (3H]thymidine labeled DNA retained by living
cells. In brief, 1 x I06 actively proliferating target cells were
labeled with 20 uCi ['H ] thymidine overnight . [ 3H ] thymidine-labeled
target cells were plated into 96 well plates (1 x 10~ cells per
well) together with unmodified or CD4r-expressing NK3.3 cells at
the effector:target ratios (E: T) ratios indicated. After a 6 hour
incubation period, cells were harvested and processed. Percentage
specific lyais was calculated from triplicate samples using the
following formula: ((S - E)/S] x 100. E - experimentally
retained DNA in the presence of CD8' effector T cells (in cpm),
S - retained DNA in the absence of CD8+ effector T cells
(spontaneous).
Raji Traasfectaats Expressing gp120
Raji is a human B cell lymphoma which expresses high
levels of class II MHC. Raji cells expressing low levels of HIV
env were generated by co-transfection with the expression vector,
pCMVenv, which encodes rev and env (gp160) from the HX82 HIV-1
clone and the selection plasmid, pIKl.lneo which confers
resistance to G418 (Roberts et al., 1994). G418-resistant clones
were isolated and analyzed for expression of the env proteins
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gp120 and gp160 by immunoblotting with an anti-gp120 mAb. Raji
clones positive by immunoblotting were then subjected FRCS
analysis to detect surface expression of gp120.
Efficient Surface Expression of CD4t in Retrovirally Traasduced
l~iic Cells
The NK cell line 3.3 waa originally isolated from human
peripheral blood mononuclear cells (PBL). NK3.3 exhibits an NK
characteristic cell surface phenotype (CD3', CD16+), and mediates
strong natural killer activity. The CD4Z chimeric receptor was
introduced into NK3.3 cells by retroviral mediated transduction
using the kat packaging system (Finer et al., 1994). After
transduction, 26% of the transduced NK population expressed CD4~
as detected by immunofluorescence of surface CD4. A population
in which greater than 85% of the cells expressed high levels of
chimeric receptor was obtained after immunoaffinity purification
of transduced NK cells with anti-CD4 mAb's. It was noted that
nmodified and CD4r-modified NK3.3 cells express comparable levels
of FcYRIIIA.
Tyrosine Phosphorylatioa Induced by CD4r Cross-linking on NR Cells
Several studies have shown that cross-linking of FcYRIIIA
on NK cells induces the tyrosine phosphorylation of the Z chain
(O'Shea et al. (199l) Proc. Natl. Acad. Sci. USA 88:350 and Vivier
et al. (199l) J. Immunol. 146:206), as well ae several additional
cellular proteins (Liao et al. (1993) J. Immunol. 150:2668, Ting
et al. (1992) J. Exp. Med. I76:1751; Azzoni et al. (l992} J. Exp.
Med. 176:1745 and Salcedo et al. (1993) J. Exp. Med. 177:1475}.
To evaluate the biochemical activity of the transduced
chimeric receptor as compared to FcyRIIIA in NK cells,
crosslinking of either receptor was achieved by incubating
unmodified (NK) or CD4r-modified NK3.3 cells (CD4(' NK) with
either OKT4A mAb to CD4 or 3G8 mAb to FcYRIIIA followed by sheep
F ( ab~ ) Z antibodies to mouse IgG.
Both CD4r and native t were immunoprecipitated from the
cell populations by treating cell lysates with anti-~ serum, and
the immunoprecipitated supernatants were subsequently analyzed on
immunoblots with an anti-phosphotyroaine antibody (4G10). Tyrosine
phosphorylation of CD4~, but not native Z, is rapidly induced by
croeslinking of the chimeric Z-receptor on NK cells. That result
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is consistent with previous studies conducted in T lymphocytes
which have shown that cross-linking of chimeric r-receptors
induces phoaphorylation of the chimeric receptor, but not of
native ( present in T cell receptor (TCR)/CD3 complexes. As
expected, cross-linking of FcyRIIIA induces rapid tyrosine
phoaphorylation of native r only, in both unmodified and
CD4(-modified NK3.3 cells.
