U.S. patent application number 09/772120 was filed with the patent office on 2001-08-16 for therapeutic multispecific compounds comprised of anti-fcalpha receptor antibodies.
This patent application is currently assigned to Mederax, Inc.. Invention is credited to Deo, Yashwant M., Graziano, Robert, Keler, Tibor.
Application Number | 20010014328 09/772120 |
Document ID | / |
Family ID | 24721785 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014328 |
Kind Code |
A1 |
Deo, Yashwant M. ; et
al. |
August 16, 2001 |
Therapeutic multispecific compounds comprised of anti-Fcalpha
receptor antibodies
Abstract
Therapeutic multispecific compounds comprised of anti-Fc.alpha.
receptor antibodies and methods of use are provided.
Inventors: |
Deo, Yashwant M.; (Audubon,
PA) ; Graziano, Robert; (Frenchtown, NJ) ;
Keler, Tibor; (Ottsville, PA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Mederax, Inc.
|
Family ID: |
24721785 |
Appl. No.: |
09/772120 |
Filed: |
January 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09772120 |
Jan 26, 2001 |
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08890011 |
Jul 10, 1997 |
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6193966 |
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08890011 |
Jul 10, 1997 |
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08678194 |
Jul 11, 1996 |
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5922845 |
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Current U.S.
Class: |
424/136.1 ;
435/7.2; 530/388.8 |
Current CPC
Class: |
C07K 2317/732 20130101;
C07K 2317/77 20130101; C07K 2317/73 20130101; C07K 14/33 20130101;
C07K 16/2863 20130101; A61P 35/00 20180101; A61P 35/02 20180101;
C07K 16/283 20130101; A61K 38/00 20130101; C07K 16/32 20130101;
C07K 16/30 20130101; A61P 31/00 20180101; C07K 16/14 20130101 |
Class at
Publication: |
424/136.1 ;
435/7.2; 530/388.8 |
International
Class: |
A61K 039/395; G01N
033/567; C07K 016/30 |
Claims
What is claimed is:
1. A bispecific binding molecule, comprising a first binding
determinant which binds an Fc.alpha. receptor and a second binding
determinant which binds one or more target antigens.
2. The bispecific binding molecule of claim 1, wherein the binding
of the first binding determinant to Fc.alpha. receptor is not
blocked by human immunoglobin A.
3. The bispecific binding molecule of claim 1, wherein the target
antigen is a cancer cell antigen.
4. The bispecific binding molecule of claim 3, wherein the cancer
cell antigen is selected from the group consisting of cancers of
the breast, ovary, testis. lung, colon, rectum, pancreas, liver,
central nervous system, head and neck, kidney, bone, blood and
lymphatic system.
5. The bispecific binding molecule of claim 1, wherein the target
antigen is an infectious disease antigen from a pathogen or
pathogen-infected cell.
6. The bispecific binding molecule of claim 3, wherein the cancer
cell antigen is a member of the human EGF-like receptor family.
7. The bispecific binding molecule of claim 6, wherein the cancer
cell antigen is an EGF receptor.
8. The bispecific binding molecule of claim 6, wherein the cancer
cell antigen is HER-2/neu.
9. The bispecific binding molecule of claim 6, wherein the cancer
cell antigen is selected from the group consisting of HER-3 and
HER-4.
10. The bispecific binding molecule of claim 6, wherein the cancer
cell antigen is a heterodimeric receptor comprised of at least one
HER subunit.
11. The bispecific binding molecule of claim 3, wherein the cancer
cell antigen is selected from the group consisting of
carcinoembryonic antigen, gastrin releasing peptide receptor
antigen, and mucine tumor antigen TAG 72.
12. The bispecific molecule of claim 1, wherein at least one of
said binding determinants is an antibody or an antibody
fragment.
13. The bispecific binding molecule of claim 12, wherein the
antibody or antibody fragment is an IgG or IgG fragment.
14. The bispecific binding molecule of claim 12, wherein the
antibody fragment is selected from the group consisting of an Fab,
Fab', F(ab').sub.2, Fv, and single chain Fv.
15. A bispecific binding molecule comprising a binding determinant
which binds an Fc.alpha. receptor and a binding determinant for a
target selected from the group consisting of a cancer cell antigen
and an antigen from a pathogen.
16. The bispecific binding molecule of claim 15, wherein the
determinant which binds an Fc.alpha. receptor is humanized.
17. The bispecific binding molecule of claim 16, wherein the
humanized determinant is derived from all or a portion of the
nucleotide sequences encoding the Fv binding determinants of
antibody A77 V.sub.78 and V.sub.H regions shown in FIG. 14 (SEQ ID
NO:5), and FIG. 15 (SEQ ID NO:7), respectively.
18. The bispecific binding molecule of claim 16, wherein the
humanized determinant is derived from all or a portion of the amino
acid residue sequences of the Fv binding determinants of antibody
A77 V.sub..kappa. and V.sub.H regions shown in FIG. 14 (SEQ ID
NO:6), and FIG. 15 (SEQ ID NO:8), respectively.
19. The bispecific binding molecule of claim 18, wherein the
humanized determinant is greater than 50% homologous to that of
antibody A77 V.sub..kappa. or V.sub.H regions shown in FIG. 14 (SEQ
ID NO:6), and FIG. 15 (SEQ ID NO:8), respectively.
20. The bispecific binding molecule of claim 15, wherein the second
binding determinant is selected from the group consisting of
antibody 520C9, antibody CC49 and finctional fragments thereof.
21. The bispecific binding molecule of claim 20 produced by
chemical linkage of the antibody A77 fragment and the second
binding determinant.
22. The bispecific binding molecule of claim 20 which is produced
recombinantly in a host cell.
23. A multispecific binding molecule, comprising a first binding
determinant which binds an Fc.alpha. receptor and a second binding
determinant which binds one or more target antigens.
24. The multispecific binding molecule of claim 23, wherein the
binding of the first binding determinant to Fc.alpha. receptor is
not blocked by human immunoglobin A.
25. The multispecific binding molecule of claim 23 selected from
the group consisting of a bispecific binding molecule and a
trispecific binding molecule.
26. The multispecific binding molecule of claim 25, comprising a
third binding determinant specific for an Fc receptor that is not
an Fc.alpha. receptor.
27. The multispecific binding molecule of claim 26, wherein the
third binding determinant binds an Fc receptor selected from the
group consisting of Fc.gamma. receptor, Fc.epsilon. receptor,
Fc.delta. receptor and Fc.mu. receptor.
28. The multispecific binding molecule of claim 23, wherein binding
of the Fc.gamma. receptor is not inhibited by human IgG.
29. The multispecific binding molecule of claim 28, wherein the
second binding determinant binds to a target antigen selected from
the group consisting of a cancer cell antigen, a pathogen antigen,
and an antigen on a pathogen-infected cell.
30. The multispecific binding molecule of claim 28 produced by
chemical linkage of said binding determinants.
31. The multispecific binding molecule of claim 23 produced
recombinantly in a host cell transfected with a nucleic acid
encoding said binding determinants.
32. The multispecific molecule of claim 23, wherein at least one of
said binding determinants is an antibody or an antibody
fragment.
33. The multispecific molecule of claim 32, wherein at least one of
said binding determinants is a humanized antibody or fragment
thereof.
34. The multispecific binding molecule of claim 29, wherein the
cancer cell antigen is from a cancer cell selected from the group
consisting of cancer cells of the breast, ovary, testis, lung,
colon, rectum, pancreas, liver, central nervous system, head and
neck, kidney, bone, blood and lymphatic system.
35. The multispecific binding molecule of claim 29, wherein the
pathogen antigen is selected from the group consisting of
bacterial, fungal, protozoal, and viral antigens.
36. The multispecific binding molecule of claim 29, wherein the
cancer cell antigen is a member of the human EGF-like receptor
family.
37. The multispecific binding molecule of claim 36, wherein the
cancer cell antigen is an EGF receptor.
38. The multispecific binding molecule of claim 36, wherein the
cancer cell antigen is HER-2/neu.
39. The multispecific binding molecule of claim 36, wherein the
cancer cell antigen is HER-3, HER-4, or a heteromultimeric receptor
comprised of at least one HER subunit.
40. The multispecific binding molecule of claim 29, wherein the
cancer cell antigen is selected from the group consisting of
carcinoembryonic antigen, gastrin releasing peptide receptor
antigen, and mucine antigen TAG 72.
41. A multispecific binding molecule comprising a functional
fragment of antibody A77 and a second binding determinant which
binds an antigen selected from the group consisting of an antigen
on a cancer cell, an antigen on a pathogen and an antigen on a
pathogen-infected cell.
42. The multispecific binding molecule of claim 41, wherein the
second binding determinant for an antigen on a cancer cell is
selected from the group consisting of antibody 520C9, antibody
CC49, and functional fragments thereof.
43. The multispecific binding molecule of claim 41, wherein the A77
antibody fragment binds to an IgA receptor on a white blood
cell.
44. The multispecific binding molecule of claim 43, wherein the
white blood cell is selected from the group consisting of a
macrophage, monocyte, neutrophil, basophil, eosinophil, and
lymphocyte.
45. The multispecific binding molecule of claim 41, wherein the
pathogen is selected from the group consisting of bacteria,
viruses, fungi and protozoans.
46. The multispecific binding molecule of claim 32, wherein the
antibody or antibody fragment is an IgG or IgG fragment.
47. The multispecific binding molecule of claim 32, wherein the
antibody fragment is selected from the group consisting of an Fab,
Fab', F(ab').sub.2, Fv, and single chain Fv.
48. The multispecific binding molecule of claim 23 comprising a
third binding determinant which binds to an antigen of a target
cell, wherein said second binding determinant binds to a different
antigen on the same target cell.
49. The multispecific binding molecule of claim 23, comprising a
third binding determinant which binds to an epitope of a target
antigen, wherein said second binding determinant binds to a
different epitope on the same target antigen.
50. The multispecific binding molecule of claim 45, wherein the
pathogen is a fungus.
51. The multispecific binding molecule of claim 50, wherein the
fungus is a species of the genus Candida.
52. The multispecific binding molecule of claim 51, wherein the
species is C. albicans.
53. The multispecific molecule of claim 50, wherein the binding
determinant to Fc.alpha. is not blocked by human immunoglobin
A.
54. A method for eliminating or reducing an unwanted cell in a
subject, comprising administering to the subject a therapeutically
effective dose of a multispecific binding molecule, comprising a
first binding determinant which binds an Fc.alpha. receptor and a
second binding determinant which binds an antigen on the unwanted
cell, in a pharmaceutically acceptable carrier.
55. The method of claim 54, wherein at least one of said binding
determinants is humanized.
56. The method of claim 55, further comprising administering to the
subject at least one agent that enhances the number or activity of
Fc.alpha. receptors on an Fc.alpha. receptor-bearing cell.
57. The method of claim 56, wherein the agent is a cytokine.
58. The method of claim 57, wherein the cytokine is selected from
the group consisting of at least one of G-CSF, GM-CSF, IFN-Y, and
TNF.
59. A method for treating a subject infected with a pathogen
wherein the subject is administered a therapeutically effective
dose of a multispecific binding molecule, said molecule comprising
a first binding determinant which binds an Fc.alpha. receptor and a
second binding determinant which binds a target antigen of the
pathogen or a pathogen-infected cell, in a pharmaceutically
acceptable carrier.
60. A method for treating a subject to remove an unwanted cell in
the subject, comprising obtaining a sample of blood or blood cells
from the subject; contacting said blood or blood cells ex vivo with
a therapeutically effective dose of a multispecific binding
molecule, said binding molecule comprising a first binding
determinant which binds an Fc.alpha. receptor and a second binding
determinant which binds one or more target antigens, in a
pharmaceutically acceptable carrier; and returning said treated
blood or blood cells to the subject.
61. The method of claim 60, wherein at least one of said
determinants is humanized.
62. The method of claim 61, wherein said blood cells are isolated
and expanded in culture.
63. The method of claim 61 for treating a subject for the presence
of an unwanted cell, wherein said blood cells are treated with at
least one agent that enhances the number or activity of Fc.alpha.
receptors.
64. A method for vaccinating a subject against a pathogen or
against a cancer, wherein the subject is administered a
multispecific binding molecule, said molecule comprising a first
binding determinant which binds an Fc.alpha. receptor and a second
binding determinant which binds an antigen selected from the group
consisting of an antigen on a cancer cell, an antigen on a pathogen
and an antigen on a pathogen-infected cell, in a pharmaceutically
acceptable carrier.
65. A method for arming effector cells of a subject against a
pathogen or against a cancer, wherein the subject is administered a
multispecific binding molecule, said molecule comprising a first
binding determinant which binds an Fc.alpha. receptor and a second
binding determinant which binds an antigen selected from the group
consisting of an antigen on a cancer cell, an antigen on a pathogen
and an antigen on a pathogen-infected cell, in a pharmaceutically
acceptable carrier.
66. A method for modulating an Fc.alpha. receptor in a subject,
comprising administering to the subject a composition comprising
antibody A77 or a functional fragment of A77, in a pharmaceutically
acceptable carrier.
67. A method for identifying for an agent which modulates Fc.alpha.
receptors on the surface of cells, comprising contacting a sample
of cells carrying Fc.alpha. receptors with the agent; determining
Fc.alpha. receptor activity in the sample with the agent, in a
control sample with an antibody that modulates Fc.alpha. receptors,
and in a control sample with cells not contacted with said agent or
with antibody; and comparing Fc.alpha. receptor activities in the
samples, such that a sample of cells contacted with said agent and
having statistically significant less Fc.alpha. receptor activity
than control cells not contacted with agent, or having
statistically significantly as low Fc.alpha. receptor activity as
cells in a sample with an antibody, identifies an agent which
modulates Fc.alpha. receptors on the surface of cells.
68. A method for designing an agent which modulates Fc.alpha.
receptors for treatment of autoimmune disease by obtaining a three
dimensional model of the A77 anti-Fc.alpha. receptor binding site
using the sequence determinants of A77 heavy and light chain
variable regions, comprising comparing the amino acid residues of
the A77 variable region with that of heavy and light chain variable
regions of antibodies of known three dimensional structure;
determining placement of non-homologous amino acid residues within
the main peptide chain of the binding region of the V.sub.H and
V.sub.78 sites, such that the size, shape and charge of the A77
anti-Fc.alpha. receptor binding site is determined; screening a
library of molecules to obtain those of suitable size, shape and
charge by computer modeling that are mimetics of the A77 binding
site; and screening such candidates of appropriate size, shape and
charge for activity as potential modulators of Fc.alpha. receptors,
such that an agent which modulates Fc.alpha. receptors is designed.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/678,194, filed Jul. 11, 1996, the contents of which is
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Receptors for the Fc portions of immunoglobulins are
important in triggering many of the protective functions of
monocytes, macrophages and polymorphonuclear cells. Receptors for
IgG (Fc.gamma. receptors or Fc.gamma.R) on these cells have been
extensively investigated and bispecific molecules targeting these
receptors have been constructed. (See e.g. European Patent No. 0
255 249 entitled "Monoclonal Antibodies to Fc Receptor for
Immunoglobulin G on Human Mononuclear Phagocytes", which is
co-owned by Applicants.) In addition, clinical trials of bispecific
molecules (BsAb) which have specificity for the Fc.gamma.R and the
HER-2/neu antigen, which is found on breast or ovarian cancers,
indicate that these molecules are both safe and efficacious
(Valone, Frank H. et al. 1995, J. of Clin. Oncol. 13(9):
2281-2292).
[0003] IgA receptors Fc.alpha. receptors (Fc.alpha.R or CD89) are
also capable of promoting effector cell function. Binding of ligand
to Fc.alpha.R triggers phagocytosis and antibody mediated cell
cytotoxicity in leukocytes and Fc.alpha.R-bearing cell lines.
Fc.alpha. receptors can also cooperate with receptors for IgG on
effector cells in enhancing the phagocytosis of target cells.
Monoclonal antibodies of the IgM (Shen, L. et al., 1989 J. Immunol.
143: 4117) and IgG (Monteiro, R. C. et al.,1992 J. Immunol, 148:
1764) classes have been developed against Fc.alpha.R.
