U.S. patent application number 08/484172 was filed with the patent office on 2002-03-14 for therapeutic compounds comprised of anti-fc receptor antibodies.
This patent application is currently assigned to MEDAREX, INC.. Invention is credited to DEO, YASHWANT M., GOLDSTEIN, JOEL, GRAZIANO, ROBERT, SOMASUNDARAM, CHEZIAN.
Application Number | 20020032312 08/484172 |
Document ID | / |
Family ID | 23923055 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020032312 |
Kind Code |
A1 |
DEO, YASHWANT M. ; et
al. |
March 14, 2002 |
THERAPEUTIC COMPOUNDS COMPRISED OF ANTI-FC RECEPTOR ANTIBODIES
Abstract
Multispecific multivalent molecules which are specific to an Fc
receptor (FcR), and therapeutic uses and therapeutic uses and
methods for making the molecules are described.
Inventors: |
DEO, YASHWANT M.; (AUDUBON,
PA) ; GOLDSTEIN, JOEL; (PISCATAWAY, NJ) ;
GRAZIANO, ROBERT; (FRENCHTOWN, NJ) ; SOMASUNDARAM,
CHEZIAN; (ALLENTOWN, PA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
|
Assignee: |
MEDAREX, INC.
|
Family ID: |
23923055 |
Appl. No.: |
08/484172 |
Filed: |
June 7, 1995 |
Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
A61P 37/02 20180101;
C07K 16/3007 20130101; C07K 2317/73 20130101; C07K 16/283 20130101;
C07K 16/2863 20130101; C07K 2317/24 20130101; A61K 38/00 20130101;
C07K 2319/00 20130101; C07K 16/32 20130101; A61P 35/00 20180101;
C07K 2317/732 20130101; C07K 14/33 20130101; C07K 14/485 20130101;
A61P 37/00 20180101 |
Class at
Publication: |
530/387.3 |
International
Class: |
C12P 021/08 |
Claims
We claim:
1. A recombinant multispecific molecule comprising an anti-Fc
receptor portion and an anti-target portion.
2. A recombinant multispecific molecule of claim 1, wherein at
least one of the anti-Fc receptor portion or the anti-target
portion is humanized.
3. A recombinant multispecific molecule of claim 2, wherein the
anti-Fc receptor portion is an antibody fragment.
4. A recombinant multispecific molecule of claim 2, wherein the
anti-target portion is an antibody fragment or a ligand.
5. A recombinant multispecific molecule of claim 2, wherein the
anti-Fc receptor portion binds an Fc receptor on an effector cell
at a site which is not bound by endogenous immunoglobulin.
6. A recombinant multispecific molecule of claim 2, wherein the
anti-Fc receptor portion binds an Fc.gamma. receptor.
7. A recombinant multispecific molecule of claim 6, wherein the
Fc.gamma. receptor is a Fc.gamma. RI receptor.
9. A recombinant multispecific molecule of claim 4, wherein the
target is a cancer cell.
9. A recombinant multispecific molecule of claim 4, wherein the
target is an infectious agent.
10. A recombinant multispecific molecule of claim 4, wherein the
target is an antibody-producing cell.
11. A recombinant multispecific molecule of claim 3, wherein the
target is a breast or ovarian cancer cell.
12. A recombinant multispecific molecule of claim 11, wherein the
target is a HER 2/neu expressing cell.
13. A recombinant multispecific molecule of claim 12, wherein the
anti-target portion is antibody 520C9.
14. A recombinant multispecific molecule of 4, wherein the ligand
is epidermal growth factor
15. A multivalent molecule comprising 1) at least one anti-Fc
receptor portion, and 2) at least one anti-target portion.
16. A multispecific molecule having one anti-FcR, one anti-target
portion and one anti-enhancement factor portion.
17. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds a cancer cell.
18. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds a carcinoma.
19. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds a sarcoma.
20. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds a pathogen.
21. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds an FcR at an epitope different and
distinct from the first FcR monoclonal antibody.
22. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds a soluble protein/peptide.
23. The multivalent molecule of claim 15 or 16, wherein the
anti-target portion binds any molecule capable of generating an
immune response or monoclonal antibody.
24. The multivalent molecule of claim 15 or 16, wherein the binding
of the anti-FcR portion to the Fe receptor is not blocked by human
immunoglobulin G
25. A multivalent molecule of claim 15 or 16 wherein the anti-FcR
portion binds specifically to FcR.
26. The multivalent molecule of claim 16, wherein the anti-EF
portion binds specifically to a T cell surface antigen.
27. The multivalent molecule of claim 16, wherein the anti-EF
portion binds CD3.
28. The multivalent molecule of claim 16, wherein the anti-EF
portion binds a second epitope on FcR.
29. The multivalent molecule of claim 16, wherein the anti-EF
portion binds a target cell.
30. The multivalent molecule of claim 16, wherein the anti-EF
portion binds a second FcR.
31. A multispecific molecule of claim 16, wherein the anti-EF
portion binds specifically to a myeloid-associated cytotoxic
trigger molecule.
32. A multispecific molecule having one portion that binds
specifically to Fc.gamma.RI, one portion that binds specifically to
one epitope of the target antigen and one portion that binds
specifically to a second site on the same target cell.
33. A method of treating cancer comprising administration of a
therapeutically effective amount of a multivalent, multispecific
molecule.
34. A method of treating autoimmune disease comprising
administration of a therapeutically effective amount of a
multivalent, multispecific molecule.
35. A method of treating removing unwanted pathogens comprising
administration of a therapeutically effective amount of a
multivalent, multispecific molecule.
Description
BACKGROUND OF THE INVENTION
[0001] Immunoglobulins (Igs) are composed of two heavy and two
light chains, each of which contains an NH.sub.2-terminal
antigen-binding variable domain and a COOH-terminal constant domain
responsible for the effector functions of antibodies. The
COOH-terminal domains of Ig heavy chains form the Fc region and are
involved in triggering cellular activities through interaction with
specific receptors known as Fe receptors (FcRs). Fc receptors for
all Ig classes, or isotypes, (e.g., IgG (Fc.gamma.R), IgE
(Fc.epsilon.R), IgA (Fc.alpha.R), IgM (Fc.mu.R) and IgD
(Fc.delta.R) have been identified. The different biological
activities of antibodies of different isotypes are based in part on
their ability to bind to different FcR expressed on different
immune (effector) cells (Fridman, W. H. (September 1991) The FASEB
Journal Vol. 5. 2684-2690). Murine antibodies, which are directed
against FcRs have been made (See e.g. U.S. Pat. No. 4,954,617
entitled Monoclonal Antibodies To Fc Receptors for Immunoglobulin G
on Human Mononuclear Phagocytes and International Patent
Application Publication No. WO 91/05871 entitled Monoclonal
Antibody Specific For IgA Receptor).
[0002] Murine monoclonal antibodies can be useful as human
therapeutics and can be produced free of contamination by human
pathogens such as the hepatitis or human inmunodeficiency virus.
However, use of murine monoclonal antibodies in some human
therapies, have resulted in the development of an immune response
to the "foreign" murine proteins. This response has been termed a
human anti-mouse antibody or HAMA response (Schroff, R. et al.
(1985), Cancer Res., 45, 879-885) and is a condition which causes
serum sickness in humans and results in rapid clearance of the
murine antibodies from an individual's circulation. The immune
response in humans has been shown to be against both the variable
and the constant regions of murine immunoglobulins.
[0003] Recombinant DNA technology can be used to alter antibodies,
for example, by substituting specific immunoglobulin regions from
one species with immunoglobulin regions from another species.
Neuberger et al. (Patent Cooperation Treaty Patent Application No.
PCT/GB85/00392) describes a process whereby the complementary heavy
and light chain variable domains of an Ig molecule from one species
may be combined with the complementary heavy and light chain Ig
constant domains from another species. This process may be used to
substitute the murine constant region domains to create a
"chimeric" antibody which may be used for human therapy. A chimeric
antibody produced as described by Neuberger et al. has a human Fc
region for efficient stimulation of antibody mediated effector
functions, such as complement fixation, but still has the potential
to elicit an immune response in humans against the murine
("foreign") variable regions.
[0004] Winter (British Patent Application Number GB2188538A)
describes a process for altering antibodies by substituting the
complementarity determining regions (CDRs) with those from another
species. This process may be used to substitute the CDRs from the
murine variable region domains of a monoclonal antibody with
desirable binding properties (for instance to a human pathogen)
into human heavy and light chain Ig variable region domains. These
altered Ig variable regions may then be combined with human Ig
constant regions to create antibodies which are totally human in
composition except for the substituted murine CDRs. The "reshaped"
or "humanized" antibodies described by Winter elicit a considerably
reduced immune response in humans compared to chimeric antibodies
because of the considerably less murine components. Further, the
half life of the altered antibodies in circulation should approach
that of natural human antibodies. However, as stated by Winter,
merely replacing the CDRs with complementary CDRs from another
antibody which is specific for an antigen such as a viral or
bacterial protein, does not always result in an altered antibody
which retains the desired binding capacity. In practice, some amino
acids in the framework of the antibody variable region interact
with the amino acid residues that make up the CDRs so that amino
acid substitutions into the human Ig variable regions are likely to
be required to restore antigen binding.
[0005] Bispecific molecules, (e.g., heteroantibodies) comprising an
anti-Fc receptor portion and an anti-target portion have been
formulated and used therapeutically, e.g., for treating cancer
(e.g. breast or ovarian) or pathogenic infections (e.g., HIV (See,
e.g., International Patent Application Publication No. WO 91/05871
entitled Bispecific Heteroantibodies With Dual Effector Functions;
and International Patent Application Publication No. WO 91/00360
entitled Bispecific Reagents for AIDS Therapy). In addition,
bispecific molecules, which recognize antigens and antigen
presenting cells can be administered to a subject to stimulate an
immune response (See, e.g., International Patent Application
Publication No. WO 92/05793 entitled Targeted Immunostimulation
With Bispecific Reagents).
