Note: Descriptions are shown in the official language in which they were submitted.
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
BISPECIFIC FUSION PROTEIN AND METHOD OF USE FOR
ENHANCING EFFECTOR CELL KILLING OF TARGET CELLS
FIELD OF THE INVENTION
This invention relates to a fusion protein comprising all or a biologically
active
portion of an interferon alpha (IFN-a) linked to an immunoglobulin protein or
polypeptide fragment thereof, which recognizes a cell surface protein
expressed by a
malignant cell. The fusion protein, when bound to a malignant cell, or a
target cell,
would bind via IFN-a to the IFN-a receptor expressed on an effector cell
(e.g., natural
killer (NK) cells, polymorphonuclear (PMNs) cells and macrophages/monocytes).
The binding of IFN-a fusion protein to its receptor on effector cells enhances
and
potentiates extracellular (e.g., antibody-dependent cell-mediated cytotoxicity
or
ADCC-type), intracellular (phagocytic) and/or direct killing of the bound
target cell.
BACKGROUND OF THE INVENTION
The cytokines, interleukin-2 (IL-2) and IFN-a, are potent activators of
natural
killer (NK) cells and other anti-tumor effector cells, but results obtained in
clinical
trials with these cytokines have proved disappointing in many forms of cancer.
The
ineffectiveness of IL-2 and IFN-a may be because intratumoral monocytes/
macrophages (MO) inhibit the cytokine-induced activation of cytotoxic effector
lymphocytes, such as NK cells, at the site of tumor growth (Hellstrand et al.,
Acta
O~col. 37: 347-53 (1998)). Nevertheless, IFN-a can regulate NIA cells and
lymphokine-activated killer (LAK) cells and can act in synergy with IL-2 to
augment
NK activity (Chikkala et al., Cancer Res. 50: 1176-82 (1990)). For example,
systemically administered IFN-a has exhibited a limited anti-tumor effect in
lymphomas, leukemia, I~aposi's sarcoma, renal cell carcinoma, melanoma,
multiple
melanoma, glioma and ovarian cancer. See Sell et al., IMMUN~LOGY,
ilVllVIUIVQPATHOLOGY & I~nu~'rY 951-2 (1996). However, systemic treatment with
either or both IFN-a or IL-2 can cause less important severe life-threatening
toxicity,
which limits their administration at higher doses leading to reduced efficacy
and
discourages their therapeutic use (Meseri-Delwail et al., Biotechrr.ol. Tlaer.
5: 47-57
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
(1994); and Sosman et al., Senain. Oncol. 17: 22-30, 38-41 (1990)). Use of IFN-
a is
further complicated as multiple species of IFN-a exist that differ
dramatically in
activity, which is explained in part by different binding affinities (Webb et
al., Cell
Inamunol. 124: 158-7 (1989); and U.S. Patent 4,780,530 (1988)). Furthermore,
systemic activation of NK cells and monocytes is less effective than
activation at the
tumor site since activated cells may not home to the tumor. This is
particularly
applicable to solid tumors including carcinomas.
Antibody dependent cellular cytotoxicity (ADCC) is viewed as an important
mechanism by which monoclonal antibodies (mAb) can exert an antitumor effect
in
vivo. ADCC can be enhanced by the cytokines IL-2 and IFN-a (Vuist et al.,
CanceY
Immunol. Immunothef°. 36: 163-70 (1993)).
Monoclonal antibodies have been designed to target specific cells or antigens.
Monoclonal antibodies therefore, have been designed to act as vectors or
delivery
vehicles for targeting foreign antigens to cells. See for general description
EP Patent
No. 553,244 (1993).
A~ati-CD19 A~atibodies. Antibodies have been raised which recognize CD19, a
signal transduction molecule restricted to the B-cell lineage. Examples of
monoclonal
anti-CD19 antibodies include anti-B4 (Goulet et al., Blood 90: 2364-75
(1997)), B43
and B43 single-chain Fv (FVS 191; Li et al., Cancer Immunol. Immunother. 47:
121-
130 (1998)). Myers et al. (Leak. L~naphoma 29: 329-38 (1998) reported
conjugating
the marine monoclonal B43 to the tyrosine kinase inhibitor (see also U.S.
Patent No.
5,872,459), genistein, to produce an immunoconjugate against CD19 antigen
positive
hematologic malignancies. Treon et al., SenZin, Oncol. 26/5 Supp: 97-106
(1999)
reported conjugation of B4 to a blocked ricin, which had no significant
activity in
patients with multiple myeloma.
Rituxirnab ahd otlaer~ A~zti-CD20 Antibodies. The FDA approved anti-CD20
antibody, Rituximab (IDEC C2B8; RITUXANO; ATCC No. HB 11388) has also been
used to treat humans. Ibritumomab, is the marine counterpart to Rituximab
(Wiseman
et al., Clin. Cafacer Res. 5: 3281s-6s (1999)). Other reported anti-CD20
antibodies
include the anti-human CD20 mAb 1F5 (Shah et al., J. Imnaunol. 162: 6589-95
(1999)), the single chain Fv anti-CD20 mouse mAb 1H4 (Haisma et al., Blood 92:
-2-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
184-90 (1998)) and anti-B1 antibody (Liu et al., J. Clin. Oncol. 16: 3270-8
(1998)).
In the instance of 1H4, a fusion protein was created reportedly fusing 1H4
with the
human (3-glucuronidase for activation of the prodrug N-[4-doxorubicin-N-
carbonyl(-
oxymethyl)phenyl~ O-(3-glucuronyl carbamate to doxorubicin at the tumor cite
(Haisma et al., 1998).
Anti-CD22 Antibodies. CD22 is a cell surface antigen expressed on normal
human B cells and some neoplastic B cells. Several monoclonal anti-CD22
antibodies
have been created, including HD6, RFB4, W22-2, TolS, 4KB 128, a humanized anti-
CD22 antibody (hLL2), and a bispecific F(ab')Z antibody linked to saporin (Li
et al.,
Cell. Immunol. 111: 85-99 (1989); Mason et al., Blood 69: 836-40 (1987); Behr
et al.,
ClifZ. Cancer Res. 5: 3304s-14s (1999); and Bonardi et al., CafZCer Res. 53:
3015-21
(1993)). Immunotoxins comprising anti-CD22 linked to the ricin A chain have
also
been prepared, which reportedly possess antitumor effects in mice (Sausville
et al.,
Blood 85: 3457-65 (1995); and Ghetie et al., Cahcef° Res. 51: 5876-80
(1991)).
AsZti-CD33 Antibodies. CD33 is a glycoprotein expressed on early myeloid
progenitor and myeloid leukemic (e.g., acute myelogenous leukemia, AML) cells,
but
not on stem cells. An IgGI monoclonal antibody was prepared in mice (M195) and
also in a humanized form (HuM195), that reportedly has antibody-dependent
cellular
cytotoxicity (Kossman et al., Glif2. Cafzcef~ Res. 5: 2748-55 (1999)).
However, Caron
et al., Clira. Cancer Res. 1: 63-70 (1995) reported that HuM195 had only
modest
ADCC capability against HL60 cells. An anti-CD33 immunoconjugate (CMA-676)
consisting of a humanized anti-CD33 antibody linked to the antitumor
antibiotic
calicheamicin reportedly demonstrated selective ablation of malignant
hematopoiesis
in some AML patients (Sievers et al., Blood 93: 3678-84 (1999). Pagliaro et
al., Clin.