[0004] IgA is abundant in the human body (Kerr, M. A. 1990,
Biochem. J. 271:285-296). A single class of IgA Fc receptor,
Fc.alpha.RI or CD89, which binds to monomeric IgA has been
identified and characterized (Albrechtsen, M. et al., 1988 Immunol.
64:201; Monteiro R., et al., 1990 J. Exp. Med., 171:597).
Fc.alpha.RI is constitutively expressed primarily on cytotoxic
immune effector cells including monocytes, macrophages,
neutrophils, and eosinophils (Morton, H. C., et al., 1996 Critical
Reviews in Immunology 16:423). Fc.alpha.RI expression on a
sub-population of lymphocytes (Morton, H. C., et al., 1996 Critical
Reviews in Immunology 16:423), and on glomerular mesangial cells
has been reported (Gomez-Guerrero, C., et al., 1996 J. Immunol.
156:4369-4376). Its expression on monocytes and PMN can be enhanced
by TNF-.alpha. (Ges1, A., et al., 1994 Scad. J. Immunol.
39:151-156; Hostoffer, R. W., et al., 1994, The J. Infectious
Diseases 170:82-87), IL-1, GM-CSF, LPS or phorbol esters (Shen L.,
et al., J. Immunol. 152:4080-4086; Schiller, C. A. et al., 1994,
Immunology, 81:598-604), whereas IFN-.gamma. and TGF-.beta.1
decrease Fc.alpha.RI expression (Reterink, T. J. F., et al., 1996,
Clin. Exp. Immunol. 103:161-166). The .alpha.-chain of human
Fc.alpha.RI is a heavily glycosylated, type one transmembrane
molecule belonging to the Ig super-gene family which also includes
receptors for IgG and IgE. One gene located on chromosome 19
encodes several alternatively spliced isoforms of the Fc.alpha.RI
alpha chain (55-110 kDa; Morton, H. C., et al., 1996 Critical
Reviews in Immunology 16:423). Myelocytic Fc.alpha.RI has been
shown to be associated with the FcR.gamma.-chain which is
implicated as playing a role in Fc.alpha.RI signal transduction
(Morton, H. C. et al. 1995, J Biol. Chem. 270:29781; Pfefferkorn,
L. C., et al. 1995, J. Immunol., 153:3228-3236, Saito, K. et al.,
1995, J. Allergy Clin. Immunol. 96:1152).
[0005] Fc.alpha.RI binds both antigen-complexed and monomeric IgA1
and IgA2 (Mazangera, R. L. et al., 1990 Biochem. J. 272:159-165),
consistent with the receptor being saturated in vivo with monomeric
IgA in the same manner as Fc.gamma.R and Fc.epsilon.RI are
saturated with IgG and IgE respectively. Cross-linking Fc.alpha.RI
on myeloid effector cells, by polymeric IgA, IgA immune complexes,
or mAb specific for epitopes within or outside the ligand binding
domain, stimulates degranulation, superoxide release, secretion of
inflammatory cytokines, endocytosis and phagocytosis (Patty, C., A.
Herbelin, A. Lihuen, J. F. Bach, and R. C. Monteiro. 1995
Immunology. 86:1-5; Stewart, W. W., R. L. Maz Yegera, L. Shen, and
M. A. Kerr. 1994 J. Leucocyte Biology. 56:481-487; Stewart, W. W.,
and M. A. Kerr. 1990. Immunology. 71:328-334; Shen, L. 1992. J.
Leukocyte Biology. 51:373-378.). These physiological responses
triggered via Fc.alpha.RI can be important in the first line of
humoral defense on mucosal surfaces (Morton, H. C., M. van Egmond,
and J. G. J. van de Winkel. 1996 Critical Reviews in Immunology.
16:423).
[0006] Thus Fc.alpha.RI is a clinically relevant trigger receptor
on cytotoxic immune effector cells and its activity can be
exploited to develop novel immunotherapies. The cytotoxic potential
of Fc.alpha.RI has not been carefully explored since almost all
monoclonal antibody (mAb) based therapies are being developed with
mAbs of IgG class which do not bind to Fc.alpha.RI.
SUMMARY OF THE INVENTION
[0007] The present invention relates to multispecific therapeutic
molecules with binding determinants for immunoglobulin A (IgA)
receptors. IgA is the predominant antibody class in fluids on
mucosal surfaces, and IgA receptors (Fc.alpha. receptors,
Fc.alpha.R or Fc.alpha.RI) are found on white blood cells including
macrophages, monocytes, neutrophils, eosinophils and lymphocytes.
The bispecific and multispecific molecules of the invention can be
used as therapeutic agents to harness the cytolysis and
phagocytosis capabilities of these white blood cells, enhancing the
attack of these cells against cancer cells, cells of infectious
microorganisms, and cells infected with pathogens.
[0008] In one aspect, the invention includes bispecific binding
molecules, comprising a first binding determinant which binds an
Fc.alpha. receptor and a second binding determinant which binds one
or more target antigens. Preferably, the first determinant binds a
site on the Fc.alpha.R that is different from the binding site for
endogenous IgA, so that binding of the molecules of the invention
is not blocked or is not substantially blocked by IgA. In a
preferred embodiment, the target antigen bound by the second
binding determinant of the bispecific molecules of the invention is
a cancer cell antigen. In a more preferred embodiment the cancer
cell antigen is an antigen of a cancer of the breast, ovary,
testis, lung, colon, rectum, pancreas, liver, central nervous
system, head and neck, kidney, bone, blood or lymphatic system. In
another preferred embodiment, the target antigen is an infectious
disease antigen from a pathogen or pathogen-infected cell. In yet
another embodiment, the invention features treatment of an
autoimmune disease with a composition that binds and modulates a
receptor for IgA, causing modulation of the receptor such that
further binding of IgA to that receptor is decreased. In a
different embodiment, the invention provides compositions that bind
and do not modulate a receptor for IgA, so that the effector cells
of a subject are armed with the bispecific and multispecific
molecules and can bind an antigen on a pathogen or on a cancer.
[0009] A preferred embodiment of bispecific molecules of the
subject invention comprise molecules with binding determinants for
a receptor of the human EGF-like receptor family, for a
carcinoembryonic antigen, for a gastrin releasing peptide receptor
antigen, and for a mucine antigen, which are overexpressed by
certain tumor cells.
[0010] The bispecific molecules of the invention encompass
molecules that are comprised in part of binding determinants of
antibodies, and the molecules of the invention include those that
are engineered to include at least one antibody or an antibody
fragment. The bispecific binding molecules of the invention
preferably comprise a binding determinant from an IgG antibody or
IgG fragment, including an Fab, Fab', F(ab').sub.2, Fv, and single
chain Fv. A binding determinant, including an Fab, Fab',
F(ab').sub.2, Fv, and single chain Fv, can be obtained also from an
IgA antibody or an antibody of another isotype. A preferred
bispecific binding molecule of the invention comprises a first
binding determinant that is at least a functional fragment of
antibody A77 and a second binding determinant that binds a cancer
cell antigen, a pathogen antigen, or an antigen on an infected
cell. Other preferred bispecific binding molecules of the invention
comprise a first binding determinant that is at least a functional
fragment of antibody A3, A59 or A62, which are similar to A77 in
affinity to the receptor for IgA and are not blocked by IgA of the
subject. The invention includes nucleic acid sequences encoding the
V.sub.H and V.sub..kappa. regions of the A77 antibody and the
predicted amino acid sequences of these regions, and these sequence
are preferably used for humanizing the A77 binding determinants for
therapeutic multispecific molecules. Preferably the second binding
determinant of the molecules of the invention is at least a
functional fragment of antibody 520C9, antibody H425 or antibody
CC49. A preferred embodiment carries one binding determinant for
Fc.alpha.R and one for the HER/neu antigen found for example on
tumors of the breast, ovary, and lung.
[0011] Several methods of producing bispecific binding molecules
are encompassed by the invention, including by chemical linkage of
the binding determinants, and by recombinant genetic methods.
Recombinant bispecific molecules encoded by nucleic acid sequences
carrying genes encoding binding determinants which are thus
genetically linked are encompassed by the invention. Further,
bispecific binding molecules of the invention are produced by cell
fusion of two antibody-producing cell lines carrying the respective
nucleic acid sequences encoding the binding determinants, such as
hybridoma cell lines, to obtain a progeny cell line producing the
bispecific molecule of the invention.
[0012] In addition to bispecific binding molecules, the instant
invention encompasses multispecific binding molecules which
comprise at least a first binding determinant which binds an
Fc.alpha. receptor and a second binding determinant which binds a
target antigen, and at least a third binding determinant. Binding
of the first determinant of these multispecific binding molecules
to Fc.alpha.R is not blocked or inhibited by human immunoglobulin
A, so there is little or no competition for binding by endogenous
IgA molecules. Multispecific binding molecules encompass bispecific
and trispecific compositions, and those with four or more binding
determinants. A preferred embodiment of a trispecific binding
molecule carries an additional binding determinant that binds to an
Fc receptor that is not an Fc.alpha. receptor, including for
example a binding determinant for CD2, CD3, Fc.gamma. receptor,
Fc.epsilon. receptor, Fc.delta. receptor and/or Fc.mu. receptor,
these determinants being in addition to the first binding
determinant to Fc.alpha. receptor. The most preferred embodiment of
an additional binding determinant for an FcR is a determinant for
Fc.gamma. receptor. For a multispecific binding molecule of the
invention carrying a binding determinant for an Fc.gamma. receptor,
binding to Fc.gamma.R is not inhibited by human IgG, since the
molecule binds Fc.gamma. at a different epitopic site from IgG
binding of Fc.gamma.R. By incorporating at least binding
determinant for each of Fc.alpha.R and Fc.gamma.R into a single
molecule, the therapeutic capability of the molecule is increased
to enhance affinity and kinetics of binding of white blood cells to
tumor cells or cells of pathogenic organisms or pathogen-infected
cells, increasing opportunities for cytolysis and phagocytosis of
these targets.
[0013] Another preferred embodiment of the multispecific binding
molecules with a determinant for Fc.alpha., is a molecule that
carries a third binding determinant that binds to a second target
antigen or a second target epitope on a cancer cell, a pathogen, or
a pathogen-infected cell. The preferred means of producing these
molecules is by chemical linkage of the binding determinants,
however the invention encompasses also multispecific binding
molecules which are recombinantly produced, or which are produced
by cell fusion of two or more cell lines each of which carries the
nucleic acid sequences encoding the binding determinants.
Preferably at least one binding determinant is an antibody or an
antibody fragment, and to improve the success of the outcome during
continued treatment of humans, the binding determinant is a
humanized antibody, which is engineered to minimize the number of
foreign epitopes born by the molecule.
[0014] A preferred embodiment of the multispecific binding
molecules of this invention is comprised of one or more binding
determinants for target cancer cell antigens, particularly cancer
cell antigens from breast, ovary, testis, lung, colon, rectum,
pancreas, liver, central nervous system, head and neck, kidney,
bone, blood and lymphatic system cancers. A different preferred
embodiment of the invented multispecific binding molecules
comprises, as the target antigen, infectious disease antigens from
pathogens or pathogen-infected cells. In one embodiment, the target
antigens are infectious disease antigens and antigens expressed on
infected cells, for example, antigens from infections by bacteria,
fungi, protozoa, and viruses. In a more preferred embodiment, the
target antigen is from a pathogenic fungus, including an antigen
from a pathogenic yeast. In a most preferred embodiment, the target
antigen is from a species of Candida, for example, Candida
albicans.
[0015] Suitable targets among cancer cell antigens are preferably
members of the human EGF-like receptor family, more preferably the
cancer cell antigen is an EGF receptor, and most preferably the
cancer cell antigen is HER-2/neu, HER-3, HER-4, or a
heteromultimeric receptor comprised of at least one HER subunit.
Additional preferred cancer cell antigens include carcinoembryonic
antigen, gastrin releasing peptide receptor antigen, and TAG
72.
[0016] A most preferred multispecific binding molecule comprises at
least a first binding determinant that is at least a functional
fragment of antibody A77 and a second binding determinant that
binds an antigen of a cancer cell, a cell of a pathogenic organism,
or a pathogen-infected cell. In preferred embodiments of
A77-derived multispecific binding molecules with a cancer antigen
binding determinant, the preferred second binding determinant is at
least a functional fragment of antibody 520C9 or antibody CC49. The
first binding determinant for an Fc.alpha. receptor preferably
binds a receptor on a white blood cell. The types of white blood
cells to which the molecules bind are preferably macrophages,
monocytes, neutrophils, eosinophils, and lymphocytes.
[0017] Yet another aspect of the invention comprises multispecific
binding molecules in which the molecule includes at least one
antigen from a pathogen or pathogen-infected cell, or an antigen
from a cancer cell. The molecules of this particular embodiment can
serve to deliver these antigens as a vaccine directly to the
antigen presenting cells of the immune system to immunize the
recipient against an infectious disease or a cancer. These antigens
can be taken from known antigenic protein sequences of bacteria,
viruses, fungi and protozoans, and from cells infected with these
pathogen, or from cancer cells, to immunize the recipient.
[0018] The multispecific binding molecules of the invention
comprise binding determinants from antibody or antibody fragment
molecules which preferably are IgG or IgG fragments. Antibody
fragments are preferably Fab, Fab', F(ab').sub.2, F(ab').sub.3, Fv,
or single chain Fv as sources of binding determinants for
construction of the multispecific binding molecules. Antibody
fragments can be obtained from an IgG isotype class of antibody,
for example from a hybridoma producing a monoclonal IgG antibody,
or from a polyclonal IgG preparation. In a preferred embodiment, a
polyclonal IgG preparation is obtained from an animal that has been
immunized with an antigen from a pathogenic organism, more
preferably from a pathogenic fungus.
[0019] Another feature of multispecific binding molecules in which
the first binding determinant binds Fc.alpha.R and the second
binding determinant binds an antigen of a target cell, encompasses
a third binding determinant which binds to a different antigen on
the same target cell as the second binding determinant. Further,
embodiments encompass a third binding determinant which binds to a
different epitope on the same target antigen as the second binding
determinant. These determinants provide a two-fold binding capacity
of the multispecific molecule to the target to link it to an immune
effector cell, for cytolysis and phagocytosis.
[0020] The invention also provides a method for eliminating an
unwanted cell in a subject, comprising administering to the subject
a therapeutically effective dose of a multispecific binding
molecule, which comprises at least a first binding determinant
which binds an Fc.alpha. receptor and a second binding determinant
which binds an antigen on the unwanted cell, in a pharmaceutically
acceptable carrier. For human subjects, the binding determinants
derived from an antibody can be humanized. Further, therapeutic
treatment can be monitored by obtaining a biological sample from
the subject during the course of treatment. Another embodiment of
the method for eliminating unwanted cells in a subject involves
treating the subject in addition with an agent that enhances the
number or activity of Fc.alpha. receptors, for example, treating
the subject with a cytokine. Preferred cytokines for administration
during treatment with the invented composition treatment include at
least one of G-CSF, GM-CSF, IFN-.gamma., and TNF, and protocols
involving treatment with the molecules of the subject invention and
more than one additional therapeutic agent are envisioned.
[0021] In another embodiment, the effector cells of the subject can
be armed against an antigen, by administering to the subject a
therapeutically effective dose of a multispecific binding molecule,
which comprises at least a first binding determinant which binds an
Fc.alpha. receptor and a second binding determinant which binds the
antigen, in a pharmaceutically acceptable carrier. The effector
cells thus armed with the multispecific molecule do not modulate or
down-regulate the Fc.alpha. receptors on their surface, and are
capable of binding to a target antigen. For human subjects, the
binding determinants derived from an antibody can be humanized.
Further, therapeutic treatment can be monitored by obtaining a
biological sample from the subject during the course of
treatment.
[0022] Another preferred embodiment of the invention is a method
for eliminating an unwanted cell in a subject, comprising obtaining
an aliquot of a sample of blood or blood cells from said subject,
treating said blood or blood cells ex vivo with a therapeutically
effective dose of a multispecific binding molecule of the invention
in a pharmaceutically acceptable carrier, said binding molecule
comprising a first binding determinant which binds an Fc.alpha.
receptor and a second binding determinant which binds one or more
target antigens, and returning said treated blood or blood cells to
the subject. Preferably, the cells of the sample of blood are
isolated and expanded in culture, and more preferably, the cells of
said sample of blood are treated with agents that enhance the
number or activity of Fc.alpha. receptors. For human subjects, the
binding determinants derived from an antibody can be humanized.