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention features multispecific,
multivalent molecules, which minimally comprise an anti-Fc receptor
portion, an anti-target portion and optionally an anti-enhancement
factor (anti-EF) portion. In preferred embodiments, the anti-Fc
receptor portion is an antibody fragment (e.g., Fab or (Fab').sub.2
fragment), the anti-target portion is a ligand or antibody fragment
and the anti-EF portion is an antibody directed against a surface
protein involved in cytotoxic activity. In a particularly preferred
embodiment, the recombinant anti-FcR antibodies, fragments or
ligand are "humanized" (e.g., have at least a portion of a
complementarity determining region (CDR) derived from a non-human
antibody (e.g., murine) with the remaining portion(s) being human
in origin).
[0007] In another aspect, the invention features methods for
generating multispecific molecules. In one embodiment, both
specificities are encoded in the same vector and are expressed and
assembled in a host cell. In another embodiment, each specificity
is generated recombinantly and the resulting proteins or peptides
are conjugated to one another via sulfhydryl bonding of the
C-terminus hinge regions of the heavy chain. In a particularly
preferred embodiment, the hinge region is modified to contain only
one sulfhydryl residue, prior to conjugation.
[0008] Recombinant antibodies and multispecific molecules generated
therefrom can be engineered to have increased affinity and
specificity. Further, humanized antibodies are typically less
immunogenic when administered to a human. Other features and
advantages of the present invention will become better understood
by reference to the following Detailed Description and Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a diagram showing the nucleotide and amino acid
residue sequences of a portion of the hinge region of a humanized
Fc.gamma. RI antibody, H22. [A] that was altered to produce a
truncated single-sulfhydryl version [B] and then altered fiber to
engineer two unique cloning sites [C]. Underlined nucleotides
indicate changes from the previous sequence. Overlined nucleotides
are the recognition sequences for the indicated restriction
sites.
[0010] FIG. 2 is a schematic representation of the generation of
anti-Fc receptor-ligand bispecific molecules.
[0011] FIG. 3 is a schematic representation of the flow cytometric
assay used for testing the activity of the humanized Fc.gamma.
receptor-epidermal growth factor fusion protein.
[0012] FIG. 4 is a graph, which plots Mean Fluorescence Intensity
(MFI) an indication of the binding of various concentrations of
epidermal growth factor (EGF) fusion protein (H22-EGF fusion) and
the fully humanized bispecific (BsAb) H447 to EGF receptor (EGFR)
expressing 1483 cells.
[0013] FIG. 5 is a graph, which plots the binding of various
concentrations of the EGF fusion protein or the BsAb H447 to A431
cells in the presence and absence of murine antibody M425, which
binds EGFR.
[0014] FIG. 6 is a graph, which plots the antibody dependent
cytotoxicity (ADCC) resulting from the binding of various
concentrations of the EGF fusion protein, BsAb H447 or the H425
antibody to A431 cells.
[0015] FIG. 7 is a a bar graph which plots the ADCC resulting from
the binding of EGF fusion protein, BsAb H447 or the H425 antibody
in the presence of media alone, media containing 25% human serum
(HS) or media containing a fab fragment of the Fc.gamma. receptor
antibody m22.
[0016] FIG. 8 is a schematic representation of the flow cytometric
assay used for testing the activity of BsAb 447 generated either by
The o-PDM or the DTNB method.
[0017] FIG. 9 is a graph, which plots the MFI of various
concentrations of o-PDM and DTNB derived BsAb 447 to EGFR and
Fc.gamma.RI expressing A431 cells.
[0018] FIG. 10 is a graph, which plots the antibody dependent
cytotoxicity resulting from the binding of o-PDM and DTNB derived
BsAb 447 to A431 cells.
[0019] FIG. 11 is a flow chart that depicts the construction of
trispecific antibodies.
[0020] FIG. 12 depicts the transformation of a bivalent, bispecific
antibody into a trivalent, bispecific antibody. The bivalent,
bispecific conjugate is reduced and mixed with o-PDM-treated 520C9
Fab' resulting in the TsAb.
[0021] FIG. 13 depicts a bifunctional fluorescence-activated cell
sorting assay for HER2/neu (panel A) and EGFR (panel B).
[0022] FIG. 14 is a graph which plots the binding of various
concentrations of antibody, either BsAb or TsAb, to target cells.
Mean Fluorescence Intensity (MFI) increases as Ab binding
increases. It shows that the TsAb bound both HER2/neu on SKBr-3
cells and soluble Fc.gamma.RI simultaneously in a dose-dependent
fashion.
[0023] FIG. 15 is a graph that shows the TsAb bound both EGFR on
A431 cells and soluble Fc.gamma.RI simultaneously in a
dose-dependent fashion. The assay is similar to that used in FIG.
14.
[0024] FIG. 16 is a graph that shows the TsAb, M22 x H425x 520C9,
and the BsAb, M22 x 520C9 were capable of inducing ADCC of SKBR-3
cells but the BsAb, M22 x H425, was not. Various concentrations of
antibodies were incubated with SKBR-3 cells and pre-activated
PMNs.
[0025] FIG. 17 is a graph that shows the TsAb, M22 x H425x 520C9,
and the BsAb, M22 x H425 were capable of inducing ADCC of A431
cells but the BsAb, M22 x 520C9, was not. The assay was performed
in a similar manner as the assay in FIG. 16.
[0026] FIG. 18 is a flow chart for a whole blood modulation assay
(panel A) and the results from the assay (panel B). This trivalent
antibody rapidly modulates F.sub.c.gamma.RI from the surface of
monocytes.
DETAILED DESCRIPTION
[0027] Multispecific Molecules
[0028] The instant invention relates to recombinantly multispecific
molecules. Multispecific molecules can include bispecific molecules
comprised of an anti-Fc receptor portion and art anti-target
portion, wherein at least one of said portions is constructed using
recombinant DNA techniques. Multispecific molecules can also
include molecules, which are comprised of more than one anti-Fc
receptor portion or anti-target portion; or molecules comprised of
at least one anti-Fe receptor, one anti-target portion and
additionally a portion or portions that recognize another molecule,
wherein at least one of said portions is constructed using
recombinant DNA techniques.
[0029] An "anti-Fc receptor portion" refers to an antibody, a
functional antibody fragment (e.g., Fab fragment) or a ligand that
recognizes and binds an Fc receptor on an effector cell. Preferred
antibodies for use in the subject invention bind the Fc receptor on
an effector cell at a site which is not bound by endogenous
immunoglobulin. Most preferably, the anti-Fc receptor portion binds
a human Fc.gamma.R (i.e., Fc.gamma.RI, Fc.gamma.RII or
Fc.gamma.RIII). Preferred humanized anti-Fc.gamma.R monoclonal
antibodies are described in PCT application WO 94/10332 and U.S.
Pat. No. 4,954,617, the teachings of which are fully incorporated
herein by reference).
[0030] An "effector cell", as used herein refers to an immune cell.
Specific effector cells express specific Fc receptors and carry out
specific immune functions. For example, monocytes, macrophages,
neutrophils and dendritic cells, which express Fc.gamma.RI are
involved in both specific killing of target cells and presenting
antigens to other components of the immune system. The expression
of a particular FcR on an effector cell can be regulated by humoral
factors such as cytokines. For example, 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 Fc.gamma.RI cells against targets.
[0031] The recombinant antibodies or antibody fragments, which
specifically bind to an Fc receptor are preferably "humanized" i.e.
derived from a human antibody, but having at least a portion of a
complementarity determining region (CDR) derived from a nonhuman
antibody. The portion being selected to provide specificity of the
humanized antibody for a human Fc receptor. The humanized antibody
has CDRs derived from a non-human antibody and the remaining
portions of the antibody molecule are human.
[0032] The antibody may be whole, i.e. having heavy and light
chains or any fragment thereof, e.g., Fab or (Fab').sub.2 fragment.
The antibody further may be a light chain or heavy chain dimer, or
any minimal fragment thereof such as a Fv or a single chain
construct as described in Ladner et al. (U.S. Pat. No. 4,946,778,
issued Aug. 7, 1990), the contents of which is expressly
incorporated by reference.
[0033] The humanized antibody or fragment may be any human antibody
capable of retaining non-human CDRs. The preferred human antibody
is derived from known proteins NEWM and KOL for heavy chain
variable regions (VHs) and REI for Ig kappa chain, variable regions
(VKs).
[0034] The portion of the non-human CDR inserted into the human
antibody is selected to be sufficient for allowing binding of the
humanized antibody to the Fc receptor. A sufficient portion may be
selected by inserting a portion of the CDR into the human antibody
and testing the binding rapacity of the created humanized antibody
using the enzyme lined immunosorbent assay (ELSA).
[0035] All of the CDRs of a particular human antibody may be
replaced with at least a portion of a non-human CDR or only some of
the CDRs may he replaced with non-human CDRs. It is only necessary
to replace the number of CDRs required for binding of the humanized
antibody to the Fe receptor. A non-human CDR derived from a murine
monoclonal antibody (mab), mab 22, is described in International
Patent Application Publication No. WO 94/10332, the contents of
which are fully incorporated herein by reference. The mab 22
antibody is specific to the Fe receptor and further is described in
U.S. Pat. No. 4,954,617, issued Sep. 4, 1988, the contents of which
are also expressly incorporated by reference. The humanized mab 22
antibody producing cell line was deposited at the American Type
Culture Collection on Nov. 4, 1992 under the designation HA022CL1
and has the accession no. CRL 11177.