FcyRIIIA is thought to mediate cellular activation
through a tyrosine-kinase dependent pathway, as indicated by the
results of previous studies demonstrating rapid tyrosine
phosphorylation of cellular proteins upon crosslinking of FcyRIIIA
(Laio et al., l993; Ting et al.) 1992; Azzoni et al., 1992; and
Salcedo et al., 1993). Rapid tyrosine phoaphorylation of cellular
proteins with molecular masses of approximately 136, 112, 97, and
32 kDa is induced upon cross-linking of either FcyRIIIA or CD4t
receptors on CD4(/NK cells. The sizes of the proteins are similar
to those previously reported as undergoing phoaphorylation upon
cross-linking of FcyRIIIA (Liao et al., l993 and Ting et al.,
1992).
Similar results were observed for unmodified NK3.3 cells
on cross-linking with mAb to FcyRIIIA, but not to CD4. Functional
and physical interaction between the ~ subunit and protein kinasea
such as ZAP-70 and the arc-related tyrosine kinase p56'''' is
supported by observations in T cells (Karnitz et al. (1992) Mol.
Cell Biol. 12:4521; Chan et al. (1992) Cell 71:649 and Wang et al.
(1992) J. Biol. Chem. 267:1685). For NK cells, similar functional
associations between p56''t and FcyRIII have been shown to be
mediated through direct interaction with t (Azzoni et al., l992
and Salcedo et al., 1993y, this subunit also acting as a substrate
for p56k'~ in vitro.
The studies described above show that the CD4~ chimeric
receptor is able to activate the tyrosine kinase signaling pathway
in a manner analogous to the FcyRIIIA/r complex in NK cells,
presumably due to retention of functional interactions between
such protein kinasea and the ( moiety of the chimeric receptor.
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CD4Z + NR Cells Mediate Cytolysis against Natural Killer-resistaat
Tusior Cells
The ability of CD4~ to confer NK cells with the ability
to kill a NK-resistant tumor cell line expressing low levels of
gp120 was evaluated to assess the anti-tumor potential of NK cells
expressing chimeric (-receptors. Target cell lines expressing
gp120 were generated from the NK-resistant human Burkitt lymphoma
cell line Raji by co-electroporation of pIKneo and pCMVenv.
G418-resistant clones were subsequently isolated and analyzed for
stable expression of the HIV env proteins gp120 and gp160 by
western immunoblotting. To detect surface expression of gp120,
it was necessary to employ a highly sensitive
allophycocyanin-streptavidin staining procedure with anti-gp120
mAb.
Unmodified and CD4r-modified NK cells were functionally
evaluated in a cytotoxicity assay against either normal Raji cells
or Raji-gp120 cells as targets, over a range of effector:target
ratios. To compare the efficiency of chimeric receptor-mediated
cytolytic activity with that of FcyRIIIA-mediated ADCC, CD4r+ NK
cells were also tested for their ability to lyre normal Raji cells
in the presence of rabbit anti-human lymphocyte serum.
The results of the studies show that whereas unmodified
NK cells exhibit little or no activity toward Raji-gp120 targets,
NK cells expressing CD4~ exhibit maximal specific lysis as high
as 50% over background levels at effector:target ratios of between
25:1 to 50:1. The specific lysis observed is highly sensitive,
with values of approximately 20% above background observed at
effector:target ratios as low as 0.4:1. Furthermore, the
efficiency of CD4~-mediated cytolysis appears to be more efficient
than FcyRIIIA-mediated ADCC, at a11 effector to target ratios
tested.
It was reported that both CD4t and scAb~ chimeric
receptors efficiently redirect primary human CD8+ T lymphocytes
to target HIV-infected cells (Roberte et al., 1994). It was
therefore of interest to compare the cytoiytic activity of CD4(+
NK cells to that of human PBMC-derived CD8+ T cells expressing
CD4( (CD4Z + CD8+ T cells) against the same Raji-gp120 target
cell line. The highly efficient cytolytic activity observed for
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CD4C + NK cells is comparable to that observed for CD4( + CD8+ T
cells.
CD4Z + NR Calls Mediate Cptolpsis agaiast HIV-infected T Cells
CD4r + NK cells can mount an efficient cytolytic response
against HIV-infected CD4' T cells. The NK-resistant CD4+ T cell
line CEM.NKR was infected by HIV-1 IIIH as previously described
(Byrn et al. (1990) Nature 344:667). When uninfected (CEM) or HIV
infected CEM-NKR cells (CEM/IIIB) were used as targets in a
cytotoxicity assay with unmodified or CD4r-modified NK cells as
effectors, specific lysis of the virally infected population was
observed at effector:target ratios as low as 1.5:1, with maximal
lysia as high as 70% above background occurring at effector:target
ratios of 50:1.
Since CD4 binds to non-polymorphic sites on MHC Class II
molecules, one concern with the use of CD4r as a chimeric receptor
for re-directing NK-mediated cytotoxicity toward HIV-infected
cells is the potential for lysis of cells expressing class II.
However, despite the fact that Raji cells express high levels of
class II MHC, no significant increase in cytolytic activity is
observed against Raji cells when NK cells expressing CD4( are
employed, even at effector:target ratios as high as 50:1. The
result is consistent with the notion that the relative affinity
of the CD4 receptor for MHC class II molecules is inadequate to
induce efficient cross-linking of the chimeric receptor, CD4r.
Chimeric Z-receptors in which the CD4 ligand binding
domain is fused to the cytoplaemic domain of the signal
transducing subunit ( of FcyRIIIA and of TCR, are expressed at
high levels on the surface of NK cells on retroviral-mediated
transduction. Furthermore, the CD4( chimeric receptor can direct
NK cells to initiate a highly effective cytolytic response against
natural killer-resistant tumor cells expressing low levels of the
relevant target ligand gp120, and against natural killer-resistant
T cells infected with HIV. The cytolytic response is highly
sensitive, and appears comparable to that previously observed for
CD4t + and scAb~ + CD8+ T lymphocytes.
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E%AMPLE 7
A humanized cc49 antibody (Shu et al. (1993) Proc. Natl.
Acad. Sci. 90:7995-7999 and Kashmiri et al. (1995) Hybridoma
14:46l-473) Was used to prepare a chimeric receptor. A acAb
consisting of the VH and V~ regions of the mAb joined through a
(G4 S)3 linker was used. A 788 by fragment obtained by NcoI and
RsrII digestion of plasmid pTAHUCC49SC1dgCH1 provided by Kashmiri
was ligated to the gl hinge and CH3 domain of human IgG, (residues
221-230 and 341-444) using an oligo linker flanked by RsrII and
NspI sites. The extracellular portion (ecAb) was attached to the
CD3~ intracellular region (residues 31-142 of t) through the human
membrane associated IgG, Ml transmembrane spanning region and
human CD4 transmembrane spanning region (residues 372-395 of CD4)
to yield the construct encoding the chimeric receptor.
The cc49-r construct was cloned in the retroviral vector
rkat43.2 (Finer et al., 1994). Virus was generated by transient
CaPO) transfection of 293 cells (ROberts et al., 1994), with the
modification of using two helper plasmids, encoding gag/pol and
env, respectively, to reduce the likelihood of generating
competent virus through recombination.
The LS174T, KLE-B, CCRF-CEM and MIP-1 cell lines were
obtained from J. Schlom of the NIH. The LS180, Snu-l, Jurkat,
NCI H716 and H508 cell lines were obtained from the ATCC.
Human T cells obtained from PBMC of buffy coats were
diluted 1:4 with Ca/Mg-free PBS. The cells obtained from a ficoll
separation were suspended in a T cell medium (TCM; ouch as, 1:1
AIM-V and RPMI with HEPES, sodium pyruvate, glutamine, pen-strep
and 10% human serum). Monocytes were removed by stationary
culture in a flask.
The non-adherent cells were exposed to anti-CD3 and
anti-CD28 antibody-coated Dynal beads in TCM. Following culture
for about 48 hours the beads were removed. The stimulated cells
then were exposed to IL-2 for about 24 hours. The cells were
resuspended in IL-2-containing medium and then mixed with
retrovirus containing the cc49-( construct and polybrene
(l00 IU/ml and 2 pg/ml final concentrations). After 24 hours,
half of the medium was removed and replaced with fresh
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virus-containing medium. That step was again repeated 24 hours
later. On day 6, the cells were removed from the transduction mix
and resuspended in TCM containing IL-2.
Cytokine expression was determined ae described herein
and using known methods. Along with IL-2, expression of IL-4,
TNF-a, IFN-Y and GM-CSF was determined using commercially
available reagents (for example, R & D Systems).
Soluble TAG-72 inhibition assays were conducted using a
2x concentration of sTAG-72 obtained from bovine submaxillary
mucin (Sigma). Cells were exposed to the carbohydrate and then
tested for cytotoxic activity as described herein.
For in vivo studies, 4-6 week old SCID-NOD mice were
injected with 1-3 x 108 cc49-Z expressing or normal T cells either
iv with 106 KLE-B cells or sc with l06 LS174T or KLE-B cells. Mice
were monitored daily for development of sc tumors or sacrificed
at various time points and examined for internal tumors.
FACS analysis using the appropriate anti-cc49 antibody
revealed that both CD4 and CD8 cells expressed the chimeric
receptor. Receptor expression was stable for at least 35 days
without an observable lose of expression. Western analysis using
an anti-r antibody indicated that the levels of chimeric receptor
expression were similar to that of the T cell receptor, that is,
about 10~ molecules per cell.
TAG-72+ cell lines, LS174T, LS180, NCI H508, Jurkat and
KLE-B were killed by the transduced cells, whereas TAS-72- cells,
MIP-1, NCI H716, CCRF-CEM and Snu-1 cells, were not lysed. KLE-B
and LS174T are positive for FAS and FASL and yet are efficiently
killed by the construct-expressing cells. The transduced cells
also killed primary TAG-72+ tumor cells obtained from patients
with advanced colon carcinoma. Labelled TAG-72' cells were not
lysed by the transduced cells.
When the transduced cells were cultured in vitro.
Stimulated cells expressed substantial levels of GM-CSF, IFN-y and
TNF-a.
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In vivo, the traneduced T cells Were immunoprotective.
LS174T, a human colon cancer cell line, introduced subcutaneously
into SCID-NOD mites produce a local palpable tumor in 2-4 weeks.
The tumors are large and encapsulated. Injection with tranaduced
cells however, prevented the development of tumors over a period
of at least 18 weeks.
The human endometrial carcinoma-derived KLE-B cell line,
when injected intraperitoneally, results in the development of
multiple tumors in the peritoneal cavity associated with the
intestine, liver, spleen, kidney and the site of injection.
Injection of cc49-Z T cells prevented the development of tumors.
As noted hereinabove, CD4+ cells transduced with a cc49
construct lysed suitable targets. CD4+ T cells transduced with
the cc49-( chimeric receptor construct lysed not only Jurkat cells
but also LS174T cells and KLE-B cells, but did not lyse the
TAG-72' cells, H716 and H508.
The transduced cells lyre targets in the same fashion as
found in normal cytotoxic T cells, that is, via perforin. When
various inhibitors of the T cell lytic pathway were tested, it was
noted that inhibitors of the perforin-mediated pathway of lysis
inhibited killing of antigen positive targets. Thus, anti-FASL
and anti-TNFa antibodies had a minimal impact on the killing
ability of transduced cells. On the other hand, concanamycin A
resulted in almost a complete blockage of killing.
When the transduced cells carrying the cc49-r construct
are stimulated with CD3 and CD28, the T cells proliferate. When
the cells are co-stimulated and exposed to anti-idiotype antibody
or a TAG-72+ target, the T cells do not proliferate or produce
substantial amounts of cytokines. The co-stimulation mechanism
might impose another mechanism on the traneduction of signalling.
All publications and patent applications mentioned in the
specification are herein incorporated by reference to the same
extent ae if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
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The invention now being fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the
spirit or scope of the appended claims.
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