Further, therapeutic treatment can be monitored by obtaining a
biological sample from the subject during the course of
treatment.
[0023] In an aspect, the invention provides a method for treatment
of a subject with an infectious disease, comprising administration
to the patient of a therapeutically effective dose in a
pharmaceutically acceptable carrier of a multispecific binding
molecule, wherein a first binding determinant binds an Fc.alpha.
receptor and a second binding determinant binds a target antigen
from a pathogen or a pathogen-infected cell, enhancing the capacity
of the immune system to eliminate the infection.
[0024] In yet another embodiment, the invention provides a method
for immunizing a subject against a cancer antigen or an antigen
found on a pathogen or a cell infected by a, comprising
administration in a pharmaceutically acceptable carrier of a
composition of a multispecific binding agent bearing one or more
antigens of a pathogenic infectious organism, or of an antigen of
infected cells, or of a cancer cell. A preferred embodiment of an
infectious organism is a pathogenic fungus, including a pathogenic
yeast; a more preferred embodiment is a species of Candida. For
human subjects, the binding determinants derived from an antibody
can be humanized. Further, therapeutic treatment can be monitored
by obtaining a biological sample from the subject during the course
of treatment.
[0025] The invention provides also a method for identifying for an
agent which modulates Fc.alpha. receptors on the surface of cells,
involving contacting a sample of cells carrying Fc.alpha. receptors
with the agentA method for identifying for an agent which modulates
Fc.alpha. receptors on the surface of cells, comprising contacting
a sample of cells carrying Fc.alpha. receptors with the agent and
determining Fc.alpha. receptor activity in the sample with the
agent, and in a control sample with an antibody that modulates
Fc.alpha. receptors such as antibody A77, and in another control
sample with cells not contacted with said agent or with antibody;
then Fc.alpha. receptor activities in the samples are compared,
such that a sample of cells contacted with the agent having
statistically significant less Fc.alpha. receptor activity than
control cells not contacted with agent, or having statistically
significantly as low Fc.alpha. receptor activity as cells in a
sample with the antibody, identifies an agent which modulates
Fc.alpha. receptors on the surface of cells.
[0026] In another embodiment, the invention provides a method for
designing an agent which modulates Fc.alpha. receptors for
treatment of autoimmune disease by obtaining a three dimensional
model of the A77 anti-Fc.alpha. receptor binding site using the
sequence determinants of A77 heavy and light chain variable
regions. This method involves comparing the amino acid residues of
the A77 variable region with that of heavy and light chain variable
regions of antibodies of known three dimensional structure,
determining placement of non-homologous amino acid residues within
the main peptide chain of the binding region of the V.sub.H and
V.sub..kappa. sites, such that the size, shape and charge of the
A77 anti-Fc.alpha. receptor binding site is determined, screening a
library of molecules to obtain those of suitable size, shape and
charge by computer modeling that are mimetics of the A77 binding
site, and screening such candidates of appropriate size, shape and
charge for activity as potential modulators of Fc.alpha. receptors,
such that an agent which modulates Fc.alpha. receptors is
designed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a gel filtration HPLC elution profile of a
A77X520C9 bispecific molecule preparation, in which the peak at
9.86 min. (.about.150KD) represents a F(ab').sub.3 heterocomplex
comprising one A77 F(ab') and two 520C9 F(ab') molecules, and the
peak at 10.43 min. represents a F(ab').sub.2 heterocomplex
comprising one F(ab') molecule each of A77 and 520C9; the gel
filtration HPLC was performed using a TSK-3000 column.
[0028] FIG. 2 is a graph showing the extent of binding of
A77X520C9, an anti-Fc.alpha.RXanti-HER2/neu bispecific antibody
(BsAb), as a function of concentration in .mu.g/ml, to neutrophils
(PMN, open squares) and to monocytes (closed squares), in which
mean fluorescence intensity analyzed by FACScan is the measure of
binding.
[0029] FIG. 3 is a cytometric analysis showing the extent of
binding of A77X520C9, an anti-Fc.alpha.RXanti-HER2/neu BsAb (at 10
.mu.g/ml, final concentration) to effector cells in heparinized
whole blood, after incubation for one hour at 0.degree. C.;
phycoerythrin-anti mouse IgG was added, erythrocytes were lysed,
and samples were analyzed by FACScan; data show substantial binding
to monocytes (middle panel) and neutrophils (bottom panel), but not
lymphocytes (top panel).
[0030] FIG. 4 is a graph showing binding of A77X520C9, the
anti-Fc.alpha.RXanti-HER2/neu BsAb (diamonds), and binding of
another BsAb, MDX210, also carrying a determinant for HER2/neu
(open squares), to target SKBR-3 breast tumor cells, as a function
of BsAb concentration in .mu.g/ml, in which mean fluorescence
intensity analyzed by FACScan is the measure of binding.
[0031] FIG. 5 is a graph showing binding to target SKBR-3 breast
tumor cells of a further preparation of A77X520C9, an
anti-Fc.alpha.RXanti-HER2- /neu BsAb, as a function of
concentration in .mu.g/ml; SKBR-3 cells were incubated with various
concentrations of A77X520C9 (circles), A77 F(ab').sub.2
(triangles), or 520C9 F(ab').sub.2 (squares) on ice; cells were
washed, stained with anti-mouse IgG conjugated with fluorescein
iso-thiocyanate (FITC), and analyzed by FACScan.
[0032] FIG. 6 is a graph showing A77XH425 BsAb binding to A431
target cells overexpressing EGF-R, in which mean fluorescence
intensity analyzed by FACScan is the measure of binding; cells were
incubated with various concentrations of A77XH425 (circles),
A77F(ab').sub.2 (triangles), or with H425 F(ab').sub.2 (squares);
cells were incubated with antibody as indicated on ice, washed,
stained with anti-human IgG conjugated with FITC, and analyzed by
FACScan.
[0033] FIG. 7 is a graph showing A77X520C9 BsAb-mediated
antibody-dependent cellular toxicity by cell lysis of SKBR-3 breast
tumor target cells by neutrophil effector cells, at a ratio of
effector to target cells of 200 to one, as a function of BsAb
concentration in .mu.g/ml, with two different preparations of
BsAb.
[0034] FIG. 8 is a graph showing A77X520C9 BsAb-mediated
antibody-dependent cellular toxicity by cell lysis of SKBR-3 breast
tumor target cells by effector cells in whole blood, using
independent duplicate BsAb preparations, as a function of BsAb
concentration in .mu.g/ml.
[0035] FIG. 9 is a bar graph showing inhibition by anti-Fc.alpha.R
A77 F(ab').sub.2 of A77X520C9 BsAb-mediated killing of breast tumor
cells by monocytes, compared to absence of inhibition by addition
of anti-Fc.gamma.R M22 F(ab').sub.2.
[0036] FIG. 10 is a graph showing A77X520C9 BsAb-mediated
cytotoxicity by lysis of target SKBR-3 cells by effector PMN cells
purified from whole blood; target SKBR-3 cells were labeled with
100 .mu.Ci of .sup.51Cr for 1 hour prior to combining with PMNs and
another preparation of BsAb indicated here as "BSM" (circles); and
this mixture of target cells, PMNs and BsAb in the presence of 50
.mu.g/ml of A77 F(ab').sub.2 (squares); and this mixture in the
presence of M22 F(ab').sub.2 (triangles), each in a `U` bottom
microtiter plate; cell and antibody mixtures were incubated for 16
hours at 37.degree. C., supernatants were collected and analyzed
for radioactivity, and cytotoxicity was calculated by the formula:
% Lysis=(experimental CPM-target leak CPM/detergent lysis of CPM
target leak CPM) X100%. BsAb-dependent lysis=% Lysis with BsAb-%
Lysis without BsAb, with an effector:target ratio of 200:1; error
bars represent +/- the standard deviation of the mean of values
obtained from triplicate experiments.
[0037] FIG. 11 is a graph showing A77X520C9 BsAb-mediated
cytotoxicity by lysis of target SKBR-3 cells, using as effector
cells monocytes purified from Leukopaks, with an effector:target
ratio of 100:1; other conditions and symbols were the same as those
in the description of FIG. 10.
[0038] FIG. 12 is a graph showing A77X520C9 BsAb-mediated
cytotoxicity by lysis of target SKBR-3 cells, using the effector
cells in whole blood, other conditions and symbols were the same as
those in the description of FIG. 10.
[0039] FIG. 13 is a graph showing cytoxicity of A431 target cells
that overexpress EGF-R, mediated by A77XH425 BsAb (indicated here
as "BSM"; circles) and PMN effector cells purified from whole
blood; cells were incubated also with BsAb in the presence of 50
.mu.g/ml of A77 F(ab').sub.2 (squares) or M22 F(ab').sub.2
(triangles); target cells were labeled with 100 .mu.Ci of .sup.51Cr
for 1 hour and cytotoxicity was determined as in the brief
description of FIG. 10.
[0040] FIG. 14 is a graph showing A77XH425 BsAb mediated lysis of
A431 target cells by monocytes purified from whole blood by
Leukopaks (E:T ratio 100:1); other conditions and symbols were the
same as those in the brief description of FIG. 13.
[0041] FIG. 15 is a graph showing A77XH425 BsAb mediated lysis of
A431 target cells by the effector cells in whole blood; other
conditions and symbols were the same as those in the brief
description of FIG. 13.
[0042] FIG. 16 is a graph showing Anti-TAG 72 x A77 BsAb-mediated
antibody-dependent cellular cytoxicity by neutrophils of TAG
72-bearing tumor cells, as a function of BsAb antibody
concentration in .mu.g/ml.
[0043] FIG. 17 is a graph showing stimulation by the BsAb
composition A77XTT of growth of tetanus toxoid (TT)-specific T
cells, measured by a spectorphotometric assay of cellular lactate
dehydrogenase, by monocyte presentation of this antigen by the
BsAb, compared to that of uncomplexed TT, as a function of
concentration of each antigen in .mu.g/ml.
[0044] FIG. 18 is a graph showing the extent of A77X520C9
BsAb-mediated phagocytosis, as a function of concentration in
.mu.g/ml, of SKBR-3 breast tumor target cells at a ratio of 15
neutrophils per target cell (circles), or 3 monocytes (squares) per
target cell.
[0045] FIG. 19 is a flow cytometric analysis of BsAb-mediated
phagocytosis of SKBR-3 cells by monocyte-derived macrophages (MDM);
SKBR-3 target cells were labeled with the lipophilic red
fluorescent dye, PKH 26, and cultured with MDM in the absence or
presence of A77X520C9 BsAb (or control antibodies) at 37.degree. C.
for 24 hours; MDM and non-phagocytized tumor cells were recovered
with trypsin, and were stained with a FITC labeled anti-CD14, and
samples were analyzed by two color fluorescence by FACScan. Percent
phagocytosis was calculated as the number of dual-positive target
cells (ingested by MDM) divided by the total number of target
cells.
[0046] FIG. 20 is a graph showing phagocytosis of SKBR-3 cells with
A77X520C9 BsAb, in which specific phagocytosis of SKBR-3 cells was
induced by A77X520C9 BsAb (circles) as compared to the presence of
both un-coupled F(ab').sub.2 fragments of A77 and 520C9
(triangles). The BsAb-mediated phagocytosis was blocked by the
addition of 10 .mu.g/ml A77 F(ab').sub.2 (squares). BsAb-dependent
phagocytosis was calculated as: % phagocytosis with BsAb-%
phagocytosis without BsAb.
[0047] FIG. 21 is a photographic and flow cytometric illustration
of neutrophil-mediated phagocytosis of the fungal pathogen, Candida
albicans, in the presence of A77Xanti-Candida (A77X.alpha.-Candida)
BsAb; panels A and B are representative photomicrographs, and
panels C and D are FACScan analyses, of neutrophils that were mixed
with C. albicans cells in the absence (panels A and C) and presence
(panels B and D) of the BsAb.
[0048] FIG. 22 is a graph showing the extent of BsAb-mediated
killing, as a function of concentration in .mu.g /ml, of SKBR-3
breast tumor target cells by neutrophils treated with G-CSF (top
panel), or with G-CSF and IFN-.gamma. (bottom panel).
[0049] FIG. 23 is a graph showing the extent of BsAb-mediated
killing, as a function of concentration in .mu.g/ml, of SKBR-3
breast tumor target cells by effector cells which are
IFN-.gamma.-treated monocytes (top right panel), by TNF-treated
monocytes (bottom panel), or by untreated monocytes (top left
panel).
[0050] FIG. 24 is a bar graph showing that pre-incubation of
effector cells with human IgA does not affect binding of A77 mAb,
compared to a control incubated in the absence of IgA.
[0051] FIG. 25 is a bar graph showing modulation of Fc.alpha.
receptors by incubation of cells with A77 mAb at concentrations
ranging from 0.001 to 10 .mu.g/ml, showing extent of change in
number of cell surface receptors as a function of incubation with
increasing A77 concentration, in comparison with control mAb 520C9
at 10 .mu.g/ml.
[0052] FIG. 26 is a bar graph showing that BsAb binding to
monocytes or to PMN effector cells does not mediate Fc.alpha.R
modulation; modulation of Fc.alpha.R after A77X520C9, A77
F(ab').sub.2 or A77 mAb binding to PMN and monocytes was examined
by flow cytometry, with various concentrations of antibodies added
directly to whole blood and incubated overnight at 37.degree. C. in
medium containing 5%CO.sub.2. Erythrocytes were lysed, and the
surface level of Fc.alpha.R expression on PMN and monocytes was
determined by incubation with an anti-hulgA-PE probe at 4.degree.
C. Modulation was calculated as: [1-(MFI of sample/MFI of no
antibody/BSM control)].times.100%.
[0053] FIG. 27 shows the DNA sequence of the light chain variable
V.sub..kappa. region of the gene encoding the A77 anti-Fc.alpha.R
antibody (SEQ ID NO: 5), and the predicted amino acid residue
sequence (SEQ ID NO: 6).
[0054] FIG. 28 shows the DNA sequence of the heavy chain variable
V.sub.H region of the gene encoding the A77 anti-Fc.alpha.R
antibody (SEQ ID NO: 7), and the predicted amino acid residue
sequence (SEQ ID NO: 8).
DETAILED DESCRIPTION OF THE INVENTION
[0055] I. Definitions
[0056] Definitions of the terms and phrases as used herein should
have the meanings indicated below. An antibody (or fragment
thereof) is used in the invention as a component of multispecific
agents which cause association of a cytolytic, phagocytic white
blood cell with a tumor cell, or unwanted infectious disease agent
or infected cell. Antibodies suitable for use in the methods of the
invention are available in the art (e.g., from the American Type
Culture Collection, Rockville, Md., or commercially, e.g., from
Becton-Dickinson or Immunotech) or can be prepared by standard
techniques for making antibodies. The term "antibody" as used
herein refers to immunoglobulin molecules and immunologically
active determinants of immunoglobulin molecules, i.e., molecules
that contain an antigen binding site which specifically binds
(immunoreacts with) an antigen. Structurally, the simplest
naturally occurring antibody (e.g., IgG) comprises four polypeptide
chains, two copies of a heavy (H) chain and two of a light (L)
chain, all covalently linked by disulfide bonds. Specificity of
binding in the large and diverse set of antibodies is found in the
variable (V) determinant of the H and L chains; regions of the
molecules that are primarily structural are constant (C) in this
set.
[0057] Structurally, a naturally occurring antibody (e.g., IgG,
M.sub.r 150 kDa) consists of four polypeptide chains, two copies of
a heavy (H) chain and two of a light (L) chain (M.sub.r 25 kDa),
the four chains being covalently linked by disulfide bonds.
Specificity of antigen binding by each molecule that comprises the
large and diverse set of antibodies is found in the variable (V)
determinant of the H and L chains; regions of the molecules that
are primarily structural are constant (C) in this set. IgA (M.sub.r
160 kDa) is the predominant antibody in secretions (saliva, tears,
mild, nasal mucus and gastrointestinal and respiratory secretions).
IgA can exist as a monomer, dimer, and higher multimeric forms (J.