[0036] An antibody can be humanized by any method which is capable
of replacing at least a portion of a CDR of a human antibody with a
CDR derived from a non-human antibody. Winter describes a method
which may be used to prepare the humanized antibodies of the
present invention (UK Patent Application GB 2188638A, filed on Mar.
26, 1987), the contents of which is expressly incorporated by
reference. The human CDRs may be replaced with non-human CDRs using
oligonucleotide site-directed mutagenesis as described in
International Patent Application Publication Number: WO 94/10332
entitled Humanized Antibodies to Fc Receptors for Immunoglobulin G
on Human Mononuclear Phagocytes.
[0037] In addition to an anti-Fc receptor portion, the claimed
multispecific molecules can comprise an "anti-target portion", i.e.
an antibody, a functional antibody fragment or a ligand that
recognizes and binds a pathogen (e.g., virus, bacteria, fungi), a
pathogen infected cell, a cancer or tumor cell (e.g., breast,
ovarian, prostate, etc.) or other unwanted cell in a subject (e.g.,
a human or animal). Additionally, the target portion may be
directed against an antigen. A preferred embodiment contains an
antigen that can be used to stimulate the immune system, for
example, in instances of chronic infection, to deplete antigen in
the circulation, and to treat tumors. A particularly preferred
embodiment has an antigen that is attached to a multivalent
molecule containing an anti-FcR antibody.
[0038] The multispecific, multivalent molecules of the invention
may also include an "anti -enhancement factor (anti-EF) portion".
The "anti-enhancement factor portion" can be an antibody,
functional antibody fragment or a ligand that binds to an antigen
and thereby results in an enhancement of the effect of the
anti-F.sub.c receptor portion or the anti-target portion. The
"anti-enhancement factor portion" can bind an F.sub.c receptor or a
target. A multivalent molecule comprising an anti-target portion
that binds to one target cell antigen and an anti-enhancement
factor portion that binds to a different target antigen is
particularly useful where the target cell undergoes antigen
modulation or antigenic variation (e.g., as has been described for
certain parasites (such as trypanosomes). Alternatively, the
anti-enhancement factor portion can bind an entity that is
different from the entity to which the anti-target or anti-F.sub.c
receptor portion binds. For example, the anti-enhancement factor
portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28,
CM4, CD40, ICAM-1 or other immune cell that results in an increased
immune response against the target).
[0039] Methods for Making Multispecific Molecules
[0040] The multispecific molecules described above can be made by a
number of methods. For example, both specificities can be encoded
in the same vector and expressed and assembled in the same host
cell. This method is particularly useful where the multispecific
molecule is a ligand x fab fusion protein as described in the
following Example 2.
[0041] Alternatively, each specificity of a multispecific molecule
can be generated separately and the resulting proteins or peptides
conjugated to one another. For example, two humanized antibodies
can be conjugated via sulfhydryl bonding of the C-terminus hinge
regions of the two heavy chains. In a particularly preferred
embodiment, the hinge region is modified to contain an odd number
of sulfhydryl residues, preferably one, prior to conjugation.
[0042] The bispecific molecules of the present invention can be
prepared by conjugating the anti-FcR and anti-target portions using
methods described in the following Example or those well-known in
the art. For example, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-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
Ins. Mitt. (1985) No. 78, 118-132); Brennan et al. (Science (1985)
229:81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0043] Therapeutic Uses for Multispecific Molecules
[0044] Based on their ability to bind FcR bearing immune cells and
specific target cells, a specific multispecific molecule can be
administered to a subject to treat or prevent a variety of diseases
or conditions, including: cancer (e.g., breast, ovarian, small cell
carcinoma of the lung), pathogenic infections (e.g., viral (such as
HIV)), protozoan (such as toxoplasma gondii), fungal (such as
candidiasis); an autoimmunity (e.g. immune thrombocytopenia purpura
and systemic lupus). The multispecific multivalent can also be
administered prophylactically to vaccinate a subject against
infection by a target cell.
[0045] For use in therapy, an effective amount of an appropriate
multispecific molecule can be administered to a subject by any mode
that allows the molecules to exert their intended therapeutic
effect Preferred routes of administration include oral and
transdermal (e.g., via a patch). Examples of other routes of
administration include injection (subcutaneous, intravenous,
parenteral, intraperitoneal, intrathecal, etc.). The injection can
be in a bolus or a continuous infusion.
[0046] A multispecific molecule can be administered in conjunction
with a pharmaceutically acceptable carrier. As used herein, the
phrase "pharmaceutically acceptable carrier" is intended to include
substances that can be coadministered with a multispecific molecule
and allows the molecule to perform its intended function. Examples
of such carriers include solutions, solvents, dispersion media,
delay agents, emulsions and the like. The use of such media for
pharmaceutically active substances are well known in the art. Any
other conventional carrier suitable for use with the molecules
falls within the scope of the instant invention.
[0047] The language "effective amount" of a multispecific molecules
refers to that amount necessary or sufficient to realize a desired
biologic effect. For example, an effective amount of a
multispecific molecule, in which the anti-target portion recognizes
a pathogenic cell could be that amount necessary to eliminate a
tumor, cancer, or bacterial, viral or fungal infection. The
effective amount for any particular application can vary depending
on such factors as the disease or condition being treated, the
particular multispecific molecule being administered, the size of
the subject, or the severity of the disease or condition. One of
ordinary skill in the art can empirically determine the effective
amount of a particular multispecific molecule without necessitating
undue experimentation.
[0048] The following examples are provided as a further
illustration of the present invention and should in no way be
construed as being limiting.
EXAMPLES
Example 1
[0049] Production of Bispecific Antibody Comprising Murine or
Humanized Antibodies Specific for an Fc Receptor and an Anti-her 2
neu Antibody
[0050] Monoclonal Antibodies
[0051] The anti-Fc.gamma.RI monoclonal antibodies (mAbs), M22,
M32.2 and 197 were purified from hybridoma supernatant by ion
exchange chromatgraphy and DZ33, a human anti-HIV-1 IgG1 mAb, was
purified from hybridoma supernatant by protein A affinity
chromatography (Pharmacia, Piscataway, N.J.) and gel filtration.
M32.2 was deposited at the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Md. 20852, on Jul. 1, 1987 and has been
designated with ATCC Accession No. HB9469.
[0052] Cell Lines
[0053] The murine myeloma NSO (ECACC 85110503) is a non-Ig
synthesizing line and was used for the expression of recombinant
mAbs. NSO cells were cultivated in DMEM plus 10% fetal bovine serum
(FBS, Gibco, Paisley, U.K.). SKBR-3 is a human breast carcinoma
cell line which overexpresses the HER2/neu protooncogene (ATCC,
Rockville, Md.) and was cultivated in Iscove's Modified Dulbecco's
Medium (IMDM, Gibco, Grand Island, N.Y.). U937 is a monocytoid cell
line that expresses Fc.gamma.RI and was obtained from ATCC and
grown in RPM-1640 plus 10% FBS (Gibco, Grand Island, N.Y.).
[0054] Cloning Murine Immunoglobulin V Region Genes
[0055] Cytoplasmic RNA from the murine hybridoma 22 was prepared as
described in Favaloro et al. (Favaloro, J., R. Treisman and R.
Kamen (1982) Transcription maps of polyoma-specific RNA: analysis
by two-dimensional S1 gel mapping. Meth. Enzymol. 65:718). The Ig V
region cDNAs were made from RNA via reverse transcription initiated
from primers CG1FOR and CK2FOR as described in International Patent
Application Publication Number WO 94/10332 entitled, Humanized
Antibodies to Fc Receptors for Immunoglobulin G on Human
Mononuclear Phagocytes. The cDNA synthesis was performed under
standard conditions using 100 U MMLV reverse transcriptase (Life
Technologies, Paisley, UK). The V.sub.H and V.sub..kappa. cDNAs
were amplified by PCR, (Orlandi, R., D. H. Gussow, P. T. Jones and
G. Winter (1989) (Cloning immunoglobulin variable domains for
expression by the polymerase chain reaction), Proc. Natl. Acad.
Sci. USA 86:3833), using the cDNA primers in concert with SH2BACK
and VK7BACK as described in International Patent Application
Publication Number WO 94/10332. Amplified V.sub.H and V.sub..kappa.
DNA were purified, cloned into M13, and sequenced by the dideoxy
method using T7 DNA polymerase (Phamacia, Piscataway, N.J.).
[0056] Construction of Chimeric Antibody Genes
[0057] To facilitate cloning of murine V region DNA into expression
vectors, restriction sites were placed in close proximity to the
termini of both M22 V region genes. For V.sub.H, a 5' PstI site and
a 3' BstEII site were introduced into a cloned murine V.sub.H gene
by PCR using VH1BACK and VH1FOR (Id.). For V.sub..kappa. a 5' PvuII
site and a 3' Bgl II site were introduced into a cloned murine
V.sub..kappa. gene by PCR using primers VK1BACK and VK1FOR (Id.).
In some instances, these primers changed one or more amino acids
from those naturally occurring. These V region genes (ChVH and
ChVK) were cut with the appropriate restriction enzymes and cloned
into M13VHPCR1and M13VKPCR1 (Id.) which contain an Ig promoter,
signal sequence and splice sites. The DNA were excised from M13 as
HindIII-BamHI fragments and cloned into the expression vectors
pSVgpt and pSVhyg containing human IgG1, (Takahashi, N. et al.,
(1982), Structure of human immunoglobulin gamma genes: implications
for evolution of a gene family, Cell, 29:671), and human kappa
constant, (Hieter, R. A. et al., (1980) Cloned human and mouse
kappa immunoglobulin constant and J region genes conserve homology
in functional segments, Cell 22:197), region genomic DNA.
