U.S. patent application number 09/735173 was filed with the patent office on 2001-06-28 for bispecific molecules for use in inducing antibody dependent effector cell-mediated cytotoxicity.
This patent application is currently assigned to Medarex, Inc.. Invention is credited to Ball, Edward D., Fanger, Michael W..
Application Number | 20010005747 09/735173 |
Document ID | / |
Family ID | 22770141 |
Filed Date | 2001-06-28 |
United States Patent
Application |
20010005747 |
Kind Code |
A1 |
Ball, Edward D. ; et
al. |
June 28, 2001 |
Bispecific molecules for use in inducing antibody dependent
effector cell-mediated cytotoxicity
Abstract
Bispecific molecules comprising a target cell specific ligand
and an effector cell specific antibody or functional antibody
fragment are disclosed.
Inventors: |
Ball, Edward D.; (Wexford,
PA) ; Fanger, Michael W.; (Lebanon, NH) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Medarex, Inc.
|
Family ID: |
22770141 |
Appl. No.: |
09/735173 |
Filed: |
December 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09735173 |
Dec 12, 2000 |
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09151893 |
Sep 11, 1998 |
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09151893 |
Sep 11, 1998 |
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08451194 |
May 26, 1995 |
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5833985 |
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08451194 |
May 26, 1995 |
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08207344 |
Mar 7, 1994 |
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Current U.S.
Class: |
530/388.8 ;
424/130.1 |
Current CPC
Class: |
A61K 47/6879 20170801;
A61K 39/395 20130101; A61K 39/395 20130101; A61K 47/68 20170801;
C07K 7/086 20130101; A61K 47/6813 20170801; C07K 2317/732 20130101;
A61K 2300/00 20130101; C07K 14/595 20130101; A61K 38/00 20130101;
C07K 2319/00 20130101; A61K 47/6857 20170801; C07K 16/283
20130101 |
Class at
Publication: |
530/388.8 ;
424/130.1 |
International
Class: |
A61K 039/395; C07K
016/00; C12P 021/08 |
Claims
1. A bispecific molecule comprising: a target cell specific ligand
and an effector cell specific antibody.
2. A bispecific molecule of claim 1, wherein the ligand is an
autocrine growth factor for a tumor cell.
3. A bispecific molecule of claim 2, wherein the tumor cell is a
human small-cell lung carcinoma cell.
4. A bispecific molecule of claim 3, wherein the ligand binds to
the gastrin-releasing peptide receptor.
5. A bispecific molecule of claim 4, wherein the ligand is selected
from the group consisting of bombesin and gastrin-releasing
peptide, or analogues thereof.
6. A bispecific molecule of claim 1, wherein the effector cell
specific antobody binds the Fc receptor of an effector cell.
7. A bispecific molecule of claim 6, wherein the effector cell
specific antibody binds the Fc.gamma. receptor at a site that is
not inhibited by endogenous immunoglobulin.
8. A bispecific molecule of claim 7, wherein the Fc.gamma. receptor
is selected from the group consisting of: Fc.gamma.RI, Fc.gamma.RII
and Fc.gamma.RIII.
9. A bispecific molecule of claim 8, wherein the effector cell
specific antibody is selected from the group consisting of: mAb22,
mAb32, mAb44, mAb62 and mAb197.
10. A bispecific molecule of claim 1, wherein the target cell
specific ligand is bombesin or an analogue thereof and the effector
cell specific antibody is a human Fc.gamma.RI-specific monoclonal
antibody.
11. A target cell-specific effector cell for inducing an antibody
dependent effector cell-mediated cytotoxicity against a target cell
comprising: (i) a target cell-specific ligand; and (ii) an effector
cell-specific antibody that is bound to an effector cell.
12. A target cell-specific effector cell of claim 11, wherein the
target cell is a tumor cell.
13. A target cell-specific effector cell of claim 12, wherein the
tumor cell is a human small-cell lung carcinoma cell.
14. A target cell-specific effector cell of claim 13, wherein the
target cell-specific ligand is an autocrine growth factor for the
human small cell lung carcinoma cell.
15. A target cell-specific effector cell of claim 14, wherein the
target cell-specific ligand is selected from the group consisting
of bombesin and gastrin-releasing peptide, and analogues
thereof.
16. A target cell-specific effector cell of claim 1, wherein the Fc
receptor of the effector cell consisting of: Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII.
17. A target cell-specific effector cell of claim 16, wherein the
effector cell-specific antibody is selected from the group
consisting of: mAb22, mAb32, mAb44, mAb62 and mAb197.
18. A method of inducing a specific antibody dependent effector
cell-mediated cytotoxicity against a target cell, in a subject,
comprising administering to the subject a bispecific molecule of
claim 1, 6, 9 or 10 in a pharmaceutically acceptable medium.
19. A method of claim 18, wherein the a target cell is a tumor
cell.
20. A method of claim 19, wherein the tumor cell is a human
small-cell lung carcinoma cell.
21. A method for stimulating an immune response in a subject
comprising administering to the subject a bispecific molecule
specific ligand in a pharmaceutically acceptable carrier.