Kendrew, Ed., The Encyclopedia of Molecular Biology, 1994,
Blackwell Science, Oxford). A further isotype is IgM can be found
as a membrane-bound monomer (M.sub.r 190 kDa) on B cells, and as a
circulating secretory form as a pentamer (M.sub.r ca. 950 kDa) that
differs from the bound form at the N-terminus of the heavy chain
due to alternative splicing. The heavy chain of the pentamer is
attached by disulfide bridges to a J chain molecule. Minor antibody
isotypes include IgD (M.sub.r 175 kDa), expressed on cell surfaces,
and IgE. IgE (total M.sub.r 190 kDa) comprises 0.0003% of serum
immunoglobulin, however it can be substantially elevated in an
allergic subject such as an asthmatic, and is medically important
as the major mediator of immediate type hypersensitivity.
[0058] The binding sites of the proteins that comprise an antibody,
i.e., the antigen-binding finctions of the antibody, are localized
by analysis of fragments of a naturally-occurring antibody. Thus,
antigen-binding fragments are also intended to be designated by the
term "antibody." Examples of binding fragments encompassed within
the term antibody include: a Fab fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; an Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; an Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546 )
consisting of a V.sub.H domain; an isolated complementarity
determining region (CDR); and an F(ab').sub.2 fragment, a bivalent
fragment comprising two Fab' fragments linked by a disulfide bridge
at the hinge region.
[0059] These antibody fragments are obtained using conventional
techniques well-known to those with skill in the art, and the
fragments are screened for utility in the same manner as are intact
antibodies. The term "antibody" is further intended to include
bispecific and chimeric molecules having at least one antigen
binding determinant derived from an antibody molecule. Furthermore,
although the H and L chains of an Fv fragment are encoded by
separate genes, a synthetic linker can be made that enables them to
be made as a single protein chain (known as single chain antibody,
sAb; Bird et al. 1988 Science 242:423-426; and Huston et al. 1988
PNAS 85:5879-5883) by recombinant methods. Such single chain
antibodies are also encompassed within the term "antibody", and may
be utilized as binding determinants in the design and engineering
of a multispecific binding molecule. Antibody fragments are also
useful for modulating the number of receptors for that antibody on
the surface of cells, and for obtaining agents that mimic this
activity, by screening for such agents in an assay for modulation
of the receptor.
[0060] Polyclonal antibodies are produced by immunizing animals,
usually a mammal, by multiple subcutaneous or intraperitoneal
injections of an immunogen (antigen) and an adjuvant as
appropriate. As an illustrative embodiment, animals are typically
immunized against a protein, peptide or derivative by combining
about 1 .mu.g to 1 mg of protein capable of eliciting an immune
response, along with an enhancing carrier preparation, such as
Freund's complete adjuvant, or an aggregating agent such as alum,
and injecting the composition intradermally at multiple sites.
Animals are later boosted with at least one subsequent
administration of a lower amount, as 1/5 to {fraction (1/10)} the
original amount of immunogen in Freund's complete adjuvant (or
other suitable adjuvant) by subcutaneous injection at multiple
sites. Animals are subsequently bled, serum assayed to determine
the specific antibody titer, and the animals are again boosted and
assayed until the titer of antibody no longer increases (i.e.,
plateaus).
[0061] Such populations of antibody molecules are referred to as
"polyclonal" because the population comprises a large set of
antibodies each of which is specific for one of the many differing
epitopes found in the immunogen, and each of which is characterized
by a specific affinity for that epitope. An epitope is the smallest
determinant of antigenicity, which for a protein, comprises a
peptide of six to eight residues in length (Berzofsky, J. and I.
Berkower, (1993) in Paul, W., Ed., Fundamental Immunology, Raven
Press, N.Y., p.246). Antibody affinities for the antigen range from
low, e.g. 10.sup.-6 M, to high, e.g., 10.sup.-11 M. The polyclonal
antibody fraction collected from mammalian serum is isolated by
well known techniques, e.g. by chromatography with an affinity
matrix that selectively binds immunoglobulin molecules such as
protein A, to obtain the IgG fraction. To enhance the purity and
specificity of the antibody, the specific antibodies may be further
purified by immunoaffinity chromatography using solid phase-affixed
immunogen. The antibody is contacted with the solid phase-affixed
immunogen for a period of time sufficient for the immunogen to
immunoreact with the antibody molecules to form a solid
phase-affixed immunocomplex. Bound antibodies are eluted from the
solid phase by standard techniques, such as by use of buffers of
decreasing pH or increasing ionic strength, the eluted fractions
are assayed, and those containing the specific antibodies are
combined.
[0062] The term "monoclonal antibody" or "monoclonal antibody
composition" as used herein refers to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
(mAb) composition displays a single binding specificity and
affinity for a particular epitope. Monoclonal antibodies can be
prepared using a technique which provides for the production of
antibody molecules by continuous growth of cells in culture. These
include but are not limited to the hybridoma technique originally
described by Kohler and Milstein (1975, Nature 256:495-497; see
also Brown et al. 1981 J. Immunol 127:539-46; Brown et al., 1980, J
Biol Chem 255:4980-83; Yeh et al., 1976, PNAS 76:2927-31; and Yeh
et al.., 1982, Int. J. Cancer 29:269-75) and the more recent human
B cell hybridoma technique (Kozbor et al., 1983, Immunol Today
4:72), EBV-hybridoma technique (Cole et al.., 1985, Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), and
trioma techniques.
[0063] Tumor specific mAb of human IgA class are not available.
Also, it is likely that serum IgA (up to 4.0 mg/ml) may interfere
with the activity of IgA mAbs under physiological conditions.
Another approach employs bispecific antibody molecules to enable
Fc.alpha.RI-dependent cell-mediated cytotoxicity of tumor targets.
Bispecific molecules (BsAb) which simultaneously bind to target
cells (tumor cells, pathogens) and a trigger receptor (e.g. CD3,
CD2, Fc.gamma.R) on immune effector cells have been described
(Michon, J., et al. 1995, Blood, 86:1124-1130; Bakcs, T., et al.
1995, International Immunology, 7,6:947-955). BsAbs can be
generated from hetero-hybridomas, or by chemically or genetically
linking F(ab') fragments of two antibodies with different
specificities or a F(ab') fragment and a ligand (Graziano, R. F.,
et al. 1995, In Bispecific Antibodies. M. W. Fanger, editor. R. G.
Landes Company/Austin, Texas; Goldstein, J. et al., 1997 J.
Immunol. 158:872-879). BsAbs produced using a trigger
receptor-specific antibody, that binds outside the natural ligand
binding domain of the trigger receptor, can circumvent interference
by serum antibodies and recruit immune effector cells in the
presence of saturating concentration of the natural ligand (Fanger,
M. et al., 1989, Immunol. Today, 10,3:92-99). This strategy has
been used to produce Fc .gamma.R-specific BsAbs, which mediate
antibody-dependent cellular cytotoxicity (ADCC) of tumor cells in
the presence of monomeric or aggregated IgG (Michon, J., et al.
1995, Blood, 86:1124-1130; Bakcs, T., et al. 1995, International
Immunology, 7,6:947-955), and have shown promising results in
clinical settings, Deo, Y. M., et al., 1997, Immunol. Today,
18:127-135. Four Fc.alpha.RI-specific mAb, identified as A3, A59,
A62 and A77, which bind Fc.alpha.RI outside the IgA ligand binding
domain, have been described (Monteiro, R. C. et al., 1992, J.
Immunol. 148:1764).
[0064] A monoclonal antibody can be produced by the following
steps. In all procedures, an animal is immunized with an antigen
such as a protein (or peptide thereof) as described above for
preparation of a polyclonal antibody. The immunization is typically
accomplished by administering the immunogen to an immunologically
competent mammal in an immunologically effective amount, i.e., an
amount sufficient to produce an immune response. Preferably, the
mammal is a rodent such as a rabbit, rat or mouse. The mammal is
then maintained on a booster schedule for a time period sufficient
for the mammal to generate high affinity antibody molecules as
described. A suspension of antibody-producing cells is removed from
each immunized mammal secreting the desired antibody. After a
sufficient time to generate high affinity antibodies, the animal
(e.g., mouse) is sacrificed and antibody-producing lymphocytes are
obtained from one or more of the lymph nodes, spleens and
peripheral blood. Spleen cells are preferred, and can be
mechanically separated into individual cells in a physiological
medium using methods well known to one of skill in the art. The
antibody-producing cells are immortalized by fusion to cells of a
mouse myeloma line. Mouse lymphocytes give a high percentage of
stable fusions with mouse homologous myelomas, however rat, rabbit
and frog somatic cells can also be used. Spleen cells of the
desired antibody-producing animals are immortalized by fusing with
myeloma cells, generally in the presence of a fusing agent such as
polyethylene glycol. Any of a number of myeloma cell lines suitable
as a fusion partner are used with to standard techniques, for
example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma
lines, available from the American Type Culture Collection (ATCC),
Rockville, Md.
[0065] The fusion-product cells, which include the desired
hybridomas, are cultured in selective medium such as HAT medium,
designed to eliminate unfused parental myeloma or lymphocyte or
spleen cells. Hybridoma cells are selected and are grown under
limiting dilution conditions to obtain isolated clones. The
supernatants of each clonal hybridoma is screened for production of
antibody of desired specificity and affinity, e.g., by immunoassay
techniques to determine the desired antigen such as that used for
immunization. Monoclonal antibody is isolated from cultures of
producing cells by conventional methods, such as ammonium sulfate
precipitation, ion exchange chromatography, and affinity
chromatography (Zola et al., Monoclonal Hybridoma Antibodies:
Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press,
1982). Hybridomas produced according to these methods can be
propagated in culture in vitro or in vivo (in ascites fluid) using
techniques well known to those with skill in the art.
[0066] For therapeutic use of antibodies of non-human origin in
humans, the non-human "foreign" epitopes elicit immune response in
the patient. If sufficiently developed, a potentially lethal
disease known as HAMA (human antibodies against mouse antibody) may
result. To eliminate or minimize HAMA, it is desirable to engineer
chimeric antibody derivatives, i.e., "humanized" antibody molecules
that combine the non-human Fab variable region binding determinants
with a human constant region (Fc). Such antibodies are
characterized by equivalent antigen specificity and affinity of
monoclonal and polyclonal antibodies described above, and are less
immunogenic when administered to humans, and therefore more likely
to be tolerated by the patient.
[0067] Chimeric mouse-human monoclonal antibodies (i.e., chimeric
antibodies) can be produced by recombinant DNA techniques known in
the art. For example, a gene encoding the Fc constant region of a
murine (or other species) monoclonal antibody molecule is digested
with restriction enzymes to remove the region encoding the murine
Fc, and the equivalent portion of a gene encoding a human Fc
constant region is substituted. (see Robinson et al., International
Patent Publication PCT/US86/02269; Akira, et al., European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison et al., European Patent Application 173,494;
Neuberger et al., International Application WO 86/01533; Cabilly et
al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent
Application 125,023; Better et al. 1988 Science 240:1041-1043); Liu
et al. 1987 PNAS 84:3439-3443; Liu et al., 1987, J. Immunol.
139:3521-3526; Sun et al. 1987 PNAS 84:214-218; Nishimura et al..,
1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446449;
and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559.)
[0068] The chimeric antibody can be further humanized by replacing
sequences of the Fv variable region which are not directly involved
in antigen binding with equivalent sequences from human Fv variable
regions. General reviews of humanized chimeric antibodies are
provided by Morrison, S. L., 1985, Science 229:1202-1207 and by Oi
et al., 1986, BioTechniques 4:214. Those methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of immunoglobulin Fv variable regions from at least one
of a heavy or light chain. Sources of such nucleic acid are well
known to those skilled in the art and, for example, may be obtained
from 7E3, an anti-GPII.sub.bIII.sub.a antibody producing hybridoma.
The recombinant DNA encoding the chimeric antibody, or fragment
thereof, can then be cloned into an appropriate expression vector.
Suitable humanized antibodies can alternatively be produced by CDR
substitution U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature
321:552-525; Verhoeyan et al. 1988 Science 239:1534; and Beidler et
al. 1988 J. Immunol. 141:4053-4060).
[0069] Human mAb antibodies directed against human proteins can be
generated using transgenic mice carrying the complete human immune
system rather than the mouse system. Splenocytes from these
transgenic mice immunized with the antigen of interest are used to
produce hybridomas that secrete human mAbs with specific affinities
for epitopes from a human protein (see, e.g., Wood et al.
International Application WO 91/00906, Kucherlapati et al. PCT
publication WO 91/10741; Lonberg et al. International Application
WO 92/03918; Kay et al. International Application 92/03917;
Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al.
1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl.
Acad. Sci. USA 81:6851-6855; Bruggeman et al.. 1993 Year Immunol
7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al..
1991 Eur J Immunol 21:1323-1326).
[0070] Monoclonal antibodies can also be generated by other methods
well known to those skilled in the art of recombinant DNA
technology. An alternative method, referred to as the
"combinatorial antibody display" method, has been developed to
identify and isolate antibody fragments having a particular antigen
specificity, and can be utilized to produce monoclonal antibodies
(for descriptions of combinatorial antibody display see e.g.,
Sastry et al. 1989 PNAS 86:5728; Huse et al. 1989 Science 246:1275;
and Orlandi et al. 1989 PNAS 86:3833). After immunizing an animal
with an immunogen as described above, the antibody repertoire of
the resulting B-cell pool is cloned. Methods are generally known
for obtaining the DNA sequence of the variable regions of a diverse
population of immunoglobulin molecules by using a mixture of
oligomer primers and PCR. For instance, mixed oligonucleotide
primers corresponding to the 5' leader (signal peptide) sequences
and/or framework 1 (FR1) sequences, as well as primer to a
conserved 3' constant region primer can be used for PCR
amplification of the heavy and light chain variable regions from a
number of murine antibodies (Larrick et al.,1991, Biotechniques
11:152-156). A similar strategy can also been used to amplify human
heavy and light chain variable regions from human antibodies
(Larrick et al., 1991, Methods: Companion to Methods in Enzymology
2:106-110).
[0071] The term "complement" refers to a set of more than 30 serum
proteins that are universally present without prior exposure to a
particular antigen (see, Liszewski, M. et al., 1993, Fundamental
Immunol., 3rd Ed., W. Paul Ed. Ch. 26 "The Complement System" p.
917). The function of the complement system is modification of the
membrane of an infectious agent, and promotion of an inflammatory
response through cell action. Complement proteins are converted to
active forms by a series of proteolytic cleavages. Production of a
reactive C3b protein can occur quickly and efficiently via the
"classical" complement pathway, or slowly and inefficiently via the
"alternative" pathway. C3 is secreted by monocytes and macrophages;
a complex of Factors B and D and properdin cleave C3 to yield the
products C3a and C3b. These products promote mast cell
degranulation, releasing inflammatory molecules such as histamine,
proteases, lysozyme, acid hydrolases, and myeloperoxidase.
Opsonization of target cell membranes promotes lysis and
phagocytosis.
[0072] As used herein, the term "cytokine" means a protein hormone
that can mediate immune defenses against "foreign" substances or
organisms. General properties of cytokines are reviewed, for
example, by Abbas, A. et al. Cell and Molecular Immunology, 2nd
Ed., 1994, Saunders, Philadelphia. Inflammatory cytokines include
tumor necrosis factor (TNF), interleukin 1.beta. (IL-1.beta.),
IL-6, and .gamma.-interferon (IFN-.gamma.). Production of cytokines
by the host can be stimulated by a microbial product, such as
lipopolysaccharide (LPS), or by a foreign antigen.
[0073] Cytokines can be produced by cells of the immune system, for
example, T cells and basophils, and can act on a nearby other cell
(paracrine action), or on the producing cell (autocrine action), or
can be released into the circulation to act on a distant cell
(endocrine). Categories of function of cytokines include: mediation
of natural immunity; regulation of lymphocyte activation, growth,
and differentiation; regulation of immune-mediated inflammation;
and stimulation of leukocyte growth and differentiation.
[0074] Cytokine function is initiated by binding to a specific
receptor on a target cell. For example, the 17kD TNF polypeptide
which functions as a trimer, is produced by phagocytes and T cells.
It binds to a specific TNF-receptor located on, for example, a
neutrophil or an endothelial cell to activate the responses of
inflammation. One such response in these target cells is production
of IL -1.beta., which in turn provokes production of IL-6. Both TNF
and IL -1.beta. act on thymocytes to initiate a signal cascade
culminating in increased expression of genes encoding Ig proteins.