[0058] Construction of Humanized Antibody Genes
[0059] Two humanized heavy chains were constructed and were based
on human V.sub.Hs of NEWM, (Poljak R. J. et al., Amino acid
sequence of the V.sub.H region of a human mycloma immunoglobulin,
(IgG New), Biochemistry, 16:3412), and KOL, (Marquat, M. et al.,
(1980) Crystallographic refinement and atomic models of the intact
immunoglobulin molecule Kol and its antigen-binding fragment at
3.0A and 1.9A resolution, J. Mol. Biol. 141:369. The humanized
light chain was derived from the human Bence-Jones protein REI,
(Epp, O. et al, (1974) Crystal and molecular structure of a dimer
composed of the vandible portion of the Bence-Jones protein REI,
Eur. J. Biochem. 45:513), with some framework region (FR) changes.
The modifications were made to make the V.sub..kappa. domain more
typical of human subgroup I, and included replacement of Thr39,
Leu104, Gln105 and Thr107 with Lys39, Val104, Glu105 and Lys107. In
addition, Met4 was changed to Leu4 to accommodate a PvuII
restriction site.
[0060] DNA containing the NEWM V.sub.H and REI V.sub..kappa. FRs
with irrelevant CDRs were cloned into the vectors M13VHPCR1 and
M13VKPCR1 (Favaloro et al. Supra). DNA encoding the KOL V.sub.H was
constructed by a series of sequential PCRs, using
oligodeoxyribonucleotides encoding KOL FR amino acids and
irrelevant CDRs. The constructs were then cloned into
M13VHPCR1.
[0061] Oligodeoxyribonucleotides were synthesized to encode the mAB
M22 CDRs which were flanked by nucleotides corresponding to the
human FRs. For the humanized V.sub.H based on NEWM, the primers
included murine FR amino acids Phe27, Ile28 and Arg71 since these
were likely to influence antigen binding, (Chothia, C. and A, M.
Lesk (1987), Canonical structures for the hypervariable regions of
immunoglobulins, J. Mol. Biol., 196:901; Tramontano, A. et al.,
(1990), Framework residue 71 is a major determinant of the position
and conformation of the second hypervariable region in V.sub.H
domains of immunoglobulins, J. Mol. Biol., 215:175). For the
humanized V.sub..kappa., murine amino acid Phe71 was similarly
included as a residue capable of affecting affinity, (Foote, J. and
G. Winter, (1992), Antibody framework residues affecting the
conformation of the hypervariable loops, J. Mol. Biol. 224:487. No
murine FR residues were included in the KOL V.sub.H.
Oligodeoxyribonucleotides were 5'-phosphorylated and with the M13
universal forward primer annealed to the human V region genes
cloned in M13 in reactions containing M13 ssDNA template. The DNA
was extended and ligated with 2.5 U T7 DNA polymerase (United
States Biochemicals, Cleveland, Ohio) and 0.5 U T4 DNA ligase
(Gibco BRL, Grand Island, N.Y.). The mutated strand was
preferentially amplified from the extension/ligation mixture using
M13 reverse sequencing primer with 1 U Vent DNA polymerase (New
England Biolabs, Beverly, Mass.) and was then amplified by PCR
using both M13 forward and reverse primers. Product DNA was cut
with BamH1 and HindIII, cloned into M13 and triple CDR-grafted
mutants identified by DNA sequencing.
[0062] M13 clones containing the humanized V regions were sequenced
in their entirety to ensure the absence of spurious mutations. RF
DNA from the confirmed clones was digested with HindIII and BamHI,
cloned into pSVgpt or pSVhyg and human IgG1 or human kappa constant
regions added exactly as described for the construction of the
chimeric antibody genes.
[0063] Expression and Purification of Recombinant mAbs
[0064] Heavy (5 .mu.g) and light (10 .mu.g) chain expression
vectors were digested with PvuI, ethanol precipitated and dissolved
in 50 .mu.l water. NSO cells (1-2.times.10.sup.7) were harvested by
centrifugation, resuspended in 0.5 ml DMEM and mixed with the DNA
in a 0.4 cm electroporation cuvette. After 5 min. on ice the cells
were given a single pulse of 170 V, 960 .mu.F (GenePulser, Bio-Rad,
Melville, N.Y.) and incubated further for 15 min. on ice. The cells
were allowed to recover in DMEM for 24-48 hours. The medium was
then made selective by the addition of mycophenolic acid (0.8
.mu.g/ml) and xanthine (250 .mu.g/ml). Aliquots of 200 .mu.l were
distributed into 96-well plates. After a further 10-12 days, cells
from the wells containing the highest levels of antibody measured
by ELISA were selected and cloned by limiting dilution.
[0065] Antibodies were purified from overgrown cultures by protein
A affinity chromatography (Boehringer Mannheim, Lewes, U.K.)
Concentrations were determined by measuring A.sub.280 nm and
confirmed by ELISA and SDS-PAGE.
[0066] ELISA for Measurement of Antibody Binding
[0067] The wells of a microtiter plate were coated with goat
anti-human IgM antibodies (Sera-Lab, Crawley Down, U.K.) in 50 mM
bicarbonate buffer, pH 9.6. The plate was blocked with 1% BSA and
followed by the addition of a soluble fusion protein consisting of
the extracellular domain of human Fc.gamma.RI and human IgM heavy
chain (sFc.gamma.RI-.mu.) obtained from transiently transfected COS
cells (the expression vector was kindly provided by Dr. Brian Seed,
Massachusetts General Hospital, Boston, Mass.). Recombinant 22 or
control mAbs were then added in the presence of excess (2.2
.mu.g/well) human IgG1 antibodies (Sigma, St. Louis, Mo.) that
contained .lambda. light chains to block the non-specific binding
of the test mAbs via their Fc portion. Bound 22 mAbs were detected
with peroxidase-labeled goat anti-human kappa chain antibodies
(Sera-Lab, Crawley Down, U.K.) and o-phenylenediamine.
[0068] Fluoresceination of Antibodies
[0069] The pH of mAb solution was adjusted to 9.3 by the addition
of 0.1 M Na.sub.2CO.sub.3. Fluorescein iso-thiocyanate (FITC)
(Sigma, St. Louis, Mo.) was dissolved in DMSO at a concentration of
2 mg/ml. Forty .mu.g of FITC was added for each milligram of mAb
and incubated for two hours at room temperature. The
fluoresceinated mAb was separated from the free FITC by G-25
chromatography.
[0070] Preparation of Blood Cells
[0071] Buffy coats were prepared from heparinized whole venous
blood. Whole blood was diluted with RPMI containing 5% dextran at a
ratio of 2.5:1 (v/v). The erythrocytes were allowed to sediment for
45 minutes on ice, then the cells in the supernatant were
transferred to a new tube and pelleted by centrifugation. The
residual erythrocytes were removed by hypotonic lysis. The
remaining lymphocytes, monocytes and neutrophils were kept on ice
until use in binding assays. For some experiments, neutrophils were
separated from mononuclear cells by ficoll hypaque (Phamacia,
Piscataway, N.J.) gradient separation. To up-regulate Fc.gamma.RI,
neutrophils and mononuclear cells were treated with cytokines.
Cultures of mononuclear cells were incubated at 37.degree. C., 5%
CO.sub.2 for 48 hours in teflon dishes at 4.times.10.sup.6 cells/ml
of RPMI containing 2.5% normal human serum type AB (Sigma, St.
Louis, Mo.) and 500 IRU/ml IFN-.gamma. (R&D Systems,
Minneapolis, Minn.). Neutrophils were cultured for 48 hours
(37.degree. C., 5% CO.sub.2) in AIM V media (Gibco, Grand Island,
N.Y.) with 50 ng/ml G-CSF (Kindly provided by R. Repp, U. of
Erlanger, Germany) and 500 IRU/ml IFN-.gamma..
[0072] Flow Cytometry
[0073] Cell binding assays were performed using 96-well microtiter
plates as previously described, (Guyre, P. M. et al., Monoclonal
antibodies that bind to distinct epitopes on Fc.gamma.R are able to
trigger receptor function. J. Immunol., 143:1650). Briefly, cells
were washed in PBS, pH 7.4 containing 2 mg/ml BSA and 0.05%
NaN.sub.3 (PBA), and adjusted to 2.0.times.10.sup.7 cells/ml with
PBA. FlTC-labeled and unconjugated antibodies were prepared in PBA.
Cells (25 .mu.l), antibody (25 .mu.l) and human serum (25 .mu.l),
or human IgG (10 mg/ml, Sigma, St. Louis, Mo.) (25 .mu.l), or PBA
(25 .mu.l) were added to the microtiter plate, and left on ice for
45-60 minutes. Unbound antibody was removed from the wells by
washing the cells 3 times with PBA. The cells were fixed with 1%
paraformaldehyde. Cell associated fluorescence was analyzed on a
Becton Dickinson FACScan.
[0074] BsAb Coupling Procedure
[0075] BsAb were constructed using the method of Glennie et al,
(Glennie, M. J. et al., (1987), Preparation and performance of
bispecific F(ab' gamma).sup.2, antibody containing thioether-linked
Fab' gamma fragments, J. Immunol., 139:2367). mAbs 22 (both murine
and humanized) and 520C9 (anti-HER2/neu) antibodies were produced
by in vitro cultivation of the respective hybridoma cells. The
antibodies were separately digested with pepsin to F(ab').sub.2,
and subsequently reduced to Fab' by addition of 10 mM
mercaptoethanolamine (MEA) for 30 minutes at 30.degree. C. The Fab'
fragments were applied to a Sephadex G-25 column equilibrated in 50
mM Na Acetate, 0.5 mM EDTA, pH 5.3 (4.degree. C.).