22. A method of claim 21 wherein the target cell specific ligand of
the bifunctional molecule is an autocrine growth factor and the
effector cell specific antibody is specific to an Fc.gamma.
receptor of an effector cell.
23. A method of claim 22 wherein the target cell specific ligand of
the bifunctional molecule is selected from the group consisting of:
insulin-like growth factor I, transferrin, vasoactive intestinal
peptide, neurotensin, neuromedin B, neurophysin, tumor necrosis
factor, transforming growth factor alpha, platelet derived growth
factor, the transferin receptor and analogues thereof.
24. A method of claim 23 wherein the target cell specific ligand of
the bifunctional molecule is selected from the group consisting of
bombesin and gastrin releasing peptide or an analogue thereof.
Description
BACKGROUND OF THE INVENTION
[0001] Several types of effector cells, such as monocytes,
neutrophils, and natural killer (NK) cells, have surface receptors
that bind the Fc portion of immunoglobulins. When such cells
encounter target cells that have been opsonized with immunoglobulin
antibodies, they form conjugates, and either lyse or phagocytose
the target cells, depending upon the effector cell type, the target
cell type and the specific Fe receptor type (FcR) involved.
[0002] It has been demonstrated that target cell conjugation with
an effector cell and lysis can also be induced by a covalently
cross-linked bispecific heteroantibody made up of both anti-Fc
receptor antibody and antibody directed against a target cell
epitope. When effector cells bind such heteroaggregates to their Fc
receptor, they can specifically bind and lyse target cells which
have not been opsonized, but which express the appropriate target
antigen (See e.g. U.S. patent application Ser. No.: 972,871;
Karpovsky et al. (1984) J. Exp. Med. 160:1686-1701). Segal et al,
have reported cytolysis of tumor cells by mouse monocytes with an
attached heteroantibody which joins the Fc receptor of the monocyte
on one end with tumor cell epitopes on the other end (See U.S. Pat.
No. 4,676,980). Recently, a variety of bispecific monoclonal
antibodies and immunotoxins have been shown to confer antitumor
effects in vitro as well in vivo (See e.g., World Patent No:
9208892; Pan et al (1990) J. Immunol., 145:267-275; Trail et al.
(1993) Science (Washington, D.C.), 261:212-215; Weiner et al.
(1993) J. Immunol., 151:2877-2886; Link et al. (1993) Blood,
81:3343-3349; and Vallera, D. A. (1994) Blood, 83:309-317).
[0003] The binding of a heteroantibody to FcR is mediated by the Fc
region of the antibody. This binding is ordinarily susceptible to
inhibition by physiological concentrations of immunoglobulin.
However, monoclonal antibodies, which bind to a site on the Fc
receptor distinct from the binding site for endogenous
immunoglobulin, have been produced (see, for example, Anderson et
al., J. Biol. Chem. 261:12856 (1986); and Shen et al., J. Immunol.
137:3378-3382 (1986)). These antibodies are useful as the
effector-specific moiety of heteroantibodies, because serum
immunoglobulin does not interfere with targeted effector cell
killing.
[0004] Heteroantibodies are large in size and therefore present
certain difficulties when used clinically. Smaller molecules
capable of binding to target cells and effector cells and
initiating ADCC would be useful.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention relates to a bispecific
molecule comprising a target cell specific ligand and an antibody
or functional antibody fragment specific for an effector cell. In a
preferred embodiment, the antibody or functional antibody fragment
is specific against the Fc receptors (FcR) of effector cells. Most
preferably the bispecific molecules of the instant invention
comprise an effector cell specific antibody or functional fragment
that binds to the FcR at a site distinct from the binding site for
endogenous immunoglobulin; and a target cell specific ligand that
binds to a tumor cell receptor, most preferably the
gastrin-releasing peptide (GRP) receptor expressed by small cell
cancer of lung (SCCL) cells.
[0006] In other aspects, the invention relates to methods for
making the novel bispecific molecules and to methods of using the
molecules therapeutically, e.g. to induce an antibody dependent
effector cell-mediated cytotoxicity (ADCC) or prophylactically, as
a vaccine.
[0007] The novel bifunctional molecules described herein are
generally of a smaller size than heteroantibodies and the target
cell specific ligand binding to target cell mimics normal
physiology. Therefore the instant bifunctional molecules offer
certain therapeutic advantages (e.g. reduced immunogenicity).
[0008] The above discussed and many other features and advantages
of the present invention will become better understood by reference
to the following detailed description when taken in conjunction
with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 shows the scheme for conjugating Lys.sup.3-bombesin
and mAb 22 or F(ab').sub.2 fragments thereof.
[0010] FIG. 2 shows a flow cytometry analysis of bispecific
molecule (Lys.sup.3-bombesin-mAb 22) binding to four small cell
cancer of lung (SCCL) cell lines, SHP77, H69, DMS273, and H345.