Similarly, IFN-.gamma. binds to specific cell receptors to
stimulate expression of different sequences. These cytokines also
bind to receptors on liver cells to activate expression of proteins
of the acute phase of immune response.
[0075] Other cytokines can be anti-inflammatory in their effects on
the immune system, for example, IL-4, IL-10, and IL-13 (Joyce, D.
et al. 1994, Eur. J. Immunol. 24: 2699-2705; Zurawski, G., et al.
1994, Immunol. Today 15: 19-26). IL-10 thus reduces the
pro-inflammatory effects of TNF by down-regulating surface TNF
receptor (TNF-R) expression, increasing production of soluble
TNF-R, and inhibiting the release of TNF.
[0076] Further, the function of human IL-13 protein, studied by
stimulation of monocytes with LPS, inhibits production of
IL-1.alpha., IL-1.beta., IL-6, IL-8, MIP-1.alpha., TNF-.alpha.,
IL-10, GM-CSF and G-CSF. Further, production of IL-1ra (receptor
antagonist), a soluble form of the IL-1 receptor, is enhanced.
These anti-inflammatory properties are similar to those of IL-4 and
IL-10.
[0077] Immune response to "foreign" antigens comprises the notion
that "self" proteins and other molecules expressed within an
organisms are not antigenic or immunogenic to that organism. In
fact, discrimination between isologous or homologous determinants
and foreign, or heterologous determinants is achieved through
maturation of the immune system of an organism during development
of the immune system. A system of selection against immune cells
bearing antibodies with binding determinants to "self" occurs, so
that when mature the immune system does not attack proteins or
other molecules native to the organism. In certain pathological
conditions known as "autoimmune diseases," however, such
discrimination is not as accurate, and endogenous structures may be
subject to attack from the immune system. Examples of autoimmune
diseases and conditions in which there is autoimmune exacerbation
of symptoms include systemic lupus erythematosus, myasthenia
gravis, multiple sclerosis, and rheumatoid arthritis. Compositions
of the instant invention which are capable of binding to a site on
the Fc.alpha. receptor, by virtue of comprising a binding
determinant of an antibody for a site on this receptor, can also
modulate the number of these receptors on the cell surface, and
accordingly are potential agents for treatment of autoimmune
diseases. Further, amino acid residue sequence data of the Fv
regions of the antibody binding determinant is the basis for
obtaining a three-dimensional model of the protein features, such
as size, charge, and shape of the set of residues which comprise
this binding site, so that agents which mimic this binding site may
be designed.
[0078] The agents of the invention are administered to subjects in
biologically compatible forms suitable for pharmaceutical
administration in vivo to produce a therapeutic response against a
cancer or an infectious disease. By "biologically compatible form
suitable for administration in vivo" is meant a form of the protein
to be administered in which any toxic effects and side effects are
outweighed by the therapeutic effects of the composition.
[0079] The term "subject," as used herein, refers to a living
animal or human in need of susceptible to a condition, in
particular a "cancer or infectious disease" as defined below. The
subject is an organism possessing leukocytes capable of responding
to antigenic stimulation and growth factor stimulation. In
preferred embodiments, the subject is a mammal, including humans
and non-human mammals such as dogs, cats, pigs, cows, sheep, goats,
horses, rats, and mice. In the most preferred embodiment, the
subject is a human. The term "subject" does not preclude
individuals that are entirely normal with respect to cancer,
infectious disease, or normal in all respects.
[0080] The term "patient," as used herein, refers to a human
subject who has presented at a clinical setting with a particular
symptom or symptoms suggesting one or more therapeutic regimens. A
patient may be in need of further categorization by clinical
procedures well-known to medical practitioners of the art (or may
have no further disease indications and appear to be in any or all
respects normal). A patient's diagnosis may alter during the course
of disease progression, such as development of further disease
symptoms, or remission of the disease, either spontaneously or
during the course of a therapeutic regimen or treatment, or
rediagnosis as being entirely of normal condition.
[0081] The term "infectious disease" is meant to include disorders
caused by one or more species of bacteria, viruses, fungi, and
protozoans, which are disease-producing organisms collectively
referred to as "pathogens." The term "fungi" is meant to include
the yeasts. In this invention, pathogens are exemplified, but not
limited to, Gram-positive bacteria such as Enterococcus fecalis,
Hemophilus pneumoniae, Listeria monocytogenes, Mycobacterium
tuberculosis, M. leprae, Proprionibacterium acnes, Staphylococcus
aureus, S. epidermis, S. intermedias, Streptococcus hemolyticus, S.
pneumoniae; Gram-negative bacteria such as Flavobacterium
meningosepticum, Helicobacter pylori, Hemophilus pneumoniae, H.
influenzae, Klebsiella pneumonia, Neisseria gonorrhoeae,
Pseudomonas aeruginosa, Shigella dysenteria, Salmonella typhi, S.
paratyphi, Escherichia coli serotype 0157, Chlamydia species,
Helicobacter species; viruses such as HIV-1,-2, and -3, HSV-I and
-II, non-A non-B non-C hepatitis virus, pox viruses, rabies
viruses, and Newcastle disease virus; fungi such as Candida
albicans, C. tropicalis, C krusei, C. pseudotropicalis, C.
parapsilosis, C. quillermondii, C. stellatoidea, Aspergillus
fumigatus, A. niger, A. nidulans, A. flavus, A. terreus, Absidia
corymbifera, A. ramosa, Cryptococcus neoforms, Histoplasma
capsulatum, Coccidioides immitis, Pneumocystis carinii, Rhizopus
arrhizus, R. oryzae, Mucor pusillus and other fungi; and protozoa
such as Entamoeba histolytica, Entamoeba coli, Giardia lamblia, G.
intestinalis, Eimeria sp., Toxoplasma sp., Cryptosporidium parvum,
C. muris, C. baileyi, C. meleagridis, C. wrairi, and C. nosarum.
Obtaining unique epitopes from these organisms by screening
proteins and by assaying peptides in vitro are commonly known to
those skilled in the art.
[0082] II. Multispecific Molecules
[0083] The instant invention relates in one embodiment to
recombinant multispecific molecules, which have affinity for and
are capable of binding at least two different entities.
Multispecific molecules can include bispecific molecules comprised
of a binding determinant for an Fc receptor and a binding
determinant for a target. The preferred multispecific molecules for
the instant invention include molecules which are comprised of at
least one copy of a binding determinant which binds specifically to
an Fc.alpha. receptor or target; or molecules comprised of at least
one binding determinant which binds an Fc.alpha. receptor, one
binding determinant for a target and additionally one or more
binding determinants that recognize other molecules. A preferred
multispecific molecule is a bispecific antibody (BsAb), which
carries at least two different binding determinants, at least one
of which is of antibody origin.
[0084] A "binding determinant for an Fc.alpha. receptor" refers to
an antibody, a functional antibody fragment (e.g., Fab fragment) or
a ligand such as an engineered binding protein that recognizes and
binds an Fc.alpha. receptor on an effector cell. Preferred
antibodies for use in the subject invention bind the Fc.alpha.
receptor on an effector cell (white blood cell) at a site which is
not bound by endogenous immunoglobulin A (IgA). Most preferably,
the anti-Fc.alpha. receptor V.sub.H and V.sub.L portion binds a
human Fc.alpha.R. Preferred humanized anti-Fc.alpha.R monoclonal
antibodies are described, the teachings of which are fully
incorporated herein by reference. The antibody that comprises the
BsAb or multispecific molecule of the invention 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.
[0085] An "effector cell" as used herein refers to an immune cell
which is a leukocyte or a lymphocyte. Specific effector cells
express specific Fc receptors and carry out specific immune
functions. For example, monocytes, macrophages, neutrophils,
eosinophils, basophils, and lymphocytes which express Fc.alpha.R
are involved in specific killing of target cells and presenting
antigens to other components of the immune system, or binding to
cells that present antigens. 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-bearing
cells against targets.
[0086] The recombinant antibodies or antibody fragments which
specifically bind to an Fc receptor, are preferably "humanized"
i.e. carry portions derived from a human antibody, but having at
least a portion of a complementarity determining region (CDR)
derived from a non-human antibody. Ordinarily that portion which is
"humanized" is selected to provide specificity of the humanized
antibody to bind a human Fc receptor. The humanized antibody has
CDR portions derived from a non-human antibody and the "constant"
portions of the antibody molecule are of human origin.
[0087] 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.alpha. receptor. A sufficient portion
may be selected by inserting a portion of the CDR into the human
antibody and testing the binding capacity of the created humanized
antibody using flow cytometry or enzyme linked immunosorbent assay
(ELISA).
[0088] 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 be replaced with non-human CDRs. It is only necessary
to replace the number of CDRs required for binding of the humanized
antibody to the Fc receptor.
[0089] 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 Application WO 94/10332 entitled, Humanized
Antibodies to Fc Receptors for Immunoglobulin G on Human
Mononuclear Phagocytes.
[0090] In addition to an anti-Fc.alpha. receptor portion, the
claimed multispecific molecules can comprise a binding determinant
for a target i.e. an antibody, a functional antibody fragment or a
ligand that recognizes and binds a pathogen (e.g., viruses,
bacteria, fungi, protozoa), a pathogen infected cell, a cancer or
tumor cell (e.g., breast, ovarian, prostate, testicular, lung,
colon, rectum, pancreas, liver, central nervous system, head and
neck, kidney, bone, blood, and lymphatic system) or other unwanted
cell in a subject (e.g., a human or animal) or an antigen or
modified form thereof. Additionally, the target portion may
comprise or be directed against an antigen. A preferred embodiment
contains an antigen that can be used to induce a specific immune
response against a chronic infection, against a tumor or cancer
cell, or to deplete antigen in the circulation. A different
particularly preferred embodiment has an antigen that is attached
to a multivalent molecule containing a binding determinant for an
FcR, which stimulates the immune system by directing the antigen to
an antigen presenting cell.
[0091] In one embodiment of the invention, the multispecific
molecule contains a binding determinant or ligand which interacts
with a molecule. In a preferred embodiment, the binding determinant
binds a protein, e.g., a protein on a target cell, such as a cancer
cell, or a cell of an infectious disease agent or the agent itself
or an infected cell. Preferred binding determinants include
antibodies, fragments of antibodies, and receptors for growth
factors or differentiation factors. For example, a multivalent
molecule can comprise an epidermal growth factor (EGF), or at least
a portion or modified form that is capable of interacting with a
receptor, e.g., an epidermal growth factor receptor EGF-R, or an
antibody to EGF-R. A particularly preferred embodiment of the
invention comprises a BsAb carrying a binding determinant for an
human EGF-like receptor, including the EGF-R, HER2/neu, HER3, HER4,
etc. In yet another preferred embodiment, the binding determinant
is for the tumor antigen TAG 72 found e.g. on tumors of the breast,
colon, and ovary.
[0092] In another preferred embodiment of the invention, the ligand
is a small peptide, such as bombesin, gastrin-releasing peptide
(GRP), litorin, neuromedin B, or neuromedin C. The sequences of the
peptides can be found, e.g., in U.S. Pat. No. 5,217,955, the
content of which is incorporated herein by reference. The ligand
can also be a modified form of any of these peptides. The
modification can increase binding to the receptor, decrease
binding, or not affect the binding to a receptor. The modification
of the ligand can also transform an agonist into an antagonist,
such that the ligand inhibits rather than stimulates cell
proliferation. Modification of the ligand can be an addition, a
deletion, a substitution, or a modification of at least one amino
acid.
[0093] In another preferred embodiment of the invention, a
multispecific or bispecific molecule comprises an antigen. As used
herein, the term "antigen" means any natural or synthetic
immunogenic substance, a fragment or portion of an immunogenic
substance, a peptidic epitope, or a hapten. In one embodiment of
the invention, a bi- or multispecific molecule is employed to
target an antigen, e.g., tetanus toxoid to the cell to enhance the
processes of internalization and presentation by these cells, and
ultimately, to stimulate an immune response therein. In a specific
embodiment, the bispecific binding agent specifically binds the
antigen (either directly, to an epitope of the antigen, or
indirectly, to an epitope attached to the antigen) and, at the same
time, binds a surface receptor of an antigen-presenting cell which
can internalize antigen for processing and presentation. In another
embodiment, the antigen is linked to the multi- or bispecific
molecule and at the same time binds a surface receptor of an
antigen-presenting cell. In a preferred embodiment the antigen is
covalently attached to the multispecific molecule by genetic or
chemical means The receptor-binding component of the bi- or
multispecific molecule (and thus the bi- or multispecific molecule,
itself) binds the receptor of the antigen-presenting cell at a site
different and distinct from the naturally-occupying ligand. Thus,
binding of the multispecific molecule occurs without competition by
the natural ligand for the receptor. As a result, binding to the
receptor will not be prevented by physiological levels of the
ligand and the targeted receptor will remain capable of binding the
molecule of the invention and the ligand.
[0094] One type of antigen can be an allergen. An "allergen" refers
to a substance that can induce an allergic or asthmatic response in
a susceptible subject. The number of allergens that elicit a
sensitive response in a proportion of a population is enormous, and
includes pollens, insect venoms, animal dander, dust mite proteins,
fimgal spores and drugs (e.g. penicillin). Examples of natural
animal and plant allergens include proteins specific to the
following genera: Felis (Felis domesticus); Canis (Canis
familiaris); Dermatophagoides (e.g Dermatophagoides farinae);
Periplaneta (e.g. Periplaneta americana); Ambrosia (Ambrosia
artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);
Cryptomeria (Cryptomeria japonica) ; Alternaria (Alternaria
alternata); Alder; Alnus (Alnus gultinosa); Betula (Betula
verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia
(Artemisia vulgaris); Plantago (e.g. Plantago lanceolata);
Parietaria (e.g. Parietaria officinalis or Parietaria judaica);
Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum);
Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and
Cupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides,
Juniperus virginiana, Juniperus communis and Juniperus ashei) Thuya
(e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa);
Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);
Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis
glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis
or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus
lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum
(e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum
(e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea);
Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum
halepensis); and Bromus (e.g. Bromus inermis).
[0095] Many allergens are found in airborne pollens of ragweed,
grasses, or trees, or in fungi, animals, house dust, or foods. As a
class, they are relatively resistant to proteolytic digestion.
Preferable allergens are those which bind to IgE on mast cells and
basophils, thereby causing a range of symptoms from inflammation
and asthma to a type I anaphylaxis hypersensitivity reaction.
[0096] In another preferred embodiment, a binding determinant is
specific for an antigen on an infectious disease agent or an
infected cell, as defined supra. A binding determinant can be
obtained from a polyclonal antibody, which can be produced by
injection of a rabbit or other suitable animal with a fragment or
component of a pathogen, for example, a cell wall component of a
pathogen. IgG can be purified from the serum of the immunized
animal, F(ab').sub.2 can be prepared, and used as the source of the
target binding determinant. For example, polyclonal antibody
prepared by immunizing a rabbit with a preparation of Candida
albicans fragments can be obtained, and used to prepare anti-C.
albicans IgG F(ab').sub.2, which can be coupled to a binding
determinant for Fc.alpha.RI by chemical methods described
herein.
[0097] In some cases, it may be desirable to couple a substance
which is weakly antigenic or nonantigenic in its own right (such as
a hapten) to a carrier molecule, such as a large immunogenic
protein (e.g., a bacterial toxin) for administration. In these
instances, the bispecific binding reagent can be made to bind an
epitope of the carrier to which the substance is coupled, rather
than an epitope of the substance itself.
[0098] The antigen that can be linked either directly, or
indirectly, to a multi- or bispecific molecule of the invention can
be soluble or particulate; it may carry B cell epitopes, T cell
epitopes or both. The antigen can be bacterial, fungal, viral or
parasitic in origin. Often, the antigen will comprise a component
of the surface structure of a pathogenic organism, or a surface
structure in a cell infected by a pathogenic organism. For example,
the antigen can comprise a viral surface structure such as an
envelope glycoprotein of human immunodeficiency virus (HIV) or the
surface antigen of hepatitis virus. In addition, the antigen can be
associated with a diseased cell, such as a tumor cell, against
which an immune response may be raised for treatment of the
disease. The antigen can comprise a tumor-specific or
tumor-associated antigen, such as the HER-2/neu proto-oncogene
product which is expressed on human breast and ovarian cancer cells
(Slamon et al. (1989) Science 244:707). Another important cancer
antigen which comprises a target of the BsAb of this invention is
TAG 72, which has been identified on about 90% of colorectal
cancers, 85% of breast tumors, and 95% of ovarian tumors (Johnson
et al. 1986 Cancer Res. 46:850-897; Bodmer, M. et al., European
Patent Specification 0 348 442 B1; Mezes, P. et al. International
Application WO 93/12231).