Ortho-phenylenedimaleimide (o-PDM, 12 mM) dissolved in dimethyl
formamide and chilled in a methanol/ice bath was added (one half
volume) to the murine 22 Fab' in the case of M 22 x 520C9, and to
520C9 Fab' in the case of H 22 x 520C9 and incubated for 30 minutes
on ice. The Fab'-maleimide was then separated from free o-PDM on
Sephadex G-25 equilibrated in 50 mM Na Acetate, 0.5 mM EDTA, pH 5.3
(4.degree. C.). For preparation of the BsAbs, the M22
Fab'-maleimide was added to the 520C9 Fab' or the 520C9
Fab'-maleimide was added to H22 Fab' at a 1:1 molar ratio. The
reactants were concentrated under nitrogen to the starting volume
using a Diaflo membrane in an Amicon chamber (all at 4.degree. C.).
After 18 hours the pH was adjusted to 8.0 with 1M Tris-HCl, pH 8.0.
The mixture was then reduced with 10 mM MEA (30 minutes, 30.degree.
C.) and alkylated with 25 mM iodoacetamide. The bispecific
F(ab').sub.2 was separated from unreacted Fab's and other products
by a Superdex 200 (Pharmacia, Piscataway, N.J.) column equilibrated
in PBS.
[0076] Antibody Dependent Cellular Cytoxicity (ADCC)
[0077] The HER2/neu over-expressing human breast carcinoma cells,
SKBR-3, were used as targets for lysis by cytokine activated
neutrophils (see preparation of blood cells). Targets were labeled
with 100 .mu.Ci of .sup.51Cr for 1 hour prior to combining with
neutrophils and antibodies in a U-bottom microtiter plate. After
incubation for 5 hours at 37.degree. C. supernatants were collected
and analyzed for radioactivity. Cytotoxicity was calculated by the
formula: % lysis=(experimental CPM-target leak CPM/detergent lysis
CPM-target leak CPM).times.100%. Specific lysis=% lysis with
antibody-% lysis without antibody. Assays were performed in
triplicate.
[0078] Superoxide Induction
[0079] U937 cells were used for measuring the ability of H22 to
trigger a superoxide burst via Fc.gamma.RI, (Pfefferkorn, L. C. and
G. R. Yeaman (1994), Association of IgA-Fc receptors (Fc x R) with
Fc.epsilon. RI.gamma. 2 subunits in U937 cells, J. Immunol.
153:3228; Hallet, H. B. and A. K. Campbell (1983). Two distinct
mechanisms for stimulating of oxygen-radical production in
polymorphonuclear leucocytes, Biochem J, 216:459). U937 cells were
cultured for five days in RPMI-1640 (Gibco, Grand Island, N.Y.)
with 10% FBS (Hyclone, Logan, Utah) in the presence of 100 U/ml
IFN-.gamma. (Genentech, S. San Francisco, Calif.) to induce
differentiation and increased expression of Fc.gamma.RI. On the day
of the experiment, these differentiated cells were incubated for 20
minutes in fresh RPMI-1640 with 10% FBS at 37.degree. C. The cells
were then pelleted and resuspended at a concentration of
3.times.10.sup.6 cells/ml in PBS supplemented with 1 mM CaCl.sub.2,
1 mM MgCl.sub.2, 11 mM glucose, and 100 .mu.g/ml BSA (Sigma, St.
Louis, Mo.). To trigger the release of superoxide, 100 .mu.l of
cells were added to 100 .mu.l of a reaction solution containing 0.1
mM luminol (Sigma, St. Louis, Mo.), 0.5 mM sodium vanadate (Sigma,
St. Louis, Mo.), and either mAb M22, H22, or 197 and placed in the
luminometer at 22.degree. C. Measurements of the spontaneous
production of superoxide were made every 30 to 40 seconds starting
immediately following the addition of the cells to the reaction
solution in the luminometer. To compare the superoxide triggered by
crosslinking Fc.gamma.RI with M22, H22 or 197, each mAb was used at
a concentration of 10 .mu.g/ml. The production of superoxide in
mV/sec was monitored for 20 minutes. MAb M22, M32.2 and 197 were
added at various concentrations to establish the
dose-responsiveness of superoxide production.
[0080] Results
[0081] Murine & V Region Genes
[0082] Ig V region cDNAs were prepared from M22 hybridoma RNA using
primers specific for murine heavy and kappa constant regions and
were amplified by PCR with the additional use of a series of
primers based on sequences of known signal and/or 5' sequences of
mature V regions. PCR products of the expected sizes for V.sub.H
and V.sub..kappa. were obtained using the SH2BACK/CG1FOR and
VK7BACK/CK2FOR primer combinations. Amplified DNA was digested with
appropriate restriction enzymes, cloned into M13 and the sequence
in both directions determined from at least 24 independent clones.
The deduced amino acid sequences are shown in SEQ. ID Nos. 29 and
30. The 4 N-terminal residues of V.sub..kappa. are encoded by the
VKBACK primer.
[0083] The M22 V.sub.H and V.sub..kappa. are members of murine
heavy chain subgroup IIID and kappa subgroup I, (Kabat, E. A. et
al., (1991), Sequences of Proteins of Immunological Interest, 5th
Ed., U.S. Department of Health and Human Services), respectively.
Apart from the residue at L97, the amino acid sequence of the M22
V.sub..kappa. is identical to that from the murine anti-IgG mAb A17
(Shlomchik, M. et al., Variable region sequences of murine IgM
anti-IgG monoclonal autoantibodies (rheumatoid factors). II
Comparison of hybridonias derived bylipopolysaccharide stimulation
and secondary protein immunization, J. Exp. Med. 165:970).
[0084] Humanized mAbs and Initial Characterization of their
Binding
[0085] M22 V.sub.H FR showed greater homology (79%) to KOL (human
subgroup III) than to NEWM (57%) (human subgroup II). To see how
this difference might affect binding, heavy chains were constructed
based either on NEWM V.sub.H including the murine residues Phe27,
Ile28 and Arg71, or on KOL V.sub.H with no murine FR amino acids.
Both humanized V.sub.H were partnered with the same REI-derived
humanized light chain.
[0086] The affinity of the humanized mAbs was initially assessed by
ELISA measuring the binding to Fc.gamma.RI/IgM heavy chain fusion
protein. The data showed that the KOL V.sub.H/REI V.sub..kappa. mAb
had the same binding as the chimeric mAb whereas the NEWM
V.sub.H/REI V.sub..kappa. mAb exhibited an approximate 5-fold lower
affinity. The low binding of a nonspecific human IgG1 mAb showed
that >95% of binding of the humanized mAbs was via the Fv
portion rather than through the Fc domain.
[0087] While additional changes to the NEWM FR would be expected to
restore binding affinity these could create novel epitopes which
might provoke an unwanted immunological response. The KOL
V.sub.H/REI V.sub..kappa. mAb, designated H22, was therefore chosen
for further examination of its binding characteristics.
[0088] Functional Characterization of mAbH22
[0089] A series of binding experiments were performed to establish
the specificity and isotype of the H22 antibody. Peripheral blood
leukocytes stained with fluorescein-conjugated M22 or H22
demonstrated specific binding to monocytes with approximately
10.sup.4 binding sites per cell. In contrast, lymphocytes or
unstimulated neutrophils had little or no specific binding (Table
1):
1TABLE 1 Specific Binding of H22 to Monocytes Antibody Monocytes
Lymphocytes PMNs M22 10,000.sup.a <1000 <1000 H22 10,500
<1000 <1000 .sup.aAntibody sites per cell, average of
duplicates
[0090] To demonstrate that the H22 binds to Fc.gamma.RI at the same
site as M22 and that it also binds as a ligand at the Fc binding
domain, competition experiments with two anti-Fc.gamma.RI murine
mAb (M22 and M32.2) and a human IgG1 mAb were performed.
Unconjugated H22 and M22 competed equivalently for either the
binding of fluoresceinated M22 or fluoresceinated H22 in the
presence of excess human IgG which saturated the Fc binding sites
on Fc.gamma.RI. As expected, the anti-Fc.gamma.RI antibody M32.2
which binds to a different site on Fc.gamma.RI than M22 (Guyre, P.
M. et al., J. Immunol. 143:1650) was also unable to compete with
the M22-FITC. In addition, the inhibition of H22-FITC by H22 and
not by an irrelevant human IgG1 mAb confirmed the specificity of
Fc.gamma.RI binding via the V regions of H22.
[0091] H22, but not M22, was able to compete for Fc mediated
binding to Fc.gamma.RI by a fluorosceinated human IgG1. This
experiment demonstrated that the Fc portion of H22 but not M22
bound to the Fc binding domain of Fc.gamma.RI. This is consistent
with the ability of the Fc portion of human IgG1 antibodies, but
not murine IgG1, to bind Fc.gamma.RI with high affinity.
[0092] Since the humanization of M22 was primarily to increase its
immunotherapeutic potential, the binding activity of H22 to
monocytes and cytokine-activated neutrophils was determined in the
presence of human serum. H22-FITC bound with similar affinity to
Fc.gamma.RI on monocytes in the presence or absence of human serum.
In contrast, the Fc-mediated binding of an irrelevant human
IgG-FITC was completely inhibited by human serum. Likewise,
H22-FITC bound with similar affinity to IFN-.gamma.-treated
neutrophils in the absence and in the presence of human serum.
Collectively, the data demonstrated that H22 binds both via its V
regions to a site distinct from the Fc binding domain and via its
Fc region to the ligand binding domain of Fc.gamma.RI. The former
binding activity effectively overcomes antibody blockade of human
IgG1.