Caface~ Res. 4: 1971-6 (1998) described a HuM195-gelonin immunoconjugate,
comprising an anti-CD33 mAb conjugated .to the single-chain plant toxin
gelonin.
AiZti-CD38 A~ztibodies. CD38 is an antigen expressed during early stages of
differentiation in normal and leukemic myeloid cells, including myeloma cells.
Ellis
et al., J. ImmuT2ol. 155: 925-37 (1995) reported a high affinity mAb (AT13/5)
against
CD38 which efficiently directed antibody-dependent cellular cytotoxicity
(ADCC)
against CD38+ cell lines, but which activated complement poorly and did not
down-
-3-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
regulate CD38 expression. Flavell et al., HenZatol. Oncol. 13: 185-200 (1995)
described an anti-CD38/anti-saporin (OKT10-Sap) immunotoxin, which purportedly
delivers the ribosome inactivating protein (rip) to the leukemic cells (HSB-2
cells).
The anti-CD38 antibody, HB7, has been chemically conjugated to a modified
ricin
molecule and can supposedly bill antigen-bearing tumor cells (Goldmacher et
al., 84:
3017-25 (1994)).
Anti EGF R A~ztibodies. Epidermal growth factor-receptor (EGF-R) binds to
EGF, a mitogenic peptide. Anti-EGF-R antibodies and methods of preparing them
can be performed as described in U.S. Patent Nos. 5,844,093; 5,558,864.
European
Patent No. 706,799A purportedly describes an immunoconjugate comprising an
anti-
EGF-R mAb fused to a C-X-C chemokine, especially IL-8. U.S. 5,824,782
describes
an imlnunoconjugate comprising an anti-EGFR antibody fused to IL-8, which
lacks at
least the first amino acid of IL-8.
Anti HM1.24 A~ztibodies. HM1.24 is a type II membrane glycoprotein is
overexpressed in multiple myeloma (MM) and Waldenstrom's macroglobulinemia
(Ohtomo et al., Bioclaem. Bioplays. Res. Cofyamun. 258: 583-91 (1999); and
Goto et al.,
Blood 84: 1922-30 (1994)). A mouse monoclonal anti-HM1.24 IgG2a/x antibody has
been demonstrated to bind to HM1.24 on MM cells and reportedly induces ADCC
(Ono et al., Mol. Imnauno. 36: 387-95 (1999)). A humanized anti-HM1.24 IgGI/K
antibody also was shown to induce ADCC against human myeloma KPMM2 and
ARH77 cells (Ono et al., Mol. In2muno. 36: 387-95 (1999)).
A~zti Her-2 Antibodies. The engB 2 gene, more cormnonly known as (Hen-
2/neu), is an oncogene encoding a transmembrane receptor. Several antibodies
have
been developed against Her-2/neu, including trastuzumab (e.g., HERCEPTIN~;
Former et al., Oncology (Huntingt) 13: 647-58 (1999)), TAB-250 (Rosenblum et
al.,
Clin. Caracen Res. 5: 865-74 (1999)), BACH-250 (Id.), TAl (Maier et al.,
Cancers Res.
51: 5361-9 (1991)), and the mAbs described in U.S. Patent Nos. 5,772,997;
5,770,195
(mAb 4D5; ATCC CRL 10463); and 5,677,171. Portions of anti-Her-2 antibodies
have also been conjugated to toxins, such as a single chain antibody domain
specific
for erbB-2 coupled to part of a Pseudonaofaas exotoxin (Skrepnik et al., Clin.
Cancer
-4-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
Res. 2: 1851-7 (1996)). U.S. Patent No. 5,855,866 reported a method of
treating the
vasculature of solid tumors using an anti-p185Her-a antibody linlced to a
cytokine.
Anti MZIC 1 Antibodies. MUC-1 is a carcinoma associated mucin. The anti-
MUC-1 monoclonal antibody, McS, was reportedly administered to mice carrying
transplanted breast tumors and purportedly suppressed tumor growth (Peterson
et al.,
Cancer Res. 57: 1103-8 (1997)). Mc5 was chemically linked in a non-cleavable
fashion to a natural IFN-a (nIFN-a) and reportedly inhibited growth of
injected tumors
in mice (Ozzello et al., Breast Cancef~ Res. Treat. 25: 265-76 (1993)). An
IgG4 anti-
MUC-1 mAb, hCTM0l, has been suggested as a suitable carrier for cytotoxic
agents
in ovarian carcinomas (Van Hof et al., Cancer Res. 56: 5179-85).
Anti phosplaatidyl serine antigen Antibodies. Phosphatidyl-serine is a
phospholipid. Antibodies have been reported which bind to phosphatidyl-serine
and
not other phospholipids (e.g., Yron et al., Clih. Exp. Immol. 97: 187-92)
(1994)).
However, anti-phospholipid antibodies appear more typically associated with
anti-
phospholipid syndrome and its diagnosis, than for use in the treatment or
diagnosis of
cancer.
Anti-TAG-72 Antibodies. TAG-72 is a tmnor-associated antigen (TAG). One
dimeric single-chain Fv antibody construct of monoclonal CC49 recognizes the
TAG-
72 epitope (Pavlinkova et al., Clifa. Cancer Res. 5: 2613-9 (1999)).
Additional anti-
TAG-72 antibodies include B72.3 (Divgi et al., Nucl. Med. Biol. 21: 9-15
(1994)) and
those disclosed in U.S. Patent No. 5,976,531. Administration of recombinant
IFN-a
reportedly increased the amount of TAG-72 expressed on tumors (Macey et al.,
ClifZ.
Caf~cey-Res. 3: 1547-55 (1997)). The CC49 antibody also has reportedly been
chemically conjugated to doxorubicin (Johnson et al., Af2ticahcey~ Res. 15:
1387-93
(1995)) and streptavidin (Ngai et al., Nucl. Med. Biol. 22: 77-86 (1995)). TAG-
72 has
also been chemically conjugated to human interleukin-2 (IL-2) (LeBerthon et
al.,
Cayacef° Res. 51: 2694-8 (1991). U.S. Patent No. 5,976,531 claimed a
human anti-
Tag-72 antibody conjugated to an interferon but no evidence was presented
indicating
its efficiency in mammals.
Interferon-a Isnnzunoconjugates. Monoclonal antibodies raised against
tumor cell lines have been covalently coupled with purified human
lymphoblastoid
-5-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
IFN-a. Administration of this coupled form of IFN-a reportedly augmented
killing of
the tumor cells and other tumor targets by peripheral blood NK cells (Flannery
et al.,
Euf°. J. Cafzcer~ Clin. Oncol. 20: 791-8 (1984)).
Techniques also have been developed to produce chimeric antibodies which
combine regions of immunoglobulin molecules from different sources (Morrison
et
al., Proc. Natl Acad. Sci. USA 81: 6851-5 (1984)). Using this technology,
chimeric
molecules comprising immunoglobulin and non-imrnunoglobulin regions have been
created. Expression vectors for fusion proteins comprising DNA sequences of an
antibody and DNA sequences encoding a cell surface antigen have been proposed,
wherein some of the suitable antigens include CD molecules (e.g., CD19, CD20,
CD22, CD33, CD38 and CD40). See, e.g., U.S. Patent No. 5,637,481 (1997) and EP
Patent No. 610,046 (1994). Fusion proteins comprising the cytokine IL-15 and
anti-
CD20 (International PCT Application 98/16254), and fusion proteins comprising
other lymphokines (e.g., IL,-2 and IL-3) and an imrnunoglobulin fragment
capable of
binding to a tumor antigen have been described (U.S. Patent Nos. 5,645,835
(1997)
and 5,314,995 (1994); Lode et al., Blood 91: 1706-1715 (1998); Hassan et al.,
Leuk.