[0099] The cells of a subject can be exposed ex vivo or in vivo to
the multispecific molecules of the invention, to target an antigen
to antigen-presenting cells. Immune cells are separated and
purified from subject blood, exposed to a multispecific molecule
comprising the antigen, or the cells can be exposed to the antigen
together with a multispecific molecule having a binding determinant
for the antigen. After stimulation, cells are returned to the
subject. Cells to be used in this procedure can also be treated
with cytokines or other factors, for the purpose of, for example,
up-regulating numbers of receptors per cell. Further, in vivo or ex
vivo therapeutic use of the molecules can be enhanced by treatment
of the subject with one or more cytokines or growth factors.
[0100] The method of this invention can be used to enhance or
reinforce the immune response to an antigen. For example, the
method is valuable for the treatment of chronic infections, such as
hepatitis and AIDS, where the unaided immune system is unable to
overcome the infection. It can also be used in the treatment of the
acute stages of infection when reinforcement of immune response
against the invading organism may be necessary.
[0101] The method can be used to reduce the dose of antigen
required to obtain a protective or therapeutic immune response or
in instances when the host does not respond or responds minimally
to the antigen. Although generally desirable, the lowering of
effective dose can be especially desirable when the antigen is
toxic to the host such as is the case for allergies. Methods and
uses for bi- or multispecific molecules comprising one or more
antigens or comprising one or more binding determinants, e.g., an
antibody interacting with an antigen, are further described in the
published PCT application PCT/US91/07283.
[0102] III. Methods for Making Multispecific Molecules
[0103] 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 multi-specific
molecule is a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2
or ligand.times.Fab fusion protein. A bispecific molecule of the
invention can also be a single chain bispecific molecule, such as a
single chain bispecific antibody, a single chain bispecific
molecule comprising one single chain antibody and a binding
determinant, or a single chain bispecific molecule comprising two
binding determinants. Multispecific molecules can also be single
chain molecules or may comprise at least two single chain
molecules. Methods for preparing bi- or multivalent antibodies are
described for example described in U.S. Pat. No. 5,260,203; U.S.
Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No.
5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.
Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.
5,482,858.
[0104] Binding of the single chain molecules to their specific
targets can be confirmed by bispecific ELISA, familiar to those
skilled in the art. Alternatively, each specificity of a
multispecific molecule can be generated separately and the
resulting proteins or peptides chemically conjugated to one
another. For example, two humanized antibodies or antibody
fragments can be conjugated via sulfhydryl bonding of the
C-terminus hinge regions of the two heavy chains as described in
Examples, infra.
[0105] The BsAbs of the present invention can be prepared by
conjugating the anti-FcR and anti-target portions using methods
described in the following Examples 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-pyridyldi- thio)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
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).
Examples of other cross-linking agents include
ortho-phylenedimaleimide (o-PDM), protein A, carbodiimide. In the
preferred embodiment for BsAb, the conjugating agent is o-PDM.
Other preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0106] Based on their ability to bind FcR bearing immune cells and
specific target cells, a particular embodiment of a multispecific
molecule can be administered to a subject to treat or prevent
reoccurrence of a variety of diseases or conditions, including:
cancer (e.g., breast, ovarian, testicular, prostate, lung, brain,
colon, rectum, pancreas, liver, central nervous system, head and
neck, kidney, bone, blood and lymphatic system), pathogenic
infections such as viral (such as HIV, HTLV and FELV), protozoan
(such as Toxoplasma gondii), fungal (such as Candida albicans); and
bacterial (such as Staphylococcus aureus, Streptococcus hemolyticus
and Mycobacterium tuberculosis). Another aspect of the invention
provides molecules that are useful for vaccination against diseases
and cancer by including an antigen from disease organisms, from
infected cells, from gene products of disease organisms or from
cancer cells. For these purposes, the invention provides
compositions which are multispecific molecules that link the useful
operative antigen to a binding determinant that directs the antigen
to the immune system. An Example provided herein describes a
molecule which functions to target tetanus toxoid directly to
Fc.alpha.R on monocytes, resulting in stimulation of T cells at
lower doses than is required by free tetanus toxoid.
[0107] 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,
transdermal (e.g., via a patch), and injection (subcutaneous,
intravenous, parenteral, intraperitoneal, intrathecal, etc.). The
injection can be in a bolus or a continuous infusion.
[0108] Further, the cells of a tissue, e.g. blood, may be removed
from a patient, fractionated and cultured if appropriate to expand
the cell number, treated ex vivo with the multispecific multivalent
composition in a pharmaceutically acceptable carrier, and returned
to the patent for therapy. During the ex vivo culture and
expansion, a particular cell type may be selected, e.g. a monocyte
population. Further, ex vivo cultured cells may be treated at
various points during ex vivo culture and expansion, with agents to
modify certain functional Fc.alpha.R molecules. Agents include but
are not limited to, growth factors, cytokines, lymphokines such as
IFN-.gamma., G-CSF, TNF, and GM-CSF, and interleukins such as IL-2,
IL-10 and IL-12.
[0109] A multispecific molecule is 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, and
are discussed infra. Any other conventional carrier suitable for
use with the molecules falls within the scope of the instant
invention. Further, therapy with the multispecific multivalent
binding molecule may be coordinated into a treatment regimen with
other similar molecules, or with traditional chemotherapeutic
agents such as cis-platin, AZT, DDI, adriamycin, tetracycline,
cefachlor, nystatin, and acyclovir.
[0110] Combinatorial libraries can be screened to obtain members of
the library with a desired binding activity, and to identify the
active species, by methods that have been described (see, e.g.,
Gordon et al, J Med. Chem., op. cit.). These include affinity
chromatography with an appropriate "receptor" to isolate ligands
for the receptor, followed by identification of the isolated
ligands by conventional techniques (e.g., mass spectrometry and
NMR). Preferably, the soluble receptor is conjugated to a label
(e.g., fluorophores, spectrophotometric enzymes, radioisotopes, or
luminescent compounds) that can be detected to indicate ligand
binding. Alternatively, immobilized compounds can be selectively
released and allowed to diffuse through a membrane to interact with
a receptor.
[0111] Combinatorial libraries of compounds can also be synthesized
with "tags" to encode the identity of each member of the library
(see, e.g., W. C. Still et al., International Application WO
94/08051). In general, this method features the use of inert but
readily detectable tags, that are attached to the solid support or
to the compounds. When an active compound is detected the identity
of the compound is determined by identification of the unique
accompanying tag. This tagging method permits the synthesis of
large libraries of compounds which can be identified at very low
levels among to total set of all compounds in the library.
[0112] Specific binding proteins with high affinities for targets
can be made according to methods known to those in the art. For
example, proteins that bind specific DNA sequences may be
engineered, and proteins that bind a variety of targets, especially
protein targets (Ladner, R. C., et al., U.S. Pat. No. 5,233,409;
Ladner, R. C., et al., U.S. Pat. No. 5,403,484) may be engineered
and used in the present invention as the Fc.alpha.R binding
determinant or as the target binding determinant, as appropriate.
Further, the methods of these libraries can be used in screens to
obtain binding determinants that are mimetics of the structural
determinants of antibodies.
[0113] In particular, the Fv binding surface of a particular
antibody molecule interacts with its target ligand according to
principles of protein-protein interactions, hence sequence data for
V.sub.H and V.sub.L (the latter of which may be of the .kappa. or
.lambda. chain type) is the basis for protein engineering
techniques known to those with skill in the art. Details of the
protein surface that comprises the binding determinants can be
obtained from antibody sequence information, by a modeling
procedure using previously determined three-dimensional structures
from other antibodies obtained from NMR studies or crytallographic
data. See for example Bajorath, J. and S. Sheriff, 1996, Proteins:
Struct., Funct., and Genet. 24 (2), 152-157; Webster, D. M. and A.
R. Rees, 1995, "Molecular modeling of antibody-combining sites," in
S. Paul, Ed., Methods in Molecular Biol. 51, Antibody Engineering
Protocols, Humana Press, Totowa, N.J., pp 17-49; and Johnson, G.,
Wu, T. T. and E. A. Kabat, 1995, "Seqhunt: A program to screen
aligned nucleotide and amino acid sequences," in Methods in
Molecular Biol. 51, op. cit., pp 1-15.
[0114] Breast and ovarian cancers are sex hormone dependent
cancers. Breast tumors may be characterized by abnormally expressed
receptors, e.g. those of the human-EGF-like receptor family (HER),
for example HER-2, -3, and 4. The invention is not limited to these
embodiments of HER antigens. The natural HER ligand, Heregulin, can
be incorporated into a bispecific antibody (BsAb) or multispecific
molecule, as a means to target a breast tumor cell expressing one
or more HER receptor during cancer. Further, a heregulin molecules
are binding determinants for heterodimeric HER receptors
containing, eg. a monomer of each of HER-2, -3 or -4 in
combination.
[0115] Additional examples of sex hormone-dependent cancer include
prostate cancer (Smith, P. (1995), Cancer Surveys Vol. 23:
Preventing Prostate Cancer, Imper. Cancer Research Fund and
testicular cancers). The growth of hormone-dependent cancer types
is promoted by male hormones (e.g., androgens such as testosterone
and dihydrotestosterone). Removal of the testes (castration) was
for many years the standard method of preventing secretion of male
hormones by the gonads, to reduce growth of the cancer. Currently,
secretion of male hormones is suppressed by chemical means by
interfering with production of luteinizing hormone (LH), which
regulates synthesis of male hormones. Similar considerations are
applicable to other sex hormone-dependent cancers, such as breast
or ovarian cancer, so that patients with these diseases or in a
population prone to these diseases, are usually not administered
sex hormones as therapeutic or replacements. Multispecific
molecules of the invention can comprise binding determinants for
sex hormones, to reduce the concentration and suppress tumor
growth.
[0116] In a preferred embodiment, the methods of this invention
include administration, for example, to a cancer patient, of a
multispecific multivalent binding molecule preparation comprising
at least one binding determinant with affinity for a tumor marker
or a tumor-specific protein of the cancer to be treated, for
example, the nestin protein for brain cancers. The nestin protein,
which is expressed during normal mammalian fetal development, is
also expressed on tumors of the central nervous system, including
most forms of brain cancer (McKay, D. G. Ronald, U.S. Pat. No.
5,338,839, Aug. 16, 1994). It is also expressed on melanomas on the
skin and on melanomas that have metastasized (V. A. Florenes, R.
Holm, O. Myklebost, U. Lendahl, O. Fodstad, Cancer Res. 54: 354-6,
1994), to other organs and are difficult to detect and treat. The
preferred site of delivery for treatment of a brain tumor with the
molecules of this invention is directly into the central nervous
system or directly, to the brain via spinal injection or fine
needle delivery. For a metastatic cancer, a preferred delivery
route would be by direct injection into the circulation, or by the
ex vivo blood methods described herein.
[0117] Other tumor types for which the methods of this invention
are exemplified by, but are not limited to, Wilm's tumor (A. J.
Buckler, et al U.S. Pat. No. 5,350,840) a pediatric kidney cancer
due to occurrence of a somatic mutation in the patient's single
copy of a gene normally found in two intact copies. Wilm's tumor
can be cured surgically in 95% of cases, and a bispecific or
multispecific multivalent binding protein is envisioned to be
suitable as an adjunct therapeutic modality for surgical patients.
Other examples of known cancer-associated proteins for which the
compositions of matter and methods of the current invention are
suitable include those associated with gastrointestinal cancer (R.
Fishel et al., International Application WO 95/14085, 05/26/95),
those characterized by development of multiple drug resistance
during chemotherapy (J. M. Croop et al., U.S. Pat. No.5,198,344),
and a large number of oncogenes well known to the skilled artisan
such as Rb, ras, and c-myc, the sequences of which are available
for analysis to those with skill in the art. The compositions of
this invention are, for example, suitable for inhibition of
secreted enzymes such as matrix metalloproteinases, which are
considered to potentiate tumor metastasis (Liotta, L. A., et al.
1991 Cell 64:327-336). In the latter embodiment, a multispecific
binding molecule with a binding determinant to the matrix
metalloproteinase and another for Fc.alpha.R would facilitate
inhibition and clearance of these enzymes from in situ activity. If
used in conjunction with standard surgical and chemotherapeutic
regimens, the compositions are foreseen to reduce cancer
re-occurrence and enhance long-term survival.
[0118] IV. Pharmaceutical Compositions
[0119] The compounds of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a
subject in vivo. In a preferred embodiment, the pharmaceutical
composition comprises either a multispecific molecule (compound, or
agent) of the invention and a pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, the
pharmaceutical composition can be administered by combination
therapy, i.e., combined with other agents. For example, the
combination therapy can include a composition of the present
invention with at least one anti-cancer agent, at least one
antibiotic, at least one cytokine, at least one vaccine, or other
conventional therapy. Exemplary anti-cancer agents include
cis-platin, adriamycin, and taxol. Exemplary antibiotics include
isoniazid, rifamycin, and tetracycline. Exemplary cytokines include
G-CSF, GM-CSF, interleukins and interferons.
[0120] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound, or for controlled
release.
[0121] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and di-carboxylic
acids, pheny-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamin- e, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0122] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. The active compounds can
be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0123] To administer a compound of the invention by certain routes
of administration, it may be necessary to coat the compound with,
or co-administer the compound with, a material to prevent its
inactivation. For example, the compound may be administered to a
subject in an appropriate carrier, for example, liposomes, or a
diluent. Pharmaceutically acceptable diluents include saline and
aqueous buffer solutions. Liposomes include water-in-oil-in-water
CGF emulsions as well as conventional liposomes (Strejan et al.,
(1984) J. Neuroimmunol. 7:27). Pharmaceutically acceptable carriers
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the pharmaceutical compositions of
the invention is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0124] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0125] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0126] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0127] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0128] For the therapeutic compositions, formulations of the
present invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal and/or
parenteral administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will vary depending upon the subject being treated, and
the particular mode of administration. The amount of active
ingredient which can be combined with a carrier material to produce
a single dosage form will generally be that amount of the
composition which produces a therapeutic effect. Generally, out of
one hundred per cent, this amount will range from about 0.01 per
cent to about ninety-nine percent of active ingredient, preferably
from about 0.1 per cent to about 70 per cent, most preferably from
about 1 per cent to about 30 per cent.
[0129] Transdermal patches offer the advantage of providing
controlled delivery of a compound of the present therapeutic
inventions to the body. Such dosage forms can be made by dissolving
or dispersing the composition in the proper medium. Absorption
enhancers can also be used to increase the flux of the composition
across the skin. The rate of such flux can be controlled by either
providing a rate controlling membrane or dispersing the composition
in a polymer matrix or gel. Devices, including patches, which
transdermally deliver a composition by iontophoresis or other
electrically-assisted methods can also be employed in the present
invention, including, for example, the devices described in U.S.
Pat. Nos. 4,708,716 and 5,372,579.
[0130] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrastemal injection and
infusion.
[0131] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0132] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0133] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given alone
or as a pharmaceutical composition containing, for example, 0.01 to
99.5% (more preferably, 0.1 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0134] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0135] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved. In general, a suitable daily dose of a compositions of
the invention will be that amount of the compound which is the
lowest dose effective to produce a therapeutic effect. Such an
effective dose will generally depend upon the factors described
above. It is preferred that administration be intravenous,
intramuscular, intraperitoneal, or subcutaneous, preferably
administered proximal to the site of the target. If desired, the
effective daily dose of a therapeutic compositions may be
administered as two, three, four, five, six or more sub-doses
administered separately at appropriate intervals throughout the
day, optionally, in unit dosage forms. While it is possible for a
compound of the present invention to be administered alone, it is
preferable to administer the compound as a pharmaceutical
formulation (composition).
[0136] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,
5,064,413, 4,941,880,4,790,824, or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. These patents are incorporated herein by
reference. Many other such implants, delivery systems, and modules
are well known to those skilled in the art.
[0137] In certain embodiments, the compounds of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs , thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134), different species of which may comprise the
formulations of the inventions, as well as components of the
invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In
one embodiment of the invention, the therapeutic compounds of the
invention are formulated in liposomes; in a more preferred
embodiment, the liposomes include a targeting moiety. In a most
preferred embodiment, the therapeutic compounds in the liposomes
are delivered by bolus injection to a site proximal to the tumor or
infection. The composition must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and
fungi.
[0138] A "therapeutically effective dosage" preferably inhibits
tumor growth or pathogen infection by at least about 20%, more
preferably by at least about 40%, even more preferably by at least
about 60%, and still more preferably by at least about 80% relative
to untreated subjects. The ability of a compound to inhibit cancer
or infectious disease can be evaluated in an animal model system
predictive of efficacy in human tumors and infectious diseases.
Alternatively, this property of a composition can be evaluated by
examining the ability of the compound to inhibit, such inhibition
in vitro by assays well-known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, prevent or delay death of infected tissues or
organs, decrease fever and while cell count, improve CD4 count or
otherwise ameliorate symptoms in a subject. One of ordinary skill
in the art would be able to determine such amounts based on such
factors as the subject's size, the severity of the subject's
symptoms, and the particular composition or route of administration
selected.
[0139] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application, are hereby
expressly incorporated by reference.
EXAMPLES
[0140] The following methodology described in the Materials and
Methods section was used throughout these Examples, set forth
below.
[0141] Materials and Methods
[0142] Cell Lines and Monoclonal Antibodies
[0143] The murine hybridoma producing cell line for the
anti-Fc.alpha.R antibody used to construct the BsAb compositions
described herein is A77 (Monteiro et al. 1992, J. Immunol. 148:
1764-1770), and other anti-Fc.alpha.RI hybridoma cell lines with
properties the same or similar to A77, such as A3, A59 and A62
((Monteiro et al., 1992), can be used also. The hybridoma cell line
for the anti-HER2/neu antibody is 520C9 (Frankel, A. et al. 1985 J.
Biol. Response Modifiers 4:273-286; available from American Type
Culture Collection, ATCC, 12301 Parklawn Drive, Rockville, Md.
20852; accession number HB8696). Antibody H425 is a humanized
anti-EGF-R antibody (Kettlesborough, C. et al. , 1991, Prot. Eng.
4: 773-783). CC49 (anti-TAG 72) hybridoma was obtained from the
ATCC (accession number HB 9459). mAb-producing cell lines were
cultivated in Iscove's Modified Dulbecco's Medium (IMDM, Gibco/BRL,
Grand Island, N.Y.) supplemented with 10% fetal bovine serum (FBS).
The mAb preparations were purified from the respective hybridoma
supernatants by protein A affinity chromatography (Bio-Rad,
Richmond, Calif.).
[0144] SKBR-3 (Backman et al. Cancer Res 54: 2456-2461), a human
breast carcinoma cell line, and SKOV-3 (ovarian carcinoma), which
overexpress the HER2/neu protooncogene, were obtained from ATCC.
EGF-R over-expressing cell lines A431 (skin carcinoma), HN5 (head
and neck carcinoma) and MDA-MB-468 (breast carcinoma), were
obtained from ATCC. Tumor cell lines were cultivated in Iscove's
Modified Dulbecco's Medium (IMDM, Gibco/BRL, Grand Island, N.Y.)
supplemented with 10% fetal bovine serum (FBS). The monocytoid cell
line U937 (J. Immunol. 136: 1641-1647, 1986) which expresses
Fc.alpha.R was obtained from the ATCC and grown in RPMI-1640 plus
10% FBS (Gibco/BRL, Grand Island, N.Y.).
[0145] Candida albicans (strain ATCC 448,585) was grown in
Sabouraud maltose broth (Difco, Detroit, Mich.).
[0146] Preparation of Blood Cells
[0147] Leukocytes were prepared from heparinized whole venous blood
or from apheresis of normal human volunteers. 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 (Pharmacia-Upjohn, Piscataway, N.J.) gradient
separation. Monocytes were enriched from mononuclear cells by cold
aggregation and settling through a cushion of fetal calf serum.
Monocyte cultures were used fresh or were incubated at 37.degree.
C., 5% CO.sub.2 for 24 to 48 hours in teflon dishes at
4.times.10.sup.6 cells/ml of MSFM containing 2.0% normal human
serum type AB (Sigma, St. Louis, Mo.) and 500 IRU/ml IFN-.gamma.
(R&D Systems, Minneapolis, Minn.). Neutrophils were cultured
for 24 to 48 hours (37.degree. C., 5% CO.sub.2) in AIM V media
(Gibco/BRL, 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..
[0148] Binding Byflow Cytometry
[0149] The binding of the BsAb to Fc.alpha.R and HER2/neu was
assessed by flow cytometry. Various concentrations of BsAb diluted
in PBS, pH 7.4 containing 2 mg/ml BSA and 0.05% NaN.sub.3 (PBA),
were incubated with SKBR-3 cells or with human leukocytes for one
hour at 0.degree. C. The cells were washed with PBA and incubated
with a phycoerythrin labeled goat anti-mouse antibody for one hour
at 0.degree. C. The cells were washed and fixed with 1%
paraformaldehyde, and cell associated fluorescence was analyzed on
a Becton Dickinson (Mountain View, Calif.) FACScan.
[0150] To assess whether IgA binding interfered with mAb A77
binding, A77 was incubated with TNF-treated cells in the presence
of an excess of human IgA, and compared to controls incubated in
the absence of IgA. A77 binding was detected with a
phycoerythrin-labeled goat anti-mouse antibody as above. Cells were
washed and fixed with 1% paraformaldehyde, and cell associated
fluorescence was analyzed on a Becton Dickinson FACScan. In
addition, binding human IgA to U937 cells was assessed in the
presence of A77 mAb, and compared to controls in the absence of
excess A77 mAb. IgA binding was detected with FITC labeled
anti-human IgA antibody
[0151] BsAb Coupling Procedure
[0152] BsAb preparations were constructed using the method of
Glennie et al. (J. Immunol. 1987 139: 2367-2375). mAbs A77
(anti-Fc.alpha.R), 520C9 (anti-HER2/neu), CC49 (anti-TAG 72) and
H425 (anti-EGF-R) antibodies were produced by in vitro cultivation
of the respective hybridoma cell lines. The antibody preparations
were each digested with pepsin to produce F(ab').sub.2
preparations, and subsequently reduced to Fab' by addition of 10 mM
mercaptoethanolamine (MEA) and incubation for 30 minutes at
30.degree. C. The Fab' fragments were applied to a Sephadex G-25
(Pharmacia-Upjohn, Piscataway, N.J.) column equilibrated in 50 mM
Na Acetate, 0.5 mM EDTA, pH 5.3 (4.degree. C.). One-half volume of
ortho-phenylenedimaleimide (o-PDM, 12 mM) dissolved in
dimethylformamide and chilled in a methanol/ice bath was added to
the 520C9 Fab', and the mixture was incubated 30 minutes at
0.degree. C. 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.).
[0153] For preparation of BsAb, the 520C9 Fab'-maleimide was added
to an equimolar solution of the A77 Fab'. The reactants were
concentrated under nitrogen to the starting volume using a Diaflo
membrane in an Amicon (Lexington, Mass.) chamber, at 4.degree. C.,
for 18 hours. The pH was then adjusted to 8.0 with 1M Tris-HCl, pH
8.0, and the mixture was reduced with 10 mM MEA (30 minutes,
30.degree. C.) and alkylated with 25 mM iodoacetamide. A77XCC49 and
A77XH425 were produced using similar procedures. The bispecific
F(ab').sub.2 reactant preparation was separated from unreacted
Fab's and other materials using a Superdex 200 column
(Pharmacia-Upjohn, Piscataway, N.J.) equilibrated in PBS.
[0154] Analysis of A77X520C9 BsAb by HPLC showed that this BsAb
comprised of two main species; 75-85% of F(ab').sub.2 heterodimer
(100 kDa, 10.43 mins) and 15-25% of F(ab').sub.3 heterotrimer
(.about.150 kDa, 9.86 mins) (FIG. 0). Based on the method of
preparation, the F(ab').sub.3 species is expected to be comprised
of two 520C9 F(ab') and one A77 F(ab'). A control experiment, in
which a F(ab')-o-PDM derivative was incubated without a
non-derivatized second F(ab'), confirmed that F(ab')-o-PDM
derivative does not cross-link with itself since all the hinge-SH
groups are occupied by o-PDM and no free -SH group is available for
linkage (data not shown). Thus the F(ab').sub.2 and F(ab').sub.3
species in these BsAb preparations are hetero-complexes of F(ab')
fragments with two different specificities.
[0155] Antibody Dependent Cellular Cytotoxicity (ADCC)
[0156] Human breast carcinoma cells, SKBR-3, which over-express
HER2/neu, were used as target cells for determination of lysis
using multispecific compositions comprising binding determinants
for HER2/neu. Other target cell lines were used in tests of
molecules with different antigen-binding determinants, for example,
A431 cells for EGF-R, KLEB for TAG 72, etc. Effector cell samples
were obtained by using heparinized whole blood, purified
neutrophils (purified as described supra), or monocytes prepared
from Leukopaks obtained from Advanced Biotechnologies Inc.
(Columbia, Md.) as previously described (Guyre, P. M. et al. 1989,
J. Immunol. 193: 1650). To prepare for use as effector cells,
monocytes were cultured in Teflon containers in Macrophage
Serum-Free Medium (Gibco/BRL) containing 2% human serum for 24 to
48 hours. Target cells were labeled with 100 .mu.Ci of .sup.51Cr
for one hour prior to combining with effector cells and antibodies
in a U-bottom microtiter plate. After incubation for 16 to 18 hours
at 37.degree. C., supernatants from each well 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%. Further, specific lysis= % lysis
with antibody-% lysis without antibody. Assays were performed in
triplicate.
[0157] Fluoresceination of Antibodies
[0158] The pH of mAb solution was adjusted to 9.3 by the addition
of 0.1M 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.
[0159] Modulation of Fc.alpha.R by mAb A 77
[0160] The ability of mAb A77 to modulate the number of Fc.alpha.R
on the surface of human monocytes was assessed by incubating
monocytes with various dilutions of MAb A77 37.degree. C. for 18
hours (or with mAb 520C9 as an isotype control). Monocytes were
then washed with PBA, and incubated for one hour at 0.degree. C. in
the presence of human serum IgA at 100 .mu.g/ml. Cells were further
washed with PBA, and IgA binding to Fc.alpha.R, was detected with
FITC-labeled anti-human IgA antibody. Percent modulation=1-(MFI of
the sample/MFI of the control).times.100%, where MFI is the mean
fluorescence intensity.
[0161] BsAb-mediated Phagocytosis
[0162] Assay of monocyte and neutrophil-mediated phagocytosis of
SKBR-3 cells was performed with SKBR-3 target cells labeled with
the lipophilic red fluorescent dye PKH 26. Buffy coat cells
purified from heparinized whole blood containing monocytes,
neutrophils, and lymphocytes were incubated with the labeled
targets at 37.degree. for 6 hours in the absence or presence of
BsAb. Monocytes and neutrophils were stained with FITC labeled
anti-CD14 mAb (AML-2-23) at 0.degree. C., and cells were washed and
analyzed by two color fluorescence by FACScan. Percent phagocytosis
is expressed as the percent of effector cells (monocytes or
neutrophils) that have PKH 26 stain associated with them.
Example 1
Bispecific Antibody A77X520C9 Binds Effector Cells
[0163] To determine efficacy of the bispecific antibody BsAb
A77X520C9 in killing breast cancer cells in a patient, ability to
specifically bind to breast cancer cells in culture was determined.
For these experiments, cells of line SKBR-3, which overexpresses
the HER2/neu oncogene, were used. The 520C9 binding determinant
derives from an anti-HER2/neu murine hybridoma (Frankel, A. et al.,
1985 J. Biol. Response Modifiers 4: 273-286).
[0164] FIG. 2 shows that BsAb A77X520C9 bound to each of two types
of effector cells, neutrophils (PMNs) and monocytes. FIG. 3 shows
that, in whole blood, A77X520C9 BsAb binds to
Fc.alpha.RI-expressing PMN and monocytes, but not to
Fc.alpha.R-negative lymphocytes. This binding activity was not
inhibited by the physiological levels of serum IgA in whole blood,
which is sufficient to saturate all the Fc.alpha.RI expressed in
vivo.
Example 2
Bispecific Antibody A77X520C9 Binds Target Breast Cancer Cells
[0165] The BsAb A77X520C9 bound to the target breast tumor cells
SKBR-3 derived from a breast tumor (FIG. 4) to the same extent as
did the previously described BsAb MBX210 (Valone et al. 1995 J.
Clin. Oncol. 13(9): 2281-2292). Mean fluorescence intensity (MFI)
as a measure of binding was found to increase as a function of BsAb
concentration when breast tumor cells were incubated with each of
0.1, 1.0 or 10 .mu.g/ml. The fraction of A77X520C9 BsAb that bound
to breast tumor target cells was equivalent to or greater than the
fraction of the control BsAb MDX210 (FIG. 4) and binding to
effector cells (FIG. 2) was similar as a function of concentration
of each of the BsAbs.
[0166] Further data showing binding of A77X520C9 BsAb to HER2/neu
positive SKBR-3 cells is shown in FIG. 5. As expected, only the
BsAb and 520C9 F(ab').sub.2, but not A77 F(ab').sub.2 control,
bound the target breast tumor cells. Data are shown in FIGS. 7-12
with a variety of effector cells, with several independent
bispecific antibody preparations, and with varying concentrations
of different preparations of BsAb.
Example 3
Bispecific Antibody A77XH425 Binds Target Skin Carcinoma Cells
[0167] The monoclonal antibody H425 is specific for the EGF
receptor (EGF-R), which is overexpressed on cell lines derived from
skin carcinoma (A431), and MDA-MB468 (breast carcinoma). The
bispecific molecule A77XH425 which carries a binding determinant
for EGF-R and for Fc.alpha.RI, was prepared by the same chemical
coupling procedure as A77X520C9, and tested for binding to target
cells by the methods described herein.
[0168] Similar to binding of A77X520C9 to HER2/neu-bearing target
cells shown in Example 2 and FIGS. 2-5, FIG. 6 shows that the EGF-R
tumor-specificity of the BsAb A77XH425 causes this molecule to be
bound to A431 tumor cells which overexpress EGF-R. Further, the
tumor binding determinant H425 F(ab')2, but not A77 F(ab').sub.2,
which bears the Fc.alpha.RI determinant, bound to the A431 target
cells which overexpress EGF-R.
Example 4
Binding of Bispecific Antibody A77X520C9 Mediates Effector
Leukocyte Cytolysis of HER2/neu-bearing Target Cells
[0169] Following determination of the ability of A77X520C9 to bind
to breast tumor cells, its ability to bind both to target cells and
to specific leukocytes such that cytolysis of the breast tumor
cells is effected was determined also. Thus, antibody-dependent
cellular cytolysis (ADCC) mediated by bispecific antibodies (BsAb;
also referred to as bispecific molecules, BSM) of the invention is
shown in FIGS. 7-16 as percent cytotoxicity or percent lysis.
[0170] As shown in FIG. 7, cytotoxicity of SKBR-3 cells mediated by
A77X520C9 and neutrophil effector cellss (PMN) varied from 6% to 9%
at 0.1 .mu.g/ml, and increased to 20% to 30% at 1.0 .mu.g/ml.
Similar data are shown for killing mediated by effector cells in
whole blood in FIG. 8. In FIG. 9 this cytotocity mediation by BsAb
is shown to be inhibited by addition also of the
Fc.alpha.RI-binding A77 F(ab').sub.2, but not that of Fc.gamma.RI
determinant of M22 F(ab').sub.2.
[0171] Therapy of subjects with multispecific multivalent chemical
compositions directly administered into the circulation requires
that these agents function in whole blood. Ability to function in
whole blood, i.e., to mediate cytolysis, is shown in FIG. 8, in
which A77X520C9 preparations were found to mediate cytolysis of
cultured breast tumor cells by blood effector cells. At 0.1
.mu.g/ml, between 15% and 20% of the tumor cells were lysed by
A77X520C9, and at 1.0 .mu.g/ml cytolysis was approximately 25% to
30% of breast tumor cells. Since whole blood contains IgA at a
concentration of 2 to 5 mg/ml, these data also show that cytotoxic
activity of this BsAb is not inhibited by IgA. These results show
that BsAb can be delivered for therapeutic application in vivo.
[0172] BsAb-mediated destruction of tumor cells by
Fc.alpha.RI-expressing cytotoxic effector cells was examined using
freshly purified effector cells (monocytes and PMN) as well as
whole blood as the source of effector cells. FIG. 10 shows that 1.0
.mu.g/ml of A77X520C9 BsAb mediated up to 37% BsAb-dependent lysis
of HER2/neu positive SKBR-3 cells by purified PMN (neutrophils).
This cytotoxic activity was dose dependent and saturated at 1.0
.mu.g/ml of the BsAb. FIG. 11 shows that A77X520C9 mediated up to
40% BsAb-dependent lysis of SKBR-3 cells when purified monocytes
were used as effector cells. Finally, A77X520C9 BsAb mediated up to
40% BsAb-dependent lysis of the same target cells when whole blood
was used as a source of effector cells (FIG. 12). In the data shown
in FIGS. 10-12, A77 F(ab').sub.2 with the determinant for
Fc.alpha.RI, inhibited the ADCC activity of this BsAb, but the
anti-CD64 M22 F(ab').sub.2 with the determinant for Fc.gamma.RI, d
id not. The background lysis (in the absence of BsAb) in these
experiments was about 10%.
[0173] Similarly, A77X520C9 BsAb mediated lysis of another HER2/neu
over-expressing tumor line, SKOV-3 (ovarian carcinoma line), in a
whole blood ADCC assay.
Example 5
Binding of Bispecific Antibody A77X5H425 Mediates Effector
Leukocyte Cytolysis of EGF-R-bearing Target Cells
[0174] Similar to the data shown for BsAb for the tumor antigen
HER2/neu, A77XH425 BsAb, with the affinity determinant for EGF-R,
mediated up to 52% BsAb-dependent lysis of A431 target tumor cells
with purified PMN as effector cells (FIG. 13), up to 55%
BsAb-dependent lysis with purified monocytes as effector cells
(FIG. 14), and up to 43% BsAb-dependent lysis with the whole blood
a source of effector cells (FIG. 15). In these experiments A77
F(ab').sub.2, but not M22 F(ab').sub.2, inhibited the cytotoxic
activity. The background lysis (without BsAb) was about 10%.
[0175] These results show that the BsAb A77X520C9 can bind both
effector and HER2/neu-bearing target cells, and can mediate target
cytolysis by purified neutrophils and monocytes, and by these
effector cells in whole blood. Lymphocytes in whole blood did not
bind the BsAb. Further, the BsAb A77XH425 can bind target cells
that overexpress EGF-R, and mediated cytolysis of these cells in
the presence of effector cells.
[0176] In whole blood ADCC assays, A77XH425 BsAb-dependent lysis
(40-50% cytolysis) of HN5 (head and neck carcinoma line) and
MDA-MB468 (breast carcinoma line), which express comparable levels
of EGF-R as A431 (skin carcinoma line), was observed.
Example 6
Bispecific Antibody anti-TAG 72XA77 Mediates Cytolysis of TAG
72-Bearing Tumor Cells
[0177] The Examples above show that BsAb preparations are effective
in mediating cytolysis of cell of cell lines derived from tumors,
bearing the tumor antigens HER2/neu, and EGF-R. To extend the
potential use of BsAb, another composition with a binding
determinant for the TAG 72 tumor marker was prepared by the
coupling method described supra. The mucine antigen TAG 72 is found
in the majority of breast, colon, ovarian and other cancers. A
variety of antibodies that specifically bind TAG 72 are available,
for example the monoclonal antibody produced by hybridoma CC49
(ATCC HB 9459, Mezes, P. et al., International Application WO
90/04410).
[0178] CC49 was coupled to A77 to produce BsAb anti-TAG 72XA77, to
target tumor cells bearing the TAG 72 antigen specifically to
effector cells bearing Fc.alpha.R. FIG. 16 shows neutrophil
mediated antibody-dependent cytotoxicity by the BsAb anti-TAG
72XA77 (constructed from CC49 and A77 mAb antibodies) of TAG
72-bearing tumor cells, as a function of concentration in .mu.g/ml.
The results in FIG. 16 show that BsAb anti-TAG 72XA77 mediated
cytolysis of tumor cells to a similar extent as BsAbs A77X520C9 for
cells bearing HER2/neu, and A77XH425 for cells bearing EGF-R, shown
in the Figures above.
[0179] Overall, Examples 1-6 demonstrate cytolysis of tumor cells
bearing three different tumor antigens mediated by the bispecific
molecules of the invention. These Examples and other studies
performed using cell lines taken from breast tumors, skin
carcinoma, gastric carcinoma, head and neck carcinoma, and ovarian
carcinoma, show that Fc.alpha.RI-directed BsAb preparations coupled
to binding determinants for a variety of tumor antigens can be
broadly applicable in a variety of malignancies. This observation
is significant because target antigen restriction of
Fc.gamma.R-mediated ADCC activity of PMN has been reported for B
cell lymphoma associated antigens (Elasser, D., et al., 1996.
Blood. 87,9:3803-3812).
Example 7
BsAb Cytolysis Requires Access to Fc.alpha.R Via the A77 Binding
Determinant
[0180] To show that BsAb-mediated cytolysis of target cells is due
to Fc.alpha.R recognition by the A77 binding determinant, BsAb
cytolysis was analyzed in the presence of A77 F(ab').sub.2 in FIGS.
9-15. If BsAb-mediated cytolysis functions by binding to Fc.alpha.R
because of the A77-derived binding determinant, then the addition
of A77 F(ab').sub.2, but not an antibody with a different receptor
binding determinant, could cause inhibition of cytolysis of breast
tumor cells.
[0181] The results shown in FIGS. 9-15 show that BsAb-mediated
cytolysis functions by binding to Fc.alpha.RI via the A77-derived
binding determinant. In FIGS. 9-12, the addition of A77
F(ab').sub.2 to the mixture of A77X520C9, target cells, and
effector cells causes inhibition of cytolysis, while similar
addition of an antibody that does not share the Fc.alpha.RI has no
effect on level of cytolysis. FIGS. 13-15 show this result also
with BsAb A77XH425-mediated cytolysis of tumor cells: inhibition
was observed in the presence 50 .mu.g/ml A77 F(ab').sub.2, and not
in the presence of 50 .mu.g/ml of M22 F(ab').sub.2. M22
F(ab').sub.2 specifically binds a different receptor, the
Fc.gamma.RI receptor (Valone et al., 1995 J Clin. Oncol. 13:
2281-2292).
[0182] These data show that BsAbs A77X520C9 and A77XH425 mediate
cytolysis of tumor cells in a manner that is dependent on specific
binding of the BsAb to Fc.alpha.RI on effector cells. Further,
inhibition of BsAb killing by A77 F(ab').sub.2 was found regardless
of the nature of the effector cells.
Example 8
Stimulation of T-cell Growth by Antigen-Presentation Using a
Bispecific Antibody
[0183] For use of a BsAb for delivery of an antigen, the following
procedure was used to couple the Fc.alpha.RI binding determinant of
mAb A77 to tetanus toxoid. Purified tetanus toxoid (TT, Accurate
Chemical and Scientific Company, Westbury, N.Y.) was reacted with
sulfo-succidimidyl, 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(s-SMCC, Pierce, Rockford, Ill.) at a molar ratio of 20 SMMC: 1 TT
for two hours at room temperature. Free SMCC was removed from
TT:SMCC by G-25 chromatography. The F(ab')2 fragment of mAb, A77,
was reduced to Fab' by incubating in the presence of 5 mM
mercaptoethylamine (MEA, Sigma, St. Louis, Mo.) for 30 minutes at
30.degree. C. Free MEA was removed from Fab' by G-25
chromatography. The A77 Fab' was incubated with the SMCC-treated TT
for two hours at room temperature followed by an overnight
incubation at 4.degree. C. The A77-TT conjugate was purified from
uncoupled Fab' by Superdex 200 gel filtration (Pharmacia-Upjohn,
Piscataway, N.J.).
[0184] To assay T cell proliferation following antigen presention,
TT-specific T cell lines were generated from immunized individuals
as previously described (Gosselin, E. J. et al. J. Immunol.
179:3477, 1992), monocytes were purified by cold aggregation as
described previously (Guyre, P. M. et al. J. Immunol. 143:1650,
1989) and T cells (50 .mu.l of 10.sup.6/ml) and autologous
monocytes (50 .mu.l of 5.times.10.sup.5/ml) were added to wells of
a 96 well tissue culture plate in the presence of various
concentrations of TT or of A77-TT and incubated at 37.degree. in a
CO.sub.2 incubator. The relative number of T cells in each well was
assessed after incubation for four days by measuring lactate
dehydrogenase (LDH) released from cells after lysis using a kit
purchased from Promega (Madison, Wis.). LDH levels were quantified
spectrophotometrically after addition of substrate and stop
solution. Optical density was read at 490 nm using an ELISA plate
reader (Molecular Devices, Palo Alto, Calif.).
[0185] Each data point presented in FIG. 17 is the mean of data
obtained from quadruplicate samples, with background value
(monocytes and T cells in the presence of media only) subtracted.
The A77-TT conjugate induced equivalent T cell proliferation at
antigen doses that were 30-100 fold lower than that of the
uncoupled TT. These data show that directing the TT antigen to
Fc.alpha.RI by coupling to mAb A77 clearly enhanced monocyte
presentation of TT to TT-specific T cells. Similar antigen
presentation can be achieved with antigens for allergies, such as
antigens of the dust mite, and cat and dog antigens, and for
vaccine antigen presentation, such as for malaria, chicken pox,
hepatitis virus C, and other non-A-non-B hepatitis viruses.
Example 9
Bispecific Antibody Binds Target Cells and Causes Phagocytosis by
Effector Cells
[0186] To demonstrate BsAb mediation of phagocytosis, the following
experiments were performed to determine the extent to which
effector cells engulf SKBR-3 breast tumor cells or cell fragments
in the presence of BsAb A77X520C9. These studies tested
phagocytosis of SKBR-3 by neutrophils, monocytes, and
monocyte-derived macrophages (MDM). Phagocytosis of tumor cells
mediated by Fc.alpha.RI has not been described.
[0187] Tumor cells were labeled prior to exposure to leukocytes
with lipophilic red fluorescent dye PKH26. Following incubation of
effector cells with target cells for 6 hours, effector monocytes
and neutrophils purified from whole blood were stained with
FITC-labeled anti-CD14 mAb, and were analyzed by cell sorting. The
results are shown in FIGS. 18-20. Percent phagocytosis is expressed
as FITC-stained effector cells associated with the lipophilic red
dye. As shown in FIG. 18, percent effector cells engaged in
phagocytosis was on the order of 65% to 75%, depending on whether
monocytes or neutrophils were tested. Increasing the bispecific
antibody concentration above 1.0 .mu.g/ml did not enhance the
percent of effector cells engaged in phagocytosis.
[0188] Monocytes differentiated into macrophages (MDM) are known to
mediate phagocytosis of tumor cells (Ely, P. et al. 1996, Blood.
87:3813-3821). To determine if A77X520C9 BsAb could induce
phagocytosis by MDM, these effector cells were incubated with
dye-labeled SKBR-3 cells in the presence of varying concentrations
of BsAb. The level of phagocytosis was determined by 2-color flow
cytometric analysis, in which one fluorescent label is denoted FL1
and the other is denoted FL2. FIG. 19 shows that the MDM (panel 1,
FL1.sup.+, FL2.sup.-) and SKBR-3 cells (panel 2, FL1.sup.-,
FL2.sup.+) are distinguished from each other in a mixture of these
two cell types by their unique fluorescence patterns (panel 3).
When the A77X520C9 BsAb was added to the mixture of these target
and effector cells, the BsAb mediated nearly a complete loss of
tumor cells (panel 5, lower right quadrant). This was confirmed by
an almost total lack of tumor cells that could be recovered from
the BsAb containing wells as determined by tumor specific ELISA
(data not shown). As expected, MDM alone mediated some phagocytosis
(.about.45%) of SKBR-3 cells without the BsAb (panel 3). However,
addition of 0.1 .mu.g/ml A77X520C9 was sufficient to enhance the
phagocytosis to >95% (panel 5). This BsAb-mediated phagocytic
activity was almost completely inhibited by A77 F(ab')2 (panel 6).
Furthermore, a mixture of uncoupled A77 F(ab')2 and 520C9
F(ab').sub.2 could not enhance phagocytosis (panel 4), indicating
the need for a conjugated A77 F(ab').sub.X 520C9 F(ab') BsAb to
target tumor cells to effector cells leading to activation of the
effector cells (MDM).
[0189] FIG. 20 shows that the BsAb-mediated phagocytic activity was
dose dependent and saturated (nearly complete loss of tumor cells)
at 0.1 .mu.g/ml of the BsAb. A77 F(ab').sub.2 almost completely
blocked this BsAb-mediated phagocytosis. Again, a mixture of
uncoupled A77 F(ab').sub.2 and 520C9 F(ab').sub.2 had no activity
to mediate phagocytosis.
[0190] The above-described studies show that, in addition to
promoting extracellular lysis, the Fc.alpha.RI-directed BsAb
mediated potent phagocytic activity. In particular, when MDM were
used as effector cells, nearly 100% of tumor cells were
phagocytosed. It is likely that BsAb-mediated both ADCC and
phagocytic activities simultaneously, and entire cells or fragments
of lysed cells were phagocytosed by the MDM via Fc.alpha.RI. These
two mechanisms have been shown to occur in concert for Fc.gamma.
RI-mediated phagocytosis (Ely, P. et al. 1996, Blood.
87:3813-3821). It has been shown that antigen presenting phagocytic
cells (such as MDM) can present antigens via both class I and class
II pathways after phagocytosis of antigen bearing particles (Falo,
L. D. et al. 1995. Nature Medicine. 1,7:649-656). Thus the potent
phagocytosis of tumor cells via Fc.alpha.RI can lead to activation
of both humoral and cellular immune functions specifically directed
to tumor associated antigens.
[0191] These cytotoxic activities of Fc.alpha.RI-directed BsAb are
of therapeutic value because Fc.alpha.RI expression is limited
primarily to immune effector cells that are herein demonstrated to
mediate BsAb-dependent cytotoxic activities, viz., PMN, monocytes
and macrophages ( Morton, H. C.et al. 1996.Critical Reviews in
Immunology. 16:423). Because distribution of Fc.alpha.RI is limited
primarily to cytotoxic effector cells and because of its potent
triggering activity, BsAb comprising a binding determinant to
Fc.alpha.RI can be of general use for cell-mediated
immunotherapies. Furthermore, the approach described herein can be
employed to prepare Fc.alpha.RI-directed BsAb utilizing the
existing high affinity tumor-specific IgG mAbs, that is, the
binding determinant components of the anti-Fc.alpha.RI BsAb can be
IgG molecules, since they are converted into F(ab').sub.2 fragments
prior to chemical coupling. Further, for construction of
recombinant BsAb, the Fc portion of the IgG can be removed by
appropriate restriction digestion and further exonuclease digestion
as appropriate. The ability to use binding determinants from the
Fab portion of IgG molecules will obviate the need to generate new
IgA class tumor specific mAbs toexploit the cytotoxic potential of
Fc.alpha.RI.
Example 10
Phagocytosis of the Fungal Pathogen Candida albicans
[0192] Bispecific and multispecific antibody compositions of the
invention can be used to direct Fc.alpha.RI-bearing leukocytes
against antigens from microbial pathogens, for example, against
bacteria, viruses, protozoan and metazoan parasites, and fungi. By
way of example, the following studies demonstrate that BsAb
mediates phagocytosis of Candida albicans, a pathogenic yeast which
causes deleterious infections in T-cell deficient immunocompromised
patients.
[0193] A bispecific antibody directed against this pathogen was
produced from rabbit polyclonal-anti Candida IgG (Biodesign,
Kennebunk, Me.). F(ab').sub.2 fragments