[0093] Functional Activity of H22 BsAb
[0094] The foremost application of anti-Fc.gamma.RI antibodies for
immunotherapy is the development of BsAbs which link
Fc.gamma.RI-bearing effector cells to a tumor cell, a virus, or a
virally-infected cell. Such BsAb have been developed with M22;
therefore, a comparison was made of the ability of the M22
anti-tumor BsAb (520C9xM22) and a corresponding H22 BsAb
(520C9xH22) to mediate cytotoxicity. These BsAbs consisted of H22
or M22 Fab' chemically conjugated to the Fab' of an anti-HER2/neu
antibody (520C9), and thus were specific for the effector cell
trigger molecule Fc.gamma.RI and the tumor antigen.
[0095] Comparison of M22-derived and H22derived BsAbs was done by
ADCC assays. M22- and H22-derived BsAbs mediated the killing of
HER2/neu overexpressing SKBR-3 cells. Both the murine and humanized
BsAbs exhibited similar levels of lysis of antigen bearing target
cells. In addition, both BsAb retained ADCC activity in the
presence of human serum, while excess M22 F(ab').sub.2 resulted in
complete inhibition of killing. Taken together these results show
that the H22 BsAb-induced lysis is mediated through the M22 epitope
and that the ADCC is Fc.gamma.RI specific.
[0096] Finally, the ability of H22 and M22 to stimulate superoxide
production by the monocyte-like cell line U937 was evaluated. M22,
which binds to the Fc.gamma.RI only by its V regions, induced a
very low level oxygen burst, presumably because it is unable to
cross-link the receptor efficiently. However, H22, which can
cross-link Fc.gamma.RI by binding as a ligand via its Fc domain
and, additionally, as an antibody via its Fv, induced a more
substantial release of superoxide.
Example 2
[0097] Generation of Functional H22-Epidermal Growth Factor Fusion
Protein (H425)
[0098] Materials and Methods
[0099] Expression Vectors and Cloning
[0100] Expression vectors for the genomic clones of the heavy
(pSVgpt) and light (pSVhyg) chains of H22 are as described in
International Patent Application Publication Number: WO 94/10332
entitled, Humanized Antibodies to Fc Receptors for Immunoglobulin G
on Human Mononuclear Phagocytes. For the Fab-ligand fusion
construct, it was unnecessary to alter the light chain. For the
heavy chain, however, the CH2 and CH3 domains had to be removed and
replaced with the coding sequences of the ligands. The heavy chain
vector contains two BamHI sites, one in the intron between V.sub.H
and CH1, and the other just downstream of CH3. Using the BamHI
restriction sites, DNA encoding the constant domains were replaced
by a truncated version encoding only CH1 and most of the hinge. To
do this, the polymerase chain reaction (PCR) was utilized to
engineer the new C-terminus of the heavy chain fragment with the
alterations shown in FIG. 1.
[0101] The construct shown in FIG. 1 [C], consisting of a
translation termination codon downstream of the cloning restriction
sites, Mol and NotI, and upstream of a BamHI site which was used to
clone the new PCR generated CH1 fragment downstream of VH, was used
to generate the fusion protein constructs. The cloning sites, which
are located downstream of most of the hinge in order to retain
flexibility between the Fd and ligand domains, was used to insert
DNA encoding EGF or other ligands. Also, the single Cys residue has
been retained from the previous construct to allow conjugation for
the formation of dimeric molecules.
[0102] DNA encoding the ligands were amplified by PCR to have a
XhoI site on the N-terminus and a NotI site on the C-terminus of
the coding region, and then inserted in the proper reading frame
into the same sites of the newly engineered H22 heavy chain
truncated fragment described above. cDNA encoding epidermal growth
factor (EGF) was obtained from the ATCC (#59957). Only DNA encoding
the 53 amino acid residues of mature EGF out of the approximately
1200 residue precursor was cloned beginning with Asn 971 and ending
with Arg 1023 (Bell, G. I., Fong, N. M., Stempien, M. M., Wormsted,
M A., Caput, D., Ku. L., Urdea, M. S., Rail, L. B. &
Sanchez-Pescador, R. Human Epidermal Growth Factor Precurser: cDNA
Sequence, Expression In Vitro and Gene Organization. Nucl. Acids
Res. 14: 8427-8446, 1986.).
[0103] Expression
[0104] The murine myeloma NSO (ECACC 85110503) is a non-Ig
synthesizing line and was used for expression of the fusion
proteins. The final expression vector, a pSVgpt construct with DNA
encoding H22 Fd fused in frame to EGF was transfected by
electroporation using a BioRad Gene Pulser to NSO which had been
previously transfected with the pSVhyg construct containing DNA
encoding H22 light chain. These polypeptides were expressed by an
Ig promoter and Ig enhancer present in the vectors, and secreted by
the mAb 22 heavy chain signal peptide located on the N-terminus of
the constructs. One or two days after transfection, mycophenolic
acid and xanthine were added to the media to select for cells that
took up the DNA. Individual growing colonies were isolated and
subcloned after binding activity was demonstrated by ELISA.
[0105] Purification
[0106] Cells expressing the H22-EGF fusion protein were subcloned
and expanded. The fusion protein-expressing clone was expanded and
grown in spinner cultures and the supernatant was clarified and
concentrated. Small scale purification was performed by affinity
chromatography on an anti-human kappa chain affinity column
(Sterogene. Carlsbad, Calif.). The purified protein was analyzed by
SDS-PAGE on a 5-15% acrylamide gradient gel under nonreducing
conditions. FIG. 2 is a schematic representation of the generation
of anti-Fc receptor-ligand fusion proteins.
[0107] Bispecific Flow Cytometry
[0108] To show that the fusion protein is capable of binding both
Fc.gamma.RI and EGFR simultaneously, a flow cytometric assay has
been developed (FIG. 3). In this assay different concentrations of
H22-EGF fusion protein or the bispecific antibody, BsAb H447 (H22 X
H425, a humanized version of the murine monoclonal antibody M425,
which binds EGFR at the ligand binding site (E. Merck) was
incubated with A431 cells, a cell line which expresses the EGF
receptor (EGFR) (ATCC, Rockville, Md.). After washing, a
supernatant containing a fusion protein consisting of the
extracellular domain of Fc.gamma.RI and the Fc portion of human IgM
was added. Finally, a Phycoerythrin (PE)-labeled mAb (32.2), that
binds Fc.gamma. RI at a site that is distinct from that bound by
mAb 22, was added. The cells were then analyzed by FACSCAN.
Alternatively, binding to EGFR was blocked by excess (100 .mu.g/ml)
whole murine mAb 425 (E. Merck), and binding of bsAb or fusion
protein was detected by PE-labeled anti-human IgG.
[0109] ADCC
[0110] ADCC mediated by the fusion protein was determined using a
.sup.51Cr killing assay. The EGFR overexpressing cell line, A431,
was used as targets for lysis by human monocytes cultured in
.gamma.-interferon (IFN-.gamma.) for 24 hours. Targets were labeled
with 100 .mu.Ci of .sup.51Cr for 1 hour prior to combining with
effector cells and antibodies in a U-bottom microtiter plate. After
incubation for 5 hours at 37.degree. C. supernatants were collected
and analyzed for radioactivity. Cytotoxicity was calculated by the
formula: % lysis=(experimental CPM-target leak CPM/detergent lysis
CPM-target leak CPM).times.100%. Specific lysis=% lysis with
antibody-% lysis without antibody. The ability of the fusion
protein to mediate ADCC was compared with that of the respective
BsAb. The assay was also performed in the presence of 25% human
serum to demonstrate that IgG or other factors found in human serum
will not inhibit fusion protein-mediated ADCC.
[0111] F. Other Fusion Proteins
[0112] Other fusion proteins, such as H22- gp30 (heregulin) (Dr.
Ruth Lupas, Georgetown University), CD4 (AIDS Repository), gp120
(AIDS Repository), and bombesin. The bombesin fusion, however, was
generated in a somewhat different manner from the others because it
is only a short peptide (14 amino acid residues). Instead of
amplifying cDNA encoding bombesin using PCR, DNA oligomers encoding
the sense and anti-sense strands of bombesin were hybridized to
create the gene. The oligomers had overlapping ends that did not
hybridize but instead created sticky ends for a XhoI site on the
N-terminus and a NotI site on the C-terminus so that it could be
cloned into the H22 heavy chain expression vector.
[0113] Results
[0114] Purification
[0115] NSO cells expressing the H22 kappa chain were transfected
with the H22-EGF heavy chain construct and clones selected for
resistance to mycophenolic acid and xanthine were expanded and the
fusion protein was affinity-purified from the supernatant on an
anti-human kappa column (Sterogene, Carlsbad, Calif.). The purified
protein was analysed by SDS-PAGE. The purified protein migrated at
an apparent molecular weight of 50-55 kDa, indicating that the
fusion protein is expressed as a monomer, not a disulfide-linked
dimer. In addition, a band was seen at an apparent molecular weight
of 25 kDa and is probably free light chain.
[0116] Binding Specificity
[0117] To demonstrate that the fusion protein could bind
Fc.gamma.RI and EGFR simultaneously a bispecific FACS assay was
devised. FIG. 4 shows that both the chemically-linked,
fully-humanized BsAb H447 (H22 (anti-Fc.gamma.RI) x H425), which
was made as described in the following Example 3, and the H22-EGF
fusion protein bound EGFR on A431 cells and soluble Fc.gamma.RI
simultaneously in a dose-dependent fashion.