[0011] FIG. 3 shows the ability of an bispecific molecule,
comprising Lys.sup.3-bombesin coupled to mAb 22, to induce lysis,
as determined by flow cytometric analysis, of four different SCCL
cell lines at various effector cell to target cell ratios. Binding
ability is expressed both as an absolute percentage of cells
stained and as a mean fluorescence intensity (MFI) of the entire
cell population.
[0012] FIG. 4 shows the ability of various concentrations of
bispecific molecule (Lys.sup.3-bombesin-mAb 22) to induce lysis of
SCCL cells from the cell line SHP-77. The peak of activity to
mediate tumor cell lysis was seen in a wide range of bispecific
molecule concentrations ranging from 25 to 25,000 ng/ml.
DETAILED DESCRIPTION OF THE INVENTION
[0013] This invention is based on the surprising finding that
ligands specific for a particular target cell can be useful for
initiating a specific antibody-dependent effector cell-mediated
cytotoxicity against the target cell (ADCC). In one aspect, the
invention features bispecific molecules comprising a ligand
specific for a target cell and an antibody or functional antibody
fragment specific for an effector cell.
[0014] As used herein, the following terms and phrases shall be
defined as follows: "bispecific molecule" shall mean a molecule
having an antibody portion that is capable of binding an Fc
receptor (FcR) on a effector cell; and a ligand portion that is
capable of being bound by a receptor or antibody on a target
cell.
[0015] "Target cell specific ligand" as used herein refers to
molecules (e.g. peptides, polypeptides or proteins) that
specifically interact with a target cell, for example by way of a
target cell surface receptor or antibody. Preferred ligands of the
present invention bind to predominantly with target cells and not
other cells when administered in vivo. Preferably a ligand is a
member of a binding pair with a receptor or antibody that is
expressed predominantly by the target cell.
[0016] In a preferred embodiment of the invention, the target cell
specific ligand is a ligand for the gastrin-releasing peptide (GRP)
receptor expressed by small cell cancer of lung (SCCL) cells. As
shown herein, GRP receptors of SCCL cells specifically bind GRP,
and analogue, bombesin, a fourteen amino acid peptide which
contains a carboxy-terminal heptapeptide sequence identical to that
of GRP. Accordingly, preferred ligands of the present invention
include bombesin, gastrin releasing peptide (GRP), and functional
fragments or analogues thereof. The term fragments or analogues
thereof is intended to include amino acid sequences which differ by
one or more amino acid substitutions, additions or deletions from
the full length native bombesin or GRP protein, such as allelic
variants. Preferred fragments and analogues of bombesin and GRP
have the ability to bind to the bombesin/GRP receptor of SCCL cells
and are at least about 50% homologous, more preferably about 60%
homologous, and most preferably at least about 70% homologous with
the amino acid sequence of native bombesin or GRP. Peptides having
the ability to bind to the bombesin/GRP receptor of SCCL cells and
having at least about 90%, more preferably at least about 95%, and
most preferably at least about 98-99% homology with the amino acid
sequence of native bombesin or GRP are also within the scope of the
invention. Homology refers to sequence similarity between two
peptides having the ability to bind to the bombesin/GRP receptor of
SCCL cells. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences.
[0017] SCCL is a neuroendrocrine tumor that in addition to
bombesin/GRP requires other hormonal growth factors for
proliferation. These other growth factors include, for example,
insulin-like growth factor I, transferrin, vasoactive intestinal
peptide, neurotensin, neuromedin B, neurophysin, tumor necrosis
factor, transforming growth factor alpha, platelet derived growth
factor, the transferin receptor and other peptides. Some of the
receptors for these growth factors have been shown to be expressed
on SCCL cell surface. Therefore these growth factors can also be
used as target cell specific ligands in the instant invention.
Bispecific molecules of the present invention formed with different
ligands specific for a particular target cell, such as those
described above for SCCL cells, can be administered alone or
concurrently with one another to induce target cell death. Because
each growth factor may stimulate a different signal transduction
pathway, concurrent administration of the bispecific molecules may
also have a synergistic effect.
[0018] Ligands of the present invention also include antagonists
against receptors of target cells. Antagonist ligands provide an
additional therapeutic advantage of inhibiting the growth of target
cells upon binding, potentially even in the absence of effector
cells. In fact, some antagonists against GRP receptors have been
shown to possess very potent activity in inhibiting the growth of
SCCL cells in vitro. However, they are quickly degraded by serum
proteases before they can reach the target site, for example a
tumor site, in vivo (see Moody et al. (1993) Life Science
52:1161-1173). However, the presence of small peptide antagonists
in a bispecific molecule of the present invention greatly retards
their degradation in vivo. Therefore, the present invention also
provides the advantage of increasing the efficacy of target cell
receptor antagonists when the antagonists are used as ligands in
the bispecific molecule of this invention. Methods for making
antagonists of the bombesin/GRP receptor are disclosed for example
in Mokotoff et al. J. Med Chem. 35:4696-4703 (1992).