Lymphoma 20: 1-15 (1995)). Chang et al., U.S. Patent 5,723,125 describes a
hybrid
molecule comprising an interferon, preferably IFN-a-2a or IFN-a-2b, which are
joined
at the carboxy terminus via a peptide to the amino terminus of a first gamma
immunoglobulin Fc fragment as a means of increasing the blood half life of the
cytokine. Chimeric immunoglobulin proteins comprising a cytokine (e.g., IL-2,
tumor
necrosis factor a, etc.) and the heavy chain of an antibody for treating viral
infections
and cancer are described in International PCT Application 92/08495, as well as
a
fusion protein of IFN-y/M-CSF. A fusion protein of RM4/IFN-tau has
demonstrated
antitumor activity in mice (Qi et al., Hum. Antibodies Hyb>"idomas 7: 21-6
(1996); and
Xiang et al., Hunt. Antibodies Hybf°idontas 7: 2-10 (1996)).
Others have proposed the creation of fusion proteins comprising non-antibody
immunomodulatory molecules, wherein IFN-a is operatively fused to a
heterologous
membrane attachment domain (IJ.S. Patent No. 5,891,432 (1999)). IFN-a-2b was
reported to be conjugated with ME31.3 and anti-carcinoembryonic antigen (CEA)
and
purportedly may improve diagnostic and therapeutic potential of monoclonal
-6-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
antibodies making them worthy of further study (Thakur et al., J. Inamuhother.
20:
194-201 (1997)).
Therefore, not withstanding what has been previously reported in the
literature, there exists a need for improved compositions and methods of using
such
compositions for the treatment of cancer. Improved immunoconjugates with
enhanced ADCC and phagocytotic activities, lower toxicity, and increased blook
serum half life would provide additional treatment therapeutics, which are
still needed
in cancer therapy.
SUMMARY OF THE INVENTION
It is one object of the invention to provide novel and improved compositions
comprising an immunoconjugate that comprises an antibody or imrnunogenic
fragment thereof that binds to an antigen expressed by a target cell that is
to be
eradicated, wherein said antibody or imrnunogenic fragment thereof possesses
human
effector function, which antibody or immunogenic fragment thereof is fused at
its
carboxy terminus to a cytokine that binds a receptor expressed on the surface
of a
natural killer cell and/or macrophage, thereby resulting in an immunoconjugate
that
facilitates extracellular (ADCC-type) and intracellular (phagocytic) killing
of a target
cell, when said immunoconjugate is administered to a host. Preferably, the
cytokine is
a interferon. Most preferably, the interferon is an a-interferon, especially
one which
has been FDA approved (e.g., IFN-a-2a, IFN-a-2b and IFN-a-nl).
It is a more specific object of the invention to provide novel
immunoconjugates which target cells expressing CD19, CD20, CD22, CD33, CD3~,
EGF-R, HM 1.24, phosphatidyl serine antigen, HER-2, TAG-72 and MUC1.
It is a further obj ect of the invention to provide novel methods of enhancing
apoptosis or treating cancer by achninistering a therapeutically effective
amount of
these immunoconjugates to a subject. The target cells to which these
immunoconjugates are directed may include malignant cells selected from the
group
consisting of a breast carcinoma cell, an ovarian carcinoma cell, a prostate
carcinoma
cell, a lung carcinoma cell, a leukemic T-cell, a leukemic B-cell, a multiple
myeloma
cell and a B-cell lymphoma cell.
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
It is another obj ect of the invention to provide a combination therapy to
treat a
malignancy in a subject comprising an immunoconjugate as described above and
at
least one chemotherapeutic agent or chemotherapeutic cocktail.
It is a further object of this invention to provide nucleic acids which encode
the immunoconjugates described herein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to IFN-a fusion proteins, specifically
immunoconjugates, and the use thereof as therapeutic agents that have the
ability to
target malignant cells and enhance the killing activity of effector cells
through the
binding of an IFN-a to its receptor on the effector cell and without systemic
administration of IFN-a and its associated toxicity. These immunoconjugates
will
have enhanced ADCC and phagocytic activity.
I. Definitions
By "interferon-a" or "IFN-a" is meant a protein or fragment thereof which can
recognize and bind to the IFN-a receptor. This includes FDA approved forms of
IFN-
a, such as IFN-a-2a (Roferon by Hoffinan-LaRoche), IFN-a-2b (INTRON~ A by
Schering Corporation) and IFN-a-nl (lymphoblastoid interferon called Wellferon
and
produced by Wellcome Foundation Ltd - Wellcome Research Laboratories), as well
as
other forms of IFN-a (e.g., IFN-a-2a and consensus IFN).
The term "fusion protein" as used herein means a hybrid protein produced
recombinantly including a synthetic or heterologous amino acid sequence. A
fusion
protein can be produced from a hybrid gene containing operatively linked
heterologous gene sequences.
By "bispecific fusion protein" is meant any immunologically reactive
molecule which specifically recognizes and binds two different targets at
alternate
times or at the same time. In particular, it will refer to an IFN-a antibody
fusion
protein, wherein the IFN is attached to an antibody, preferably anti-tumor
antibody, at
the carboxy terminus of the antibody. In a preferred embodiment, the antibody
portion may recognize and bind to a target antigen expressed on a targeted
cell. In a
more preferred embodiment, the antigen-binding, Fc receptor binding, Clq and C
_g_
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
activation, and the ability of interferon to bind to its receptor and activate
effector
cells and macrophages are substantially maintained activities of the
immunoconjugate.
This is preferably effected by attachment of the interferon directly or
indirectly to the
antibody hinge, CHl, CH2 or CH3 domain carboxy-terminus.
The terms "effector cell" and "effector function" as used herein means a cell
which expresses an IFN-a receptor and can thereby bind to an IFN-a fusion
protein.
Effector cells can include natural killer (NK) cells, LAK cells, monocytes,
macrophages and polymorphonuclear (PMNs) cells. Preferred effector cells
include
NK cells and macrophages.
An "expression vector" means a nucleic acid molecule comprising (1) a
promoter and other sequences (e.g., leader sequences) necessary to direct
expression
of a desired gene or DNA sequence, and (2) the desired gene or DNA sequence.
Optionally, the nucleic acid molecule may comprise a poly A signal sequence to
enhance the stability of the gene transcript and/or to increase gene
transcription and
expression.
By "binding domain," "binding region" means a binding site which recognizes
and binds the entire binding area of a target or any portion thereof. Examples
for
antibodies or immunoglobulin fragments include: (1) single variable region of
an
antibody VL or VH; (2) two or more variable regions (e.g., VL and VH, VL and
VL; or
VH and VH) or the complementary determining region (CDR) thereof; (3) antibody
fragments such as Fabl, Fab2, SFV, single chain antibodies, domain-deleted
antibodies
and minibodies; or (4) an IFN-a or a segment of IFN-a which binds to an IFN-a
receptor on an effector cell.
By "minibody" is meant an antigen binding protein which includes VL and VH
domains of a native antibody fused to the hinge region and CH3 domain of an
immunoglobulin or which encodes in a single chain comprising the essential
elements
of a whole antibody. The single chain comprises the antigen binding region,
CH3
domain to permit assembly into a bivalent molecule, and the antibody hinge to
accommodate dimerization by disulfide linkages. For methods of preparing
minibodies, see, e.g., U.S. Patent No. 5,837,821.
-9-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
By "antibody" is intended to refer broadly to any immunologic binding agent
such as IgG (including IgGI, IgG2, IgG3, and IgG4), IgM, IgA, IgD, IgE, as
well as
antibody fragments. Antibodies in the broadest sense covers intact monoclonal
antibodies, polyclonal antibodies, as well as biologically active fragments of
such
antibodies such as those discussed above. In particular, domain-deleted
antibodies are
included within the scope of the present invention, such as CH2 domain-deleted
antibodies.
By "monoclonal antibody" is meant an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the
population are identical except for possible naturally occurring mutations
that may be
present in minor amounts. Monoclonal antibodies are highly specific, being
directed
against a single antigenic site. Furthermore, in contrast to conventional
(polyclonal)
antibody preparations, which typically include different antibodies directed
against
different determinants (epitopes), each monoclonal antibody is directed
against a
single determinant on the antigen. In addition to their specificity, the
monoclonal
antibodies are advantageous in that they are synthesized by the hybridoma
culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates
the
character of the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring production
of the
antibody by any particular method. For example, the monoclonal antibodies to
be used
in accordance with the present invention may be made by the hybridoma method
first
described by Kohler et al., Nature 256: 495 (1975), or may be made by
recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may
also be isolated from phage antibody libraries using the techniques described
in
Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. lVlol. Biol.,
222:
581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies
(immunoglobulins) in which a portion of the heavy and/or light chain is
identical with
or homologous to corresponding sequences in antibodies derived from a
particular
species or belonging to a particular antibody class or subclass, while the
remainder of
the chains) is identical with or homologous to corresponding sequences in
antibodies
-10-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
derived from another species or belonging to another antibody class or
subclass, as
well as fragments of such antibodies, so long as they exhibit the desired
therapeutic
activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.
USA, 81:
6851-5 (1984)).
"Humanized" forms of non-htunan (e.g., marine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab')2 or other antigen-binding subsequences of antibodies), which contain
minimal
sequence derived from a non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which residues
from a
complementarity-determining region (CDR) of the recipient are replaced by
residues
from a CDR of a non-human species (donor antibody) such as mouse, rat or
rabbit
having the desired specificity, affinity, and capacity. In some instances, Fv
framework
region (FR) residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies may comprise residues
which are found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine and
optimize
antibody performance. In general, the humanized antibody will comprise
substantially
all of at least one, and typically two, variable domains, in which all or
substantially all
of the CDR regions correspond to those of a non-human immunoglobulin and all
or
substantially all of the FR regions are those of a human immunoglobulin
sequence.
The humanized antibody optimally also will comprise at least a portion of an
irmnunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For
further details, see Jones et al., Nature, 321: 522-5 (1986); Reichmann et
al., Natuf°e,
332: 323-9 (1988); and Presta, Curs. ~p. StYZICt. Biol., 2: 593-6 (1992).
By "target antigen" is meant the antigen recognized by the antibody or
immunoglobulin fragment portion of the immunoconjugate. Preferred target
antigens
include: 5E10, CDl, CD2, CD3, CDCDS, CD7, CD13, CD14, CD15, CD19, CD20,
CD21, CD23, CD25, CD33, CD34, CD38, CEA, EGFR, HER-2, HLA-DR, HM 1.24,
HMB 45, Ia, Leu-M1, MUC1, phosphatidyl serine antigen, PMSA and TAG-72.
-11-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
By "nucleic acid" is meant to include an oligonucleotide, nucleotide,
polynucleotide and fragments and portions thereof, and a DNA or a RNA of
genomic
or synthetic origin, which may be single or double stranded.
By "interferon a" or "IFN-a" preferably is meant to include all members of the
interferon-a family of proteins. Fragments of IFN-a are also included, as long
as the
fragment is capable of recognizing and binding to the IFN-a receptor and
thereby
activating effector cells. Preferred forms of IFN-a include such FDA approved
forms
of IFN-a as IFN-a-2a, IFN-a-2b and IFN-a-ln.
By "purified" and "isolated" is meant, when referring to a polypeptide or
nucleotide sequence, that the indicated molecule is present in the substantial
absence
of other biological macromolecules of the same type. The term "purified" as
used
herein preferably means at least 75% by weight, more preferably at least 85%
by
weight, more preferably still at least 95% by weight, and most preferably at
least 98%
by weight, of biological macromolecules of the same type are present. An
"isolated
nucleic acid molecule wluch encodes a particular polypeptide" refers to a
nucleic acid
molecule which is substantially free of other nucleic acid molecules that do
not
encode the subject polypeptide; however, the molecule may include some
additional
bases or moieties which do not deleteriously affect the basic characteristics
of the
composition. Thus, for example, an isolated nucleic acid molecule which
encodes a
particular CDR polypeptide consists essentially of the nucleotide coding
sequence for
the subject molecular recognition unit.
By "therapeutically effective" is meant the ability of the immunoconjugate to
inhibit in vitro growth of a target cell by greater than about 20%, at a
concentration of
about 0.1 to about 3.0 ~,g/ml of the immunoconjugate, wherein said target
cells (e.g.,
tumor cells) are cultured in an appropriate culture medium and said growth
inhibition
is determined about 4, 5, 6, 7, 8, 9, or 10 days after exposure of the target
cells to the
immunoconjugate.
By "subject" is meant a living animal or human susceptible to a condition,
especially cancer. In the preferred embodiments, the subject is a mammal,
including
human and non-human marmnals. Non-human mammals include dogs, cats, pigs,
cows, sheep, goats, horses, rats and mice.
-12-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
II. Method of Mal~in~ an IFN-a Fusion Protein
Nucleic acids encoding the desired fusion protein can be inserted into
expression vectors for expression. Expression vectors useful in the invention
include
prokaryotic and eukaryotic expression vectors. Such expression vectors,
including
plasmids, cosmids, and viral vectors such as bacteriophage, baculovirus,
retrovirus
and DNA virus vectors, are well known in the art (see, for example, Meth.
Enzymol.,
Vol. 185, D. V. Goeddel, ed. (Academic Press, Inc., 1990), and Kaplitt and
Loewy
(Ed.), VIRAL VECTORS: GENE THERAPY AND NEUROSCIENCE APPLICATIONS
(Academic Press, Inc., 1995), each of which are incorporated herein by
reference).
Expression vectors contain the elements necessary to achieve constitutive or
inducible
transcription of a nucleic acid molecule encoding an IFN-a fusion protein. One
form
of IFN-a is described in Pitha et al., J. Immunol. 141: 3611-6 (1988).
Preferred
embodiments of the invention will utilize FDA approved forms of IFN-a.
The recombinant nucleic acid molecules encoding an IFN-a fusion protein, an
immunoglobulin polypeptide, or a specific IFN-a species or fragment thereof
may be
obtained by any method known in the art (see, e.g., Maniatis et al., MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, 1982 and 1989) or obtained from publicly available clones,
such
as pGL2BIFN (ATCC No. 53371) and pALCAISIFN (ATCC No. 53369).
Alternatively, a nucleic acid encoding an IFN-a or an antibody which
recognizes a
tumor-associated antigen (TAA) may be obtained as follows. A population of
cells
known to actively express an IFN-a or a specific antibody may be obtained, and
total
cellular RNA harvested therefrom. The amino acid sequence of the IFN-a or
antibody
may be used to deduce the sequence of a portion of its nucleic acid so as to
design
appropriate oligonucleotide primers; or, alternatively, the oligonucleotide
primers may
be obtained from a known nucleic acid sequence encoding an antibody or an IFN-
a.
The oligonucleotide fragment may then be used in conjunction with reverse
transcriptase to produce a cDNA corresponding to the immunoglobulin and/or IFN-
a
encoding nucleotide sequence (Okayama et al., Methods Erazymol. 154: 3-29
(1987)).
The cDNA can then be cloned, and/or portions of the antibody or IFN-a coding
region
-13-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
amplified from this cDNA using polymerase chain reaction (PCR) and appropriate
primer sequences (Saiki et al., Science 239: 487-491 (1988)).
In preferred embodiments of the invention, a recombinant vector system may
be created to accoimnodate sequences encoding IFN-a, wherein the IFN-a
sequence is
attached to the sequences encoding the C-terminus of an antibody or
immunogenic
fragment that recognizes a tumor-associated antigen. The resultant fusion
protein will
preserve the ability of the IFN-a molecule to bind to its receptor on the
effector cell
and enhance the effector cell's killing ability. The immunoconjugate will also
preserve the antigen-binding Fc receptor-binding, Clq binding and C'
activation
regions of the antibody molecule.
Nucleic acid sequences encoding the various components of the IFN-a based
fusion proteins of the invention may be joined together using any techniques
known in
the art, including restriction enzyme methodologies and the use of synthetic
linker
sequences.
To provide for adequate transcription of the recombinant constructs of the
invention, a suitable promoter/enhancer sequence may be incorporated into the
recombinant vector. Promoters which may be used to control the expression of
the
antibody-based fusion protein include, but are not limited to, the SV40 early
promoter
region (Bernoist et al., Nature 290: 304-310 (1981)), the promoter contained
in the 3'
long terminal repeat of Rous sarcoma viruses (Yamamoto et al., Cell 22: 787-
797
(1980)), the herpes thymidine kinase (tk) promoter (Wagner et al., Proc. Natl.
Acad.
Sci. USA 78: 144-1445 (1981)), the regulatory sequences of the metallothionine
gene
(Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression systems
such as the
LAC, or (3-lactamase promoters (Villa-Kamaroff et al., Proc. Natl. Acad. Sci.
USA 75:
3727-3731 (1978)), or the tac lambda phage promoter (DeBoer et al., P~oc.
Natl.
Acad. Sci. USA 80: 21-25 (1983)). Other suitable promoters would be apparent
to the
skilled artisan.
Additionally, it may be desirable to include, as part of the recombinant
vector
system, nucleic acids corresponding to the 3' flanking region of an
immunoglobulin
gene, including RNA cleavage/polyadenylation sites and downstream sequences.
Furthermore, it may be desirable to engineer a signal sequence upstream of the
IFN-a
- 14-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
fusion protein-encoding sequences to facilitate the secretion of the fused
molecule
from a cell transformed with the recombinant vector.
Creation of IFN-a containing fusion proteins can also utilize sequences
encoding conservative amino acid substitutions in an IFN-a sequence, as well
as
substitutions in the antibody or immunoglobulin region of the fusion protein.
Such
changes include substituting an isoleucine, valine and leucine for any other
of these
hydrophobic amino acids; aspartic acid for glutamic acid and vice versa;
glutamine for
asparagine and vice versa; and serine for threonine and vice versa. Other
substitutions
can also be considered conservative, depending on the environment of the
particular
amino acid and its role in the three-dimensional structure of the protein. For
example,
glycine for alanine can frequently be interchangeable, as well as alanine for
valine due
to structural and charge similarities.
Successful incorporation of IFN-a based fusion gene constructs may be
identified by three general approaches: (a) DNA-DNA hybridization, (b)
presence or
absence of "marker" gene functions, and (c) expression of inserted sequences.
In the
first approach, the presence of a foreign gene inserted in an expression
vector can be
detected by DNA-DNA hybridization using probes comprising sequences that are
homologous to the inserted antibody IFN-a fusion protein DNA. In the second
approach, the recombinant vector/host system can be identified and selected
based
upon the presence or absence of certain "marker" gene functions (e.g.,
thymidine
kinase activity, resistance to antibiotics such as 6418, transformation
phenotype,
occlusion body formation in baculovirus, etc.) caused by the insertion of
foreign genes
in the vector. For example, if the IFN-a fusion gene is inserted so as to
interrupt the
marker gene sequence of the vector, recombinants containing the antibody
fusion gene
~ insert can be identified by the absence of the marker gene function. In the
third
approach, recombinant expression vectors can be identified by assaying the
foreign
gene product expressed by the recombinant. Such assays can be based, for
example,
on the physical or functional properties of the IFN-a fusion gene product in
bioassay
systems.
The cytokine can be any cytokine or analog or fragment thereof which
activates effector cells. The preferred cytokine is an interferon, with the
preferred
-15-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
interferon being IFN-a, especially FDA approved forms. The gene encoding the
cytoleine can be cloned de novo, obtained from an available source, or
synthesized by
standard DNA synthesis from a known nucleotide sequence as discussed above.
The heavy chain constant region for the conjugates can be selected from any of
the five isotypes: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu
(IgM).
Heavy chains or various subclasses (such as the IgG subclasses 1-4) can be
used. The
light chains can have either a kappa or lambda constant chain. DNA sequences
for
these immunoglobulin regions are well known in the art. (See, e. g., Gillies
et al., J.
Immunol. Metla. 125: 191 (1989)).
In preferred embodiments, the variable region is derived from an antibody
specific for the target antigen, and the constant region includes the CH1, CH2
and
CH3 domains. The gene encoding the cytokine is joined, in frame to the 3' end
of the
gene encoding the constant region (e.g., CH1, CH2 or CH3 exon depending on
domain-deleted form desired), either directly or through an intergenic region.
The nucleic acid construct can include an endogenous promoter and enhancer
for the variable region-encoding gene to regulate expression of the chimeric
immunoglobulin chain. For example, the variable region encoding genes can be
obtained as DNA fragments comprising the leader peptide, the VJ gene
(functionally
rearranged variable (V) regions with joining (J) segment) for the light chain
or VDJ
gene for heavy chain, and the endogenous promoter and enhancer for these
genes.
Alternatively, the gene coding for the variable region can be obtained apart
from
endogenous regulatory elements and used in an expression vector that provides
these
elements.
Variable region genes can be obtained by standard DNA cloning procedures
from cells that produce the desired antibody. Screening of the genomic library
for a
specific functionally rearranged variable region can be accomplished with the
use of
appropriate DNA probes, such as DNA segments containing the J region DNA
sequence and sequences downstream. Identification and confirmation of correct
clones
are then achieved by DNA sequencing of the cloned genes and comparison of the
sequence to the corresponding sequence of the full length, properly spliced
mRNA.
-16-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
The target antigen preferably can be a cell surface antigen of a tumor cell,
but
also includes viral antigens or other disease associated antigens expressed on
the cell
surface. Genes encoding appropriate variable regions can be obtained generally
from
immunoglobulin producing lymphoid cells. For example, hybridoma cell lines
producing immunoglobulin specific for tumor associated antigens or viral
antigens
can be produced by standard somatic cell hybridization techniques (see, e.g.,
U.S. Pat.
No. 4,96,265.). These inununoglobulin producing cell lines provide the source
of
variable region genes in functionally rearranged form. The variable region
genes will
typically be of marine origin, because the marine system lends itself to the
production
of a wide variety of imrnunoglobulins of desired specificity.
The DNA fragment containing the functionally rearranged variable region
gene is linked to a DNA fragment containing the gene encoding the desired
constant
region (or a portion thereof). Immunoglobulin constant regions (heavy and
light
chain) can be obtained from antibody-producing cells by standard gene cloning
techniques. Genes for the two classes of human light chains and the five
classes of
human heavy chains have been cloned, and thus, constant regions of human
origin are
readily available from publically available clones.
The fused gene encoding the hybrid immunoglobulin heavy chain is assembled
or inserted into expression vectors for incorporation into a recipient cell.
The
introduction of gene construct into plasmid vectors can be accomplished by
standard
gene splicing procedures.
Recipient cell lines are generally lymphoid cells. The preferred recipient
cell
is a myeloma (or hybridoma). Myelomas can synthesize, assemble, and secrete
immunoglobulins encoded by transfected genes, and they post-tra~islationally
modify
the protein. A particularly preferred recipient cell is the Sp2/0 myehoma
which
normally does not produce endogenous immunoglobulin. When transfected, the
cell
will produce only innnunoglobulin encoded by the transfected gene constructs.
Transfected myelomas can be grown in culture or in the peritoneum of mice
where
secreted immunoconjugate can be recovered from ascites fluid. Other lymphoid
cells,
such as B lymphocytes, also can be used as recipient cells.
-17-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
There are several methods for transfecting lymphoid cells with vectors
containing the nucleic acid constructs encoding the chimeric Ig chain. A
preferred way
of introducing a vector into lymphoid cells is by spheroblast fusion, as
described by
Gillies et al., Biotechn.ol. 7: 798-804 (1989). Alternative methods include
electroporation or calcium phosphate precipitation. See also, the methods in
MANJATIS, ET AL. (1989).
IFN-a based fusion protein produced by the host cell may be collected using
any technique known in the art, including, but not limited to, affinity
chromatography
using target antigen or antibody specific for any portion of the fusion
protein. The
activity of the fused IFN-a or antibody (e.g., anti-CD20) may be confirmed
using
biological assays, which detect or measure the activity of the lymphokine or
cellular
factor. For example, and not by way of limitation, the presence of IFN-a
activity may
be confirmed in assays which detect receptor binding, virus neutralization and
enhanced killing ability of the effector cells.
Preferred methods of detecting such enhanced effector cell ability can utilize
receptor binding assays and virus neutralization assays. These assays are
described
generally below.
Receptor Binding Assay
Receptor binding assays, such as those provided below, can be utilized to
determine whether the immunoconjugate binds to the target antigen or to an IFN-
a
receptor.
Virus Neutralization Assay
A virus neutralization assay is one form of receptor binding assay which can
be utilized to determine the efficacy of which an immmloconjugate to
neutralize virus-
infected cells when the antibody targets a viral antigen expressed on the cell
surface.
For example, virus neutralizations can be determined using the method
described by
Ho et al., J. Virol. 65: 489-93 (1991), for HIV-1 neutralization using a p24
assay.
Neutralization is defined as the percent reduction in the amount of target
antigen
-18-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
released into the culture supernatants or detected in cells from wells treated
with the
immunoconjugate compared with control wells not treated with the
immunoconjugate.
ADCC Assay
The ability of the fusion protein to induce ADCC can be assessed using a
chromium release assay. Generally, antibodies of the IgGZa and IgG3 subclass
and
occasionally of the IgGI subclass mediate ADCC. Antibodies of the IgG3, IgG2a
and
IgM classes bind and activate serum complement. To assess the ability of the
immunoconjugates described herein to mediate ADCC and complement activation,
one can use a SICr-release assay. Briefly, a cell line expressing the antigen
being
targeted for lysis by effector cells are labeled with 100 ~,Ci of SICr for
about 1 hour
prior to combining effector cells and antibodies in a U-bottom microtiter
plate. After
incubation for about 5 hours at 37°C, supernatants axe collected and
analyzed for
radioactivity. Cytotoxicity can be calculated by the formula: % lysis =
[((experimental CPM) - (target lealc CPM)) / ((detergent lysis CPM) - (target
leak
CPM))] X 100%. Specific lysis is calculated using the formula: Specific lysis
=
(%lysis with antibody) - (% lysis without antibody).
Complement Binding Assay
To assess the ability of the immunoconjugates to bind complement, the
following assay can be utilized. Cells expressing the target antigen
recognized by the
immunoconjugate are incubated with the immunoconjugate at a concentration of
10
~,g/ml. After incubating the plates containing the cells and immunoconjugate
for 15
minutes at room temperature, the plates are washed three times. After the
third wash,
the cells are resuspended in 50 ~,l of a 1/10 dilution of complement (e.g.,
guinea pig
complement from ICN) and incubated at 37°C for varying times. Then 50
~,l of
0.25% (w/v) trypan blue is added and cell number and plasma integrity of the
cells are
estimated.
-19-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
Pha~ocytosis Assay
To assess the phagocytosis enhancing ability of a particular immunoconjugate,
the following assay can be used. Cells expressing the target antigen are
labeled with
lipophilic red fluorescent dye PITH 26. Buffy coat cells purified from
heparinized,
whole blood containing effector cells are incubated with the labeled taxgets
at 37°C
for about 6 hours in the absence or in the presence of the immunoconjugate.
Effector
cells are then stained with FITC (fluorescein isothiocyanate) labeled
antibody, which
binds to the effector cell at 0°C. Cells are washed and analyzed using
two color
fluorescence by FACScan or other scanning method. Percent phagocytosis is
expressed as the percent of effector cells (NK cells, monocytes, neutrophils
or
macrophages) that have PKH 26 stain associated with them.
III. Methods of Administering IFN-a Fusion Protein
A fusion protein of the invention is administered to subjects in a
biologically
compatible form suitable for pharmaceutical administration ih vivo. By
"biologically
compatible form suitable for administration ifa vivo" is meant a form of the
immunogonjugate to be administered in which any toxic effects are outweighed
by the
therapeutic effects of the protein. An immunogonjugate can be administered in
any
pharmacological form, optionally in a pharmaceutically acceptable carrier.
Administration of a therapeutically effective amount of the innnunoconjugate
is
defined as an amount effective, at dosages and for periods of time necessary
to
achieve the desired result (e.g., inhibition of the progression or
proliferation of the
disease being treated). For example, a therapeutically active amount of an
immunoconjugate may vary according to such factors as the disease stage (e.g.,
stage I
versus stage IV), age, sex, medical complications, and weight of the
individual, and
the ability of the immunoconjugate to elicit a desired response in the
individual. The
dosage regimen may be adjusted to provide the optimum therapeutic response.
For
example, several divided doses may be administered daily, or the dose may be
proportionally reduced as indicated by the exigencies of the therapeutic
situation.
The active compound, an immunoconjugate, by itself or in combination with
other active agents, such as chemotherapeutic anti-cancer drugs. The
-20-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
immunoconjugate, alone or in combination with other agents, may be
administered in
a convenient manner such as by injection (subcutaneous, intramuscularly,
intravenous,
etc.), inhalation, transdermal application or rectal achninistration.
Depending on the
route of administration, the active compound may be coated with a material to
protect
the active compound from the action of enzymes, acids and other natural
conditions
which may inactivate the compound. A preferred route of administration is by
intravenous (LV.) injection.
To achninister an immunoconjugate by other than parenteral administration, it
may be necessary to coat the IFN-a fusion protein with, or co-administer the
IFN-a
fusion protein with, a material to prevent its inactivation. For example, an
IFN-a
fusion protein can be administered to an individual in an appropriate carrier
or diluent,
co-administered with enzyme inhibitors or in an appropriate carrier or vector,
such as
a liposome. Pharmaceutically acceptable diluents include saline and aqueous
buffer
solutions. Liposomes include water-in-oil-in-water emulsions, as well as
conventional liposomes (Strejan et al., J. Neuroirn~uszol. 7: 27 (1984)).
Additional
pharmaceutically acceptable carriers and excipients are known in the art or as
described in IZEMINGTON'S PHARMACEOTICAL SCIENCES (18th ed. 1990).
The active compound may also be administered parenterally or
intraperitoneally. Dispersions of the active compound can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary
conditions of storage and use, these preparations may contain one or more
preservatives to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersions. In
all cases,
the composition must be sterile and must be fluid to the extent that easy
syringability
exists. It must be stable under the conditions of manufacture and storage and
must be
preserved against the contaminating action of microorganisms, such as bacteria
and
fungi. The carrier can be a solvent or dispersion medium containing, for
example,
water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
-21 -
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
can be maintained, for example, by the use of a coating such as lecithin, by
the
maintenance of the required particle size in the case of dispersion, and by
the use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
ascorbic acid, thimerosal and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars, polyalcohols, such as manitol, sorbitol,
or
sodium chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition an agent
which
delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating an active
compound (e.g., an IFN-a fusion protein) in the required amount in an
appropriate
solvent with one or a combination of ingredients enumerated above, as
required,
followed by filtered sterilization. Generally, dispersions are prepared by
incorporating
the active compound into a sterile vehicle, which contains a basic dispersion
medium
and the required other ingredients from those enumerated above. In the case of
sterile
powders for the preparation of sterile injectable solutions, the preferred
methods of
preparation are vacuum drying and freeze-drying, which yields a powder of an
active
ingredient plus any additional desired ingredient from a previously sterile-
filtered
solution thereof.
When the active compound is suitably protected, as described above, the
protein may be orally administered, for example, with an inert diluent or an
assimilable edible carrier. As used herein, "pharmaceutically acceptable
carrier"
includes any and all solvents, dispersion media, coatings, antibacterial and
antifungal
agents, isotonic and absorption delaying agents, and the like. The use of such
media
and agents for pharmaceutically active substances is well kno~cm in the art.
Except
insofar as amy conventional media or agent is incompatible with the active
compound,
use thereof in the therapeutic compositions is contemplated. All compositions
discussed above for use with an IFN-a fusion protein may also comprise
supplementary active compounds in the composition.
It is especially advantageous to formulate parenteral compositions in dosage
unit form for ease of administration and uniformity of a dosage. "Dosage unit
form,"
-22-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
as used herein, refers to physically discrete units suited as unitary dosages
for the
mammalian subjects to be treated; each unit containing a predetermined
quantity of
active compound is calculated to produce the desired therapeutic effect in
association
with the required pharmaceutical carrier. The specification for the dosage
unit forms
of the invention are dictated by and directly dependent on: (A) the unique
characteristics of the active compound and the particular therapeutic effect
to be
achieved; and (B) the limitations inherent in the art of compounding such an
active
compound for the treatment of sensitivity in individuals.
- 23 -
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
IV. Method of Treating Cancer
The immunoconjugates described herein can be targeted to a variety of
malignant cells which express a tumor-associated antigen (TAA) expressed on
the
surface of the cell. IFN-a fusion proteins comprising antibodies can be
prepared
which recognize B and T cell leukemias and lymphomas, multiple myelomas, and
solid tumors (e.g., prostate carcinoma, colon carcinoma, lung carcinoma,
breast
carcinoma and ovarian carcinoma). In preferred embodiments, the immunoglobulin
portion of the IFN-a fusion protein may recognize B cell markers (e.g., CD19,
CD20,
CD22), multiple myeloma antigens (e.g., CD38, HM1.24), leukemia markers (e.g.,
CD33), and phosphatidyl-serine antigen. Additional markers affiliated with
certain
malignancies and to which the immunoglobin portion of the fusion protein can
include, but are not limited to, the following:
Table 1
Cancer Tumor Associated Anti ens
Acute Lymphocytic Leukemia HLA-Dr, CD34, CD19, CD20,
(ALL) CD1,
CD2, CDS, CD7
Acute Myelogenous Leukemia HLA-Dr, CD34, CD13, CD14,
(AML) CD15,
CD33, CD7
Breast Cancer EGFR, HER-2, MUC1, TAG-72
Carcinoma CEA, TAG-72, MUC1
Chronic Lymphocytic Leukemia CD3, CD21, CD20, CD19, CD23,
(CLL) HLA-Dr
Hairy Cell Leukemia (HCL) HLA-Dr, CD19, CD20, CD21,
CD25
Hodgkin's Disease Leu-M1
Melanoma HMB 45
Non-Hodgkin's Lymphoma CD20, CD19, Ia
Prostate PSMA, SE10
-24-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
David A. Scheinberg et al., "The Leukemias" in HARRISON'S PRINCIPLES of
INTER~IaL
MEDICINE 1764-1774 (Kurt J. Isselbacher et al., eds., 13th ed. 1994).
V. Drugs to be Used in Combination with the Fusion Protein
The immunoconjugates described above can be used in combination with one
or more different cancer treatment modalities, such as radiotherapy,
immunotherapy,
chemotherapy, and surgery. The combination of treatments used on any
particular
subject will vary depending on cancer type, stage of disease, family history,
age, sex,
weight and condition of the subject. Preferably, the innnunoconjugates are
administered in combination with one more chemotherapeutics. Preferably,
chemotherapeutic or chemotherapeutic cocktail is administered in combination
with
the interferon immunoconjugate described herein, and include those listed in
the table
below:
TABLE 2
Cancer Chemothera
Acute Lymphocytic Ara-C alone or with L-asparaginase, doxorubicin,
Leukemia (ALL) idarubicin, mitoxantrone
Acute Myelogenous hydroxyurea and busulfan; chlorambucil,
melphalan, 6-
Leukemia (AML) mercaptopurine, 6-thioguanine, dibromomannitol,
ara-C,
IFN-a
Breast Cancer CMF, CAF, CEF, CMFVP, AC, VAT, VATH,
CDDP +
VP-16, Tam
Chronic Lymphocytic CAP, CVP, CMP, CHOP
Leukemia (CLL)
Hairy Cell Leukemia 2-chlorodeoxyadenosine, deoxycoformycin,
(HCL) IFN-a
Hodgkin's Disease VABCD, ABDIC, CBVD, PCVP, CEP, EVA,
MOPLACE, MIME, MINE, MTX-CHOP, CEM, CEVD,
CAVP, EVAP, EPOCH, MOPP, MVPP, ChIVPP,
AVD,
MOPP + ABVD, MOPP + ABV
Melanoma (metastatic)dacarbazine, cisplatin, IFN-a-2b, caxmustine,
lomustine,
-25-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
Cancer Chemothera
tauromustine, fotemustine, carboplatin,
vincristine,
vinblastine, vindesine, taxol, dibromodulcitol,
detorubicin, and iritrexim
Non-Hodgkin's LymphomaIMVP-16, MIME, DHAP, ESHAP, CEPP(B),
CAMP,
CHOP, CAP-BOP, CHOP-B, ProMACE-MOPP,
m-
BACOD, MACOP-B, ProMACE-CytaBOM
Prostate (hormonallydoxorubicin, doxorubicin + ketoconazole
or
relapsed patients) cyclophosphamide, estramustine, vinblastine,
paclitaxel,
navelbine, prenisolone, mitoxantrone
or combinations
thereof.
These chemotherapeutic drugs and drug cocktails (e.g., more than one
chemoterapeutic agent) can be administered according to the regimens described
in
CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY (Vincent T. DeVita, Jr. et al. eds.,
Stn
ed. 1997) or as would be known to the skilled artisan.
The abbreviations for the chemotherapeutic cocktail and chemotherapeutic
acronyms are as follows:
AC doxorubicin, cyclophosphamide
ABDIC doxorubicin, bleomycin, dacarbazine, lomustine, prednisone
ABVD doxorubicin, bleomycin, vinblastine, dacarbazine
Ara-C cytarabine
AVD doxorubicin, vinblastine, dacarbazine
CAF cyclophosphamide, doxorubicin, 5-fluorouracil
CAMP lomustine, mitoxantrone, cytarabine, prednisone
CAP cyclophosphamide, doxorubicin, prednisone
CAP-BOP cyclophosphamide, doxorubicin, procarbazine,
bleomycin,
vincristine, prednisone
CAVP lomustine, melphalan, etoposide, prednisone
CEVD lomustine, etoposide, vindesine, dexamethasone
CDDP+VP-16 cisplatin, etoposide, mitomycin C + vinblastine
CEF cyclophosphamide, epirubicin, 5-fluorouracil
-26-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
CEM lomustine, etoposide, methotrexate
CEP lomustine, etoposide, prechumustine
CEPP(B) cyclophosphamide, etoposide, procarbazine, prednisone,
bleomycin
CEVD lomustine, etoposide, vindesine, dexamethasone
ChIVPP chlorambucil, vinblastine, procarbazine,
prednisone
CHOP cyclophosphamide, doxorubicin, vincristine,
prednisone
CHOP-B CHOP plus bleomycin
CMF cyclophosphamide, methodtrexate, 5-fluorouracil
CMP cyclophosphamide, melphalan, prednisone
CMVP cyclophosphamide, methotrexate, 5-fluorouracil,
vincristine, prenisone
CVP cyclophosphamide, vincristine, prednisone
DHAP dexamethasone, high-dose .Ara-C, cisplatin
ESHAP etoposide, methylpredisolone, high-dose
cytarabine,
cisplatin
EPOCH etopsoide, vincristine, doxorubicin, cyclophospharilide,
prednisone
EVA etoposide, vinblastine, doxorubicin
EVAP etoposide, vinblastine, cytarabine, cisplatin
IFN-a interferon a
IMVP-16 ifosfamide, methotrexate, etoposide
MACOP-B methodtrexate, doxorubicin, cyclophosphamide,
vincristine, prednisone, bleomycin, leucovorin
m-BACOD methotrexate, bleomycin, doxorubicin, cyclophosphamide,
vincristine, dexamethasone, leucovorin
MIME methyl-gag, ifosfamide, methotrexate, etoposide
MINE mitoquazone, ifosfamide, vinorelbine, etoposide
MOPLACE cyclophosphamide, etoposide, prednisone,
methotrexate,
cytarabine, vincristine
-27-
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
MOPP nitrogen mustard, vincristine, procarbazine, prednizone
MOPP+ABV MOPP plus doxorubicin, bleomycin, vinblastine
MOPP+ABVD alternating months of MOPP and ABVD
MVPP nitrogen mustard, vinblastine, procarbazine, prednisone
MTX-CHOP methotrexate + CHOP
PCVP vinblastine, procarbazine, cyclophosphamide, prednisone
ProMACE-CytaBOM prednisone, doxorubicin, cyclophosphamide, etoposide,
cytarabine, bleomycine, vincristine, methotrexate,
leucovorin
ProMACE-MOPP prednisone, methotrexate, doxorubicin,
cyclophosphamide, etoposide, leucovorin + standard
MOPP
Tam ~ tamoxifen
VABCD vinblastine, doxorubicin, dacarbazine, lomustine, bleomycin
VAT vinblastine, doxorubicin, thiotepa
VATH vinblastine, doxorubicin, thiotepa, fluoxymesterone
EXAMPLES
The examples and methods provided below serve merely to illustrate particular
embodiments of the invention and are not meant to limit the invention.
EXAMPLE 1
Immunoconiu~ate Comnrisin~ IFN-a and Rituximab
The nucleic acid encoding an IFN-a (e.g., IFN-a-2a, IFN-a-2b or IFN-a-nl) is
operably linl~ed to the nucleic acid encoding Rituximab such that when
translated the
IFN-a would form the carboxy terminus of the fusion protein. The antigen-
binding Fc
receptor-binding, C 1 q binding and complement (C') activation, as well as the
ability
of IFN-a to bind to NIA cells and macrophages are characteristics possessed by
the
agents. In addition to the nucleic acid encoding Rituximab, other nucleic
acids
encoding anti-CD20 antibodies can be operably attached to the nucleic acid
encoding
IFN-a. The other anti-CD20 antibodies include Ibritumomab, IFS, B1 and 1H4.
_2g_
CA 02412901 2002-12-18
WO 01/97844 PCT/USO1/40835
EXAMPLE 2
ha Yitro Testing of an Immunoconlu~ate
A tumor cell line (cell expressing a target antigen) expressing Her2/neu
(e.g.,
human breast carcinoma cells, SKBR-3) is selected to determine lysis using the
immunoconjugates described herein. Effector cell samples are obtained by using
heparinized whole blood or obtained from a third party. To prepare for use as
effector
cells, monocytes are cultured in Teflon containers in Macrophage Serum-Free
Medium (Gibco/BRL) containing 2% human serum for 24 to 48 hours. Target cells
are labeled with 100 ~,Ci of 5lCr for one hour prior to incubation with the
effector
cells and immunoconjugate in a U-bottomed microtiter plate. After incubation
for
about 16 to 18 hours at 37°C, supernatants from each well are collected
and analyzed
for radioactivity. Cytotoxicity and specific lysis can be calculated as
previously
described.
EXAMPLE 3
ha hivo Testing of Immunoconlu~ate
For assessment of anti-tumor activity of the immunoconjugates, a mouse
model can be used. In the instance of solid tumors, about 1x10' cells in
culture media
are injected subcutaneously into the right anterior flank of BALBIc, nulnu or
SCm
mice. Approximately, fourteen days later or when the tumors have grown to
about 0.8
to 1.2 cm in diameter, the mice are separated into groups of 5-10 animals and
injected
intravenously with 200 ~,I of immunoconjugate at concentrations of 1 ~,g to 10
mg.
Perpendicular tumor diameters are measured at regular intervals and tumor
volumes
can be calculated. Alternatively, animals can be euthanized and sections of
tumor
prepared to determine the impact on tumor progression by the immunoconjugate
as
compared to the control animals (untreated).
Although the present invention has been described in detail with reference to
examples above, it is understood that various modifications can be made
without
departing from the spirit of the invention. All cited patents and publications
referred
to in this application are herein incorporated by reference in their entirety.
-29-