[0118] The EGFR-specificity of the fission protein was demonstrated
by the ability of the murine mAb, M425, which binds EGFR at the
ligand binding site, to inhibit fusion protein or H22 x H425
binding. Various concentrations of either the BsAb H447, or of the
H22-EGF fission protein were incubated with A431 cells in either
the presence or absence of an excess of M425. FIG. 5 shows that
binding of both the BsAb and the fusion protein were inhibited by
M425, demonstrating the specificity of the fusion protein for
EGFR.
[0119] ADCC
[0120] The ability of the fission protein to mediate ADCC was
analyzed using A431 cells as targets. Human monocytes cultured for
24 hours in the presence of IFN-.gamma. were used as effector
cells. FIG. 6 demonstrates the whole antibody, H425, the BsAb H447
(H22 x H425) and the fission protein mediated dose-dependent lysis
of A431 cells. FIG. 7 demonstrates that while ADCC mediated by the
whole antibody is inhibited by 25% human serum (25%HS), ADCC
mediated by the fusion protein was not inhibited by human serum
and, in this particular experiment, fission protein-mediated ADCC
was enhanced by human serum. These results support the clinical
utility of these molecules by demonstrating that the fusion protein
was capable of killing EGFR-overexpressing cells, even in the
presence of Fc.gamma. RI-expressing effector cells as would be
present in vivo.
Example 3
[0121] Production of Bispecific Antibodies from Modified Humanized
Antibody Fragments
[0122] Materials and Methods
[0123] Expression Vectors and Cloning
[0124] Expression vectors for the genomic clones of the heavy
(pSVgpt) and light (pSVhyg) chains of H22 were as described in
International Patent Application Publication Number: WO 94/10332
entitled, Humanized Antibodies to Fc Receptors for Immunoglobulin G
on Human Mononuclear Phagocytes. For the Fab' construct, it was
unnecessary to alter the light chain. For the heavy chain, however,
the CH2 and CH3 domains had to be removed and replaced with a
termination codon. The heavy chain vector contains two BamHI sites,
one in the intron between VH and CH1, and the other just downstream
of CH3. Using the BamHI restriction sites, DNA encoding the
constant domains were replaced by a truncated version encoding only
CH1 and most of the hinge. To do this, The polymerase chain
reaction (PCR) was utilized to engineer the new C-terminus of the
heavy chain fragment with the alterations shown in FIG. 1. FIG. 1
[B] shows the alterations for generation of a truncated
single-sulfhydryl version.
[0125] Expression
[0126] The murine myeloma NSO (ECACC 85110503) is a non-Ig
synthesizing line and was used for expression of the modified H22
antibody. The final expression vector, a pSVgpt construct with DNA
encoding H22 Fd was cotransfected with the pSVhyg construct
containing DNA encoding H22 light chain by electroporation using a
BioRad Gene Pulser. These polypeptides were expressed by an Ig
promoter and Ig enhancer present in the vectors, and secreted by
the mAb 22 heavy chain signal peptide located on the N-terminus of
the constructs. One or two days after transfection, mycophenolic
acid and xanthine were added to the media to select for cells that
took up the DNA Individual growing colonies were isolated and
subcloned after Fc.gamma.RI binding activity was demonstrated.
[0127] Purification
[0128] The single sulfhydryl form of the H22 antibody and the whole
H425 (anti-EGFR) antibody were produced by in vitro cultivation of
the respective transfected NSO cells. The H425 was purified by
protein A affinity chromatography. The single sulfydryl form of the
antibody H22 was purified by ion exchange chromatography using
Q-Sepharose followed by SP-Sepharose (Pharmacia, Piscataway, N.J.).
The purity of the single sulfhydryl form of the H22 antibody was
assessed by SDS-PAGE.
[0129] Generation of Bispecific Antibody (BsAb)
[0130] BsAb was constructed using the method of Glennie et al.
(Glennie, J. J. et al., (1987), Preparation and performance of
bispecific F(ab' gamma).sup.2, antibody containing thioether-linked
Fab' gamma fragments, J. Immunol., 139:2367). The F(ab').sub.2 of
H425 was generated by limited pepsin proteolysis in 0.1 M citrate
buffer, pH 3.5 and the F(ab').sub.2 purified by ion exchange
chromatography. The mAbs were reduced by addition of 20 mM
mercaptoethanolamine (MEA) for 30 minutes at 30.degree. C. The Fab'
fragments were applied to a Sephadex G-25 column equilibrated in 50
mM sodium acetate, 0.5 mM EDTA, pH 5.3 (4.degree. C.).
Ortho-phenylenedimaleimide (o-PDM, 12 mM) dissolved in dimethyl
formamide and chilled in a methanol/ice bath was added (one half
volume) to the H22 Fab' and incubated for 30 minutes on ice. The
Fab'-maleimide was then separated from free o-PDM on Sephadex G-25
equilibrated in 50 mM Na Acetate, 0.5 mM EDTA, pH 5.3 (4.degree.
C.). For preparation of the BsAbs, the H22 Fab'-maleimide was added
to the H425 Fab' at a 1.2:1 molar ratio. The reactants were
concentrated under nitrogen to the starting volume using a Diaflo
membrane in an Amicon chamber (all at 4.degree. C.). After 18 hours
the pH was adjusted to 8.0 with 1M Tris-HCl, pH 8.0. The mixture
was then reduced with 10 mM MEA (30 minutes, 30.degree. C.) and
alkylated with 25 mM iodoacetamide. The bispecific F(ab').sub.2 was
separated from unreacted Fab's and other products by a Superdex 200
(Pharmacia, Piscataway, N.J.) column equilibrated in PBS.
[0131] Bispecific Flow Cytometry
[0132] To show that BsAb generated by the o-PDM method as well as
that generated by the DTNB method are capable of binding both
Fc.gamma.RI and EGFR simultaneously, a flow cytometric assay has
been developed (FIG. 8). In this assay different concentrations of
the two BsAbs were incubated with A431 cells, a cell line which
expresses the EGF receptor (EGFR). After washing, a supernatant
containing a fusion protein consisting of the extracellular domain
of Fc.gamma.RI and the Fc portion of human IgM was incubated with
the cells. Finally, the cells were incubated with a FITC-labeled
anti-human IgM-specific antibody. The cells were then analyzed by
FACSCAN.
[0133] ADCC
[0134] BsAb-mediated ADCC was determined using a .sup.51Cr killing
assay. The EGFR overexpressing cell line, A431, was used as targets
for lysis by human monocytes cultured in .gamma.-interferon for 24
hours. Targets were labeled with 100 .mu.Ci of .sup.51Cr for 1 hour
prior to combining with effector cells and antibody in a
flat-bottomed microtier plate. After incubation for 16 hours at
37.degree. C. supernatants were collected and analyzed for
radioactivity. Cytotoxicity was calculated by the formula: %
lysis=(experimental CPM-target leak CPM/detergent lysis CPM-target
leak CPM).times.100%. Ab-dependent lysis=% lysis with antibody-%
lysis without antibody.
[0135] Results
[0136] Purification
[0137] NSO cells were cotransfected with the truncated H22 heavy
chain construct and the intact kappa chain construct. Clones
selected for resistance to mycophenolic acid and xanthine were
expanded and the protein was purified from the supernatant by
Q-Sepharose followed by SP-Sepharose ion exchange chromatography.
The purified protein was analyzed a by SDS-PAGE. The purified
protein migrated at an apparent molecular weight of 50 kDa,
indicating that the protein is expressed as a monomer, not a
disulfide-linked dimer.
[0138] Construction and Characterization of a BsAb Composed of
Single Sulfhydryl H22 Linked to Fab' of H425 (Anti-EGFR)
[0139] A BsAb was constructed where the single sulfhydryl form of
H22 was linked to the Fab' fragment of H425, a humanized anti-EGFR
mAb. The BsAb was generated using o-PDM as a linker by the method
of Glennie et al. (Glennie, M. J. et al., (1987), Preparation and
performance of bispecific F(ab' gamma).sup.2, antibody containing
thioether-linked Fab' gamma fragments, J. Immunol., 139:2367). The
activity of this BsAb was compared to one a generated by the DTNB
method using Fab' fragments made from pepsin digestion and
reduction of whole H22. To demonstrate that these BsAbs could bind
Fc.gamma.RI and EGFR simultaneously a bispecific FACS assay was
devised. FIG. 9 shows that both the o-PDM-linked BsAb and the BsAb
made by the DTNB method bound EGFR on A431 cells and soluble
Fc.gamma.RI simultaneously in a dose-dependent fashion.
[0140] The ability of the two BsAbs to mediate ADCC was analyzed
using A431 cells as targets. Human monocyte cultured for 24 hours
in the presence of IFN-.gamma. were used as effector cells. FIG. 10
demonstrates the two BsAbs mediated dose-dependent lysis of A431
cells in a comparable fashion. These results demonstrated that BsAb
generated from the truncated, single sulfhydryl form of H22 was
capable of killing EGFR-overexpressing cells in the presence of
Fc.gamma.RI-expressing effector cells.
Example 4
[0141] Production of Trivalent Antibodies
[0142] Materials and Methods
[0143] Cell Lines and Antibodies, M22, 520C9, H425, SKBR3 and
A431
[0144] M22 and 520C9 were purified from hybridoma supernatant by
ion exchange chromatography (Pharmacia, Piscataway, N.J.) and 520C9
was further purified by protein A affinity chromatography
(Pharmacia, Piscataway, N.J.). H425 was purified from hybridoma
supernatant by protein A affinity chromatography (Pharmacia,
Piscataway, N.J.). The M22- and 520C9-producing murine hybridoma
were described previously (Guyre et al., (1989) Monoclonal
antibodies that bind to distinct epitopes on FcgRI are able to
trigger receptor function, J. Immunol. 143:5, 1650-1655; Frankel et
al., (1985) Tissue distribution of breast cancer-associated
antigens defined by monoclonal antibodies, J. Biol. Response
Modifiers, 4:273-286). The murine myeloma NSO (ECACC 85110503) is a
non-Ig synthesizing line and was used for the expression of the
humanized mAb, H425 (Kettleborough et al., (1991) Humanization of a
mouse monoclonal antibody by CDR-grafting: the importance of
framework residues on loop conformation, Protein Eng., 4:773).