[0019] In addition to SCCL other "target cells" include tumor any
cell which expresses a specific receptor or antibody to which a
ligand can be generated. Such target cells can for example, myeloid
leukemia, ovarian carcinoma or colon carcinoma cells. Other types
of undesirable cells that can be targeted by the bispecific
molecule of the present invention include, for example,
auto-antibody producing lymphocytes for treatment of an autoimmune
disease or an IgE producing lymphocyte for treatment of an allergy.
The target can also be a microorganism (bacterium or virus) or a
soluble antigen (such as rheumatoid factor or other
auto-antibodies).
[0020] The phrase "effector cell specific antibody" as used herein
refers to an antibody or functional antibody fragment. Preferred
antibodies for use in the subject invention bind the Fc receptor of
effector cells at a site which is not bound by endogenous
immunoglobulin. Most preferably, the anti-Fc.gamma. receptor
antibody is a human monoclonal antibody, the binding of which is
not blocked by human immunoglobulin G (IgG). The production and
characterization of these preferred monoclonal antibodies are
described by Fanger et al. in PCT application WO 88/00052 and in
U.S. Pat. No. 4,954,617, the teachings of which are fully
incorporated by reference herein. These antibodies bind to an
epitope of Fc.gamma.RI, Fc.gamma.RII or Fc.gamma.RIII at a site
which is distinct from the Fc.gamma. binding site of the receptor
and, thus, their binding is not blocked substantially by
physiological levels of IgG. Specific anti-Fc.gamma.RI antibodies
useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb
197. The hybridoma producing mAb 32 is available from the American
Type Culture Collection, ATCC Accession No. HB9469.
Anti-Fc.gamma.RI mAb 22, F(ab').sub.2 fragments of mAb 22, and can
be obtained from Medarex, Inc. (Annandale, N.J.).
[0021] Fragments of anti-FcR antibodies can also be used in the
bispecific molecule of the present invention. For example, as shown
in the following example, bispecific molecules between
Lys.sup.3-bombesin and F(ab').sub.2 fragments of mAb 22 have been
constructed and found to exhibit a similar binding profile to both
target and effector cells and are only slightly less active in
inducing cytotoxicity against SCCL cells as compared to bispecific
molecules between Lys.sup.3-bombesin and the whole mAb 22 (See
Tables 1, 4, and 5). Furthermore, since antibody fragments, such as
F(ab').sub.2 fragments, are smaller than whole antibody molecules,
they may more readily reach tumor sites in vivo and therefore be of
greater clinical utility.
[0022] The bispecific molecules of the present invention can be
prepared by conjugating (e.g. ionically or covalently) the ligand
and the antibody or functional antibody fragment using any method
known in the art. For example, a variety of coupling or
cross-linking agents can be used to covalently conjugate the target
cell specific ligand and the effector cell specific antibody.
Examples of cross-linking agents include protein A, carboimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M. A. et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648. Other methods include those described by Paulus (Behring
Inst. Mitt. (1985) No. 78, 118-132); Brennn et al. (Science (1985)
229:81-83), and Gennie et al. (J. Immunol. (1987) 139:2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0023] Effector cells for inducing ADCC against a target cell
include human leukocytes, macrophages, monocytes, activated
neutrophils, and possibly activated natural killer (NK) cells and
eosinophils. Preferred effector cells express Fc.gamma.RI and
include, for example, monocytes and activated neutrophils.
Expression of Fc.gamma.RI has been found to be up-regulated by
interferon gamma (IFN-.gamma.. This enhanced expression increases
the cytotoxic activity of monocytes and neutrophils against target
cells, such as SCCL cells. Accordingly, effector cells are
preferably activated with (IFN-.gamma.), or other cytokines (e.g.
as tumor necrosis factor, lymphotoxin, colony stimulating factor,
and interleukin-2) to increase the presence of Fc.gamma.RI on the
surface of the cells prior to being contacted with a bispecific
molecule of the present invention.
[0024] The bispecific molecules of the present invention can be
used to induce antibody-dependent effector cell-mediated
cytotoxicity (ADCC) against the target cell. To this end,
bispecific molecules of the present invention can be administered
freely in a physiologically acceptable solution or can first be
coupled to an effector cell, forming an "activated effector cell",
prior to being administered to a subject. "Activated effector
cell", as used herein, is intended to include an effector cell, as
previously defined, linked to a bispecific molecule, as previously
defined, so that the effector cell is brought into contact with a
particular target cell via a specific ligand-mediated linkage.
[0025] Activated effector cells can be administered in vivo as a
suspension of cells in a physiologically acceptable solution. The
number of cells administered can be in the order of
10.sup.8-10.sup.9, but will vary depending on the therapeutic
purpose. In general, the amount will be sufficient to obtain
localization of the effector cell at the target cell, and to effect
killing of the cell by ADCC and/or phagocytosis. The term
physiologically acceptable solution, as used herein, is intended to
include any carrier solution which stabilizes the targeted effector
cells for administration in vivo including, for example, saline and
aqueous buffer solutions, solvents, antibacterial and antifungal
agents, isotonic agents, and the like.
[0026] Accordingly, another aspect of the present invention
provides a method of inducing a specific ADCC against a cell in a
subject, comprising administering to the subject the bispecific
molecule or activated effector cell of the invention in a
physiologically acceptable medium. Routes of administration can
vary and include intravenous, intramuscular, and intraperitoneal
administration. Prior to or concurrent with administration of the
bispecific molecule, the subject may be treated in a manner
resulting in increased expression in the target cells of the
particular receptor or antibody to which the target cell specific
ligand of the bispecific molecule will bind. For example, the
subject may be given an agent that upregulates expression of the
particular receptor or antibody on the target cell surface. In a
preferred embodiment of the invention, a bispecific molecule
comprising bombesin or an analogue thereof coupled to a human
anti-FcR monoclonal antibody, is administered alone or coupled to
an effector cell (i.e. an activated effector cell) to a subject
afflicted with small-cell lung cancer to induce ADCC against SCCL
cells.
[0027] A further aspect of the invention provides a method for
using the bispecific molecules as an immunogen. For example, where
the target specific ligand is an autocrine growth factor, a
bispecific molecule comprising the autocrine growth factor ligand
can be administered prophylactically to prevent or retard
proliferation of the target cell. For use as a vaccine, bispecific
molecules of the instant invention can be administered in a
pharmaceutically acceptable solution at a dosage that will evoke an
immune response against the target specific ligand. The optimum
dose may vary depending on factors such as the immune status of the
host. In most cases, the dose of target specific ligand required to
elicit an immune response (as determined by any standard method for
assessment of immune response) should be lower than that which
would be required if the target cell specific ligand were
administered alone.
[0028] The instant invention is further illustrated by the
following Example, which is not intended to limit the invention in
any manner.
EXAMPLE
Construction of Bifunctional Molecule Lys.sup.3-bombesin and mAb22
and Use Thereof in Inducing Monocyte-Mediated Lysis of Small Cell
Cancer of Lung (SCCL) Cells
[0029] Bispecific molecules comprising Lysine.sup.3-bombesin
coupled to the human anti-Fc.gamma.RI monoclonal antibody, mAb 22,
were prepared and assayed for their ability to induce antibody
dependent effector cell-mediated cytotoxicity (ADCC) against
small-cell lung carcinoma (SCCL) cells as follows:
I MATERIALS AND METHODS
[0030] Cell lines: SCCL cell lines, NCI-h69, NCI-H345, and SHP-77
were maintained in RPMI-1640 medium (GIBCO/BRL, Grand Island, N.Y.)
supplemented with 5% fetal calf serum (FCS), 2 mM of L-glutamine,
100 units/ml of penicillin, and 100 .mu.g/ml of streptomycin
(GIBCO/BRL, Grand Island, N.Y.) at 37.degree. C. in a humidified
atmosphere with 5% CO.sub.2. Another SCCL cell line, DMS 273 (Ball,
E. D. unpublished observation) was maintained in Waymouth's MB
752/1 medium (GIBCO/BRL, Grand Island, N.Y.) supplemented with 10%
FCS.
[0031] Antibodies and Reagents: Anti-Fc.gamma.RI (mAb 22),
F(ab').sub.2 fragments of mAb 22, and FITC-labeled mAb 22, were
obtained from Medarex, Inc. (Annandale, N.J.). SCCL-1, an IgG2a mAb
that reacts with the tranferrin receptor on the surface of SCCL
cells was produced according to the method of Petroni, et al (1988)
J. Immunol. 140:3467-3472. Lysine.sup.3-bombesin (Lys-BN), a
bombesin (BN) analog with similar binding affinity to the BN/GRP
receptor (McDonald, et al. (1979) Biochem. Biophys. Res. Commun.
90:227-233, and Spindel, et al. (1984) Proc. Natl. Acad. Sci. USA.
81:5699-5703), and hydroxylamine were purchased from Sigma Chemical
Company (St. Louis, Mo.). Conjugation chemicals,
N-succinimidyl-S-acetyl-thioacetate (SATA) and sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), were
obtained from Pierce Chemical Co. (Rockford, Ill.).
[0032] Protein conjugation. FIG. 1 is a schematic illustration of
the process used to conjugate .sup.3-Lysine and bombesin. The
resulting conjugate, Lys-BN, was freshly dissolved in 0.1 M sodium
phosphate buffer (pH 7.4) containing 2.5 mM EDTA and the SATA was
freshly dissolved in 100% dimethylformamide. The SATA was mixed
with Lys-BN in a final molar ratio of 10:1. After thirty minutes of
reaction at room temperature, the Lys-BN-SATA conjugate was
separated from non-reacted Lys-BN and SATA by reverse phase high
pressure liquid chromatography (R-HPLC) on a Vydac C18 analytical
column. The R-HPLC eluent containing the Lys-BN-SATA was adjusted
to pH 4.0-5.0 by adding 1 M sodium phosphate (pH 8.0). The free
sulfhydryl group was generated by deacetylation with hydroxylaminc
at 4.degree. C. for two hours. A second R-HPLC was performed to
separate Lys-BN-SH. The fraction containing Lys-BN-SH was collected
and neutralized to pH 7.0. The presence of free sulfhydryl group
could be determined via reaction with Ellman's reagent. At the same
time, mAb22 and F(ab').sub.2 fragments of mAb22 were reacted with
Sulfo-SMCC to produce a maleimide-activated antibody. The activated
antibody was separated from unreacted Sulfo-SMCC by centrifugation
through a Centricon 30 apparatus (Amicon, Beverly, Mass.). The
final conjugation between Lys-BN-SH and the activated antibody was
carried out by mixing at equal molar amount at room temperature
overnight. The unreacted Lys-BN-SH and other by-products were
removed by centrifugation through a Centricon 30 apparatus. The
concentration of the bispecific molecule was quantified using a
Bio-Rad DC protein assay (Bio-Rad Laboratories, Richmond, Calif.)
and its purity was checked by SDS-PAGE.
[0033] Immunofluorescence staining. SCCL cells were washed with
ice-cold phosphate buffered saline containing 0.1% bovine serum
albumin and 0.1% sodium azide (PBA solution) twice and incubated
with different amounts of the bispecific molecule at 4.degree. C.
for 1 h in the presence of 100 ug/ml human IgG. The amount of
bispecific molecule added was 1, 5, and 10 .mu.g per
5.times.10.sup.5 cells. After washing three times with PBA
solution, the cells were resuspended and incubated with
FITC-labeled goat F(ab').sub.2 anti-mouse Ig (Caltag Lab., South
San Francisco, Calif.) for 30 min at 4.degree. C. After washing,
the cells were fixed by addition of PBA solution and 2%
paraformaldehyde at 1:1 ratio. Monocytes before and after
IFN-.gamma. stimulation were stained directly with FITC-labeled mAb
22 to evaluate the expression of Fc.gamma.RI.
[0034] The binding of the bispecific molecule to SCCL cell lines
was analyzed by FACScan flow cytometry (Becton-Dickinson, San Jose,
Calif.). The mAb 22 and its F(ab').sub.2 fragments did not stain
the SCCL cells by themselves. A typical flow cytometric analysis
using the bispecific molecule with four SCCL cell lines is
illustrated in FIG. 2. The binding was directly proportional to the
amount of bispecific molecule used to stain the cells. This was
manifested both by an increase in the absolute percentage of cells
stained positively and by an augmentation of the mean fluorescence
intensity (MFI) of the entire cell population, as shown in Table 1.
As the amount of bispecific molecule was increased from 2.5
.mu.g/ml to 25 .mu.g/ml, the percentage of positive cells increased
from 50% to 85%, and the MFI increased from less than 100 to
greater than 200. In general, the bispecific molecule prepared
between the whole antibody of mAb 22 and Lys-BN had a higher MFI
than the one prepared between the F(ab')2 fragments of mAb 22 and
Lys-BN.
1TABLE 1 Conc. Type of IC Cell line (.mu.g/ml) % pos .+-. SD MFI
.+-. SD mAb 22 - BN NCI-H69 2.5 49.7 .+-. 16.6 84.90 .+-. 58.8 (4)
10 67.9 .+-. 13.9 195.2 .+-. 108.0 (4) 25 75.0 .+-. 7.5 234.9 .+-.
121.5 (4) F(ab').sub.2-BN NCI-H69 2.5 43.9 .+-. 9.4 51.40 .+-. 15.9
(4) 10 69.7 .+-. 9.6 112.1 .+-. 37.9 (4) 25 75.0 .+-. 7.7 130.8
.+-. 30.4 (4) mAb 22 - BN NCI-H35 2.5 63.6 .+-. 9.3 86.50 .+-. 24.4
(3) 10 81.9 .+-. 7.0 224.9 .+-. 121.1 (3) 25 84.5 .+-. 3.3 233.7
.+-. 87.0 (3) F(ab').sub.2-BN NCI-H35 2.5 67.5 .+-. 6.3 57.50 .+-.
17.7 (3) 10 80.2 .+-. 3.3 94.10 .+-. 29.8 (3) 25 84.9 .+-. 6.6
129.8 .+-. 10.2 (3) mAb 22 -BN SHP-77 2.5 60.0 .+-. 7.8 64.50 .+-.
14.9 (2) 10 80.6 .+-. 5.4 204.8 .+-. 38.4 (2) 25 85.9 .+-. 1.6
220.6 .+-. 37.0 (2)
[0035] The binding of the bispecific molecule to normal peripheral
lymphocytes and to two leukemia cell lines were also tested. The
results are shown in Table 2. The bispecific molecules did not bind
to normal peripheral lymphocytes because these cells did not
express the Fc.gamma.RI. The mAb 22 and F(ab').sub.2 fragments of
mAb 22 stained both HL-60 and NB4 cells with very dim fluorescence.
There was no significant increase in the MFI when they were stained
with the bispecific molecule, although the percentage of positive
cells increased slightly.
2 TABLE 2 Antibody or IC Cell (25 .mu./ml) % pos MFI NB4 cells mAb
22 63.5 19.6 mAb 22 - BN 62.8 20.4 F(ab').sub.2 19.2 15.1
F(ab').sub.2- BN 45.3 20.1 HL-60 cells mAb 22 17.0 20.4 mAb 22 - BN
19.5 21.8 F(ab').sub.2 1.80 18.7 F(ab').sub.2- BN 17.0 20.4 Normal
lymphocytes mAb 22 1.8 5.9 mAb 22 - BN 3.8 5.7 F(ab').sub.2 0.9 6.1
F(ab').sub.2- BN 2.2 5.8
[0036] Isolation of peripheral monocytes. Leuko-Packs were obtained
from the Pittsburgh Central Blood Blank. Peripheral mononucleated
cells were isolated using Ficoll-Hypaque gradient centrifugation.
The mononuclear cells were washed twice with Hanks' balanced salt
solution (GIBCO/BRL, Grand Island, N.Y.) containing 1 mM EDTA and
then cultured in flask with RPMI-1640 medium containing 10% FCS for
2 h at 37.degree. C. The nonadherent cells were removed. The
adherent cells were detached and the purity of isolated monocytes
was determined by staining with anti-CD14, anti-CD45, anti-CD3,
anti-CD13, and anti-CD56 (Becton-Dickinson). The results were
analyzed by FACScan flow cytometry.
[0037] Activation of monocytes. Human rIFN-.gamma. was a gift from
Dr. Paul Guyer (Dartmouth Medical School, Lebanon, N.H.). The
concentration of rIFN-.gamma. used in this study (200 units/ml) has
been shown to saturate the receptor for rIFN-.gamma. and to induce
a maximal increase in the expression of Fc.gamma.RI on the surface
of monocytes (See Petroni et al. (1988) J. Immunol., 140:3467-3472;
Mendel (1990) J. Immunol., 145:267-275). Isolated monocytes were
incubated with rIFN-.gamma. in RPMI-1640 medium containing 19% FCS
for 18 h at 37.degree. C. before the ADCC assay. The expression of
Fc.gamma.RI on monocytes before and after rIFN-.gamma. incubation
was determined by staining with FITC-labeled mAb 22 and analyzed by
FACScan flow cytometry.
[0038] The binding of the bispecific molecule to peripheral
monocytes before and after incubation with 200 units/ml of
rIFN-.gamma. for 18 h was also tested. The results are shown in
Table 3. rIFN-.gamma. dramatically increased the expression of
Fc.gamma.RI on human monocytes as defined by the increase of MFI
from less than 30 to more than 120. In contrast, there was no
change in the expression of Fc.gamma.RI on human peripheral
lymphocytes. The conjugation of Lys-BN to the antibody did not
interfere with its binding to Fc.gamma.RI.
3 TABLE 3 Before rIFN-.gamma. I incubation After rIFN-.gamma.
incubation % pos .+-. SD MFI .+-. SD % pos .+-. SD MFI .+-. SD
mAb22 83.5 .+-. 2.2 52.0 .+-. 26.0 (2) 85.2V16.7 210.1 .+-. 46.7
(2) F(ab')2 70.7 .+-. 15.6 29.9 .+-. 14.6 (2) 84.7 .+-. 17.2 124.6
.+-. 25.5 (2) mAb22 - BN 86.7 .+-. 3.4 25.7 .+-. 0.10 (2) 92.1 .+-.
7.60 188.0 .+-. 85.1 (2) F(ab')2 - BN 72.3 .+-. 10.7 26.7 .+-. 8.57
(2) 85.8 .+-. 16.3 119.6 .+-. 20.9 (2)
II ANTIBODY-DEPENDENT EFFECTOR CELL-MEDIATED ASSAY
[0039] The assay was performed in 96-well round-bottomed microtiter
plates (Rainin Instrument Co., Woburn, Mass.). The target SCCL
cells were washed once with RPMI-1640 medium and incubated with
sodium [.sup.51Cr] chromate (New England Nuclear, Boston, Mass.)
for 1 h at 37.degree. C. After washing several times, cells were
resuspended in RPMI-1640 medium containing 10% FCS to a
concentration of 1.times.10.sup.5/ml. Activated monocytes serving
as effector cells were suspended in RPMI-1640 medium in a final
concentration of 2.times.10.sup.7/ml. Then, 100 .mu.l of effector
cells was added to the first row of wells and serial dilution was
performed with equal volume of RPMI-1640 medium. 100 .mu.l of
target cells was then added in the wells to yield a final effector:
target cell ratio of 100:1, 50:1, 25:1, and 12:1. In a standard
assay, 5 .mu.g of the bispecific molecule was finally added. The
mAb SCCL-1 was included in each assay as a positive control to
measure the activity of the monocytes. Several other controls were
also incorporated, including incubation of target and effector
cells without any antibody, with irrelevant mouse IgG1, with
unconjugated mAb 22, and incubation of target cells with bispecific
molecule alone. In some assays, 10-fold excessive amounts of Lys-BN
and unconjugated mAb 22 along with the bispecific molecule were
incubated together to determine whether the tumor cell lysis could
be blocked by either of the parental substance.
[0040] The incubation was carried out at 37.degree. C. for 4 h. The
microplates were contrifuged and the supernatant was collected for
estimation of .sup.51 Cr release. Maximal lysis was achieved by the
addition of 100 .mu.l of 5% NP-40 to 100 .mu.l of target cells. The
percentage of cell lysis was calculated as 100.times. (experimental
cpm - spontaneous release mean cpm)/(maximal release mean cpm -
spontaneous mean cpm). In all the assays, spontaneous release from
the target cells was less than 20% of maximal release. Results were
expressed as the mean of triplicate wells.
[0041] For dose-response assays, the bispecific molecule was
serially diluted and added. The effector to target cell ratio in
those assays was 100:1. Since the amount of bispecific molecule
added in a standard assay was 25 .mu.g/ml, we defined the
percentage of tumor cell lysis achieved with that amount of
bispecific molecule as 100% activity. The tumor cell lysis achieved
with diluted bispecific molecule was calculated accordingly.
[0042] The ability of the bispecific molecule to direct
monocyte-mediated tumor cell lysis was tested by a series of
chromium-releasing assays. The results of three experiments using
SHP-77 cell line as target cells are presented in Table 4. Results
are expressed as a percentage of total tumor cells lysed. Since the
source and preparation of effector cells had an impact on cell
lysis, the potency of lysis varied in each experiment. Lysis was
dependent on pretreatment of monocytes with rIFN-.gamma.. Without
such pretreatment, tumor cell lysis was totally abolished. It was
also dependent on effector to target cell ration (E/T ratio). As
shown in FIG. 3, an E/T ratio of 100:1, about 60% of tumor cells
were consistently lysed. This cell lysis decreased to about 25% at
an E/T ratio of 6:1. The mAb 22 itself could induce some
nonspecific lysis of SCCL cells in the presence of stimulated
monocytes at the highest E/T ratio of 100:1. The addition of Lys-BN
did not further increase this nonspecific lysis.
[0043] As shown in Table 5, the bispecific molecule induced SCCL
cell lysis could be blocked by adding excessive amounts of
unconjugated mAb 22 or Lys-BN. Table 5 shows the effect which
conjugation of bombesin and mAb 22 and fragments thereof has on
tumor cell lysis (SCCL cell line SHP-77), as compared with
administration of free bombesin, mAb 22 and fragments thereof. The
presence of irrelevant mouse IgG1 had no effect on the results of
the assay.
[0044] Dose response assays were also performed to test the ability
of various concentrations of bispecific molecule to induce lysis of
SCCL cells. The results of two such experiments are shown in FIG.
4. The peak of activity to mediate tumor cell lysis was seen in a
wide range of concentrations of the bispecific molecule between 25
to 25000 ng/ml.
4 TABLE 4 Type of IC E:T ratio % lysis .+-. SD Experiment 1
mAb22-BN 100:1 60.9 .+-. 7.7 50:1 54.6 .+-. 12.9 25:1 46.6 .+-. 8.3
12.1 44.6 .+-. 4.1 6:1 39.4 .+-. 2.3 Experiment 2 mAb22-BN 100:1
56.4 .+-. 13.1 50:1 49.7 .+-. 14.3 25:1 38.4 .+-. 7.9 12:1 31.8
.+-. 1.5 6:1 39.4 .+-. 2.3 Experiment 3 mAb22-BN 100:1 56.2 .+-.
4.7 50:1 49.1 .+-. 1.8 25:1 33.7 .+-. 5.3 12:1 27.7 .+-. 7.7 6:1
22.5 .+-. 0.9 F(ab)2-BN 100:1 51.9 .+-. 6.4 50:1 43.5 .+-. 8.6 25:1
39.5 .+-. 6.1 12:1 32.8 .+-. 7.6 6:1 28.7 .+-. 1.6
[0045]
5 TABLE 5 Incubation condition % lysis .+-. SD No monocytes 0.1
.+-. 0.1 mAb 22 + monocytes 27.0 .+-. 1.6 mAb 22 + BN + monocytes
24.4 .+-. 2.6 SCCL-1 + monocytes 50.9 .+-. 0.4 mAb 22-BN +
monocytes 49.1 .+-. 1.8 mAb 22-BN + BN + monocytes 33.6 .+-. 3.2
mAb 22-BN + mAb 22 + monocytes 35.0 .+-. 2.0 F(ab')2 + monocytes
14.0 .+-. 1.9 F(ab')2 + BN + monocytes 21.0 .+-. 1.3 SCCL-1 +
monocytes 38.0 .+-. 0.7 F(ab')2-BN + monocytes 43.5 .+-. 8.6
F(ab')2-BN + BN + monocytes 24.2 .+-. 0.6 F(ab')2-BN + F(ab')2 +
monocytes 31.8 .+-. 5.6
EQUIVALENTS
[0046] Although the invention has been described with reference to
its preferred embodiments, other embodiments can achieve the same
results. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific embodiments described herein. Such
equivalents are considered to be within the scope of this invention
and are encompassed by the following claims.
* * * * *