SKBR-3, (ATCC, Rockville, Md.) a human breast carcinoma cell line
that overexpresses the HER2/neu protooncogene, and A431 (ATCC,
Rockville, Md.), a human squamous carcinoma cell line that
overexpresses EGFR (ATCC, Rockville, Md.) were cultivated in
Iscove's Modified Dulbecco's Medium (IMDM, Gibco, Grand Island,
N.Y.).
[0145] Neutrophil Preparation
[0146] Neutrophils are separated from mononuclear cells by ficoll
hypaque (Pharmacia, Piscataway, N.J.) gradient separation. To
up-regulate F.sub.c.gamma.RI, neutrophils are treated with
cytokines. Neutrophils are cultured for 24-48 hrs (37.degree. C.,
5% CO.sub.2) in AIM V media (Gibco, Grand Island, N.Y.) containing
2.5% normal human serum type AB (Sigma, St. Louis, Mo.), 50 ng/ml
G-CSF (Kindly provided br R. Repp, U. of Erlanger, Germany) and 100
IRU/ml IFN-.gamma..
[0147] Conjugation Method
[0148] BsAb were constructed using the method of Glennie et al
(Glennie, M. J. et al., (1987), Preparation and performance of
bispecific F(ab' gamma).sup.2, antibody containing thioether-linked
Fab' gamma fragments, J. Immunol., 139:2367). mAbs M22, 520C9
(anti-HER2/neu, 33), and H425 (anti-EGFR) antibodies were produced
by in vitro cultivation of the respective hybridoma cells. The
F(ab').sub.2 of each antibody were generated by limited pepsin
proteolysis in 0.1 M citrate buffer, pH 3.5 and the F(ab').sub.2
purified by ion exchange chromatography. mAbs M22 and H425 were
reduced to Fab' by addition of 20 mM mercaptoethanolamine (MEA) for
30 minutes at 30.degree. C. The Fab' fragments were applied to a
Sephadex G-25 column equilibrated in 50 mM Na Acetate, 0.5 mM EDTA,
pH 5.3 (4.degree. C.). Ortho-phenylenedimaleimide (o-PDM, 12 mM)
dissolved in dimethyl formamide and chilled in a methanol/ice bath
was added (one half volume) to the murine 22 Fab' and incubated for
30 minutes on ice. The Fab'-maleimide was then separated from free
o-PDM on Sephadex G-25 equilibrated in 50 mM Na Acetate, 0.5 mM
EDTA, pH 5.3 (4.degree. C.). For preparation of the BsAbs, the M22
Fab-maleimide was added to the H425 Fab' at a 1:1 molar ratio. The
reactants were concentrated under nitrogen to the starting volume
using a Diaflo membrane in an Amicon chamber (all at 4.degree. C.).
After 18 hours the pH was adjusted to 8.0 with 1M Tris-HCl, pH 8.0.
The mixture was then reduced with 10 mM MEA (30 minutes, 30.degree.
C.) and alkylated with 25 mM iodoacetamide. The bispecific
F(ab').sub.2 as separated from unreacted Fab's and other products
by a Superdex 200 (Pharmacia, Piscataway, N.J.) column equilibrated
in phosphate buffered saline (PBS). The BsAb M22 x 520C9 was made
in a similar fashion except that 520C9 was used instead of
H425.
[0149] Trispecific antibody composed of M22 x H425 x 520C9 was made
in two stages (FIG. 11). In the first stage, M22 was linked to H425
as described above to create the M22 x H425 BsAb except that rather
than a final reduction and alkylation, the reactants were treated
with DTNB to block the remaining free sulfhydryl groups. The
bivalent BsAb was purified by gel filtration on a Superdex 200
column, reduced to F(ab').sub.2(SH) and mixed in a 1:1 molar ratio
with o-PDM-treated 520C9. The resulting trispecific F(ab)3 was
purified on a Superdex 200 column. The TsAb was analyzed by HPLC
size exclusion chromatography using a TSK 3000 column (ToJo Haas,
Japan). Using the same procedure as above another TsAb comprising
m22 Fab' x 32.2 Fab' x m22 Fab' has been constructed.
[0150] Bispecific Flow Cytometry
[0151] The TsAb can bind to EGFR and F.sub.c.gamma.RI
simultaneously or to HER2/neu and F.sub.c.gamma.RI simultaneously.
Either A431 cells (high EGFR-expressing cells) or SKBR-3 cells
(high HER2/neu-expressing cells) were incubated with various
concentrations of BsAbs (M22 x 520C9 or M22 x H425) or with the
TsAb, M22 x H425 x 520C9. The cells were washed and then incubated
with the soluble F.sub.c.gamma.RI. Soluble F.sub.c.gamma.RI binding
was detected with mAb 32.2-FITC which binds F.sub.c.gamma.RI at a
site that is distinct from the 22 binding site. The cells were then
analyzed by FACSCAN.
[0152] ADCC
[0153] Either SKBR-3 cells or A431 cells were used as targets for
lysis by cytokine activated neutrophils. Targets were labeled with
100 .mu.Ci of .sup.51Cr for 1 hour prior to combining with
neutrophils and antibodies in a U-bottom microtiter plate. After
incubation for 16 hours at 37.degree. C. supernatants were
collected and analyzed for radioactivity. Cytotoxicity was
calculated by the formula: % lysis=(experimental CPM-target leak
CPM/detergent lysis CPM-target leak CPM).times.100%. Specific
lysis=% lysis with antibody-% lysis without antibody. Assays were
performed in triplicate.
[0154] Fc.gamma.RI Modulation Assay
[0155] The M22 x 32.2 x M22 BsAb was used for modulation of
Fc.gamma.RI on monocytes in whole blood. The assay procedure is
shown in the enclosed flow chart (see FIG. 18A). FIG. 18B shows
that treatment with 10 .mu.g/mL of this BsAb decreased the
Fc.gamma.RI expression on monocytes to approximately 50% of the
level prior to BsAb treatment.
[0156] Results
[0157] Construction and Biochemical Characterization of the
TsAb
[0158] TsAb was made according to the flow chart depicted in FIG.
11. In the first stage of the procedure, M22 was coupled to H425,
treated with DTNB, and the resulting bispecific F(ab').sub.2
purified by gel filtration. In the second stage, this bispecific
F(ab').sub.2 was reduced and mixed with o-PDM-treated 520C9 Fab'
resulting in the TsAb, M22 x H425 x 520C9. This TsAb is depicted
schematically in FIG. 12. In this figure, Fab'-A represents M22,
Fab'-B represents H425, and Fab'-C represents 520C9.
[0159] Binding (Bs FACS)
[0160] To demonstrate that the TsAb, M22 x H425 x 520C9, could bind
F.sub.c.gamma.RI and HER2/neu simultaneously a bispecific FACS
assay was devised. This assay is depicted schematically in FIG.
13A. FIG. 14 shows that both the TsAb bound HER2/neu on SKBR-3
cells and soluble F.sub.c.gamma.RI simultaneously in a
dose-dependent fashion. The BsAb, M22 x H425, generated negligible
signal in this assay over a wide range of concentrations. To
demonstrate that the TsAb, M22 x H425 x 520C9, could bind
F.sub.c.gamma.RI and EGFR simultaneously a similar assay was
devised using the EGFR-overexpressing cell line, A431, in the case.
This assay is depicted schematically in FIG. 13B. FIG. 15 shows
that both the TsAb and the BsAb, M22 x H425, bound EGFR on A431
cells and soluble F.sub.c.gamma.RI simultaneously in a
dose-dependent fashion. The BsAb, M22 x 520C9, generated negligible
signal in this assay over a wide range of concentrations.
[0161] ADCC
[0162] The ability of the TsAb to mediate ADCC was analyzed using
either SKBR-3 or A431 cells as targets. Human neutrophils cultured
for 24-48 hours in the presence of IFN-.gamma. and G-SF were used
as effector cells. FIG. 16 demonstrates the both the BsAb, M22 x
520C9, and the TsAb, M22 x H425 x 520C9, mediated lysis of SKBR-3
cells, whereas the BsAb, M22 x H425, did not. On the other hand,
FIG. 17 demonstrates the BsAb, M22 x H425, and the TsAb, mediated
lysis of SKBR-3 cells, whereas the BsAb, M22 x 520C9, did not.
These results demonstrated that the TsAb was capable of killing
both HER2/neu and EGFR-overexpressing cells in the presence of
F.sub.c.gamma.RI-expressing effector cells.
[0163] The trispecific antibody described above included M22, the
murine version of the anti-Fc.gamma.RI mAb. Such a trispecific
antibody could be constructed using the single-sulfhydryl form of
the humanized anti-Fc.gamma.RI mAb, H22. The only difference being
that single-sulfhydryl form is secreted as a F(ab').sub.2 fragment
of this antibody. The single-sulfhydryl form is purified from
culture supernatants utilizing ion exchange chromatography using
Q-Sepharose followed by SP-Sepharose (Pharmacia, Piscataway, N.J.),
Once the single-sulfhydryl form of H22 is purified, the creation of
a trispecific antibody using this reagent would be identical to
that described above using the F(ab').sub.2 fragment of M22.
[0164] Equivalents
[0165] Those skilled in he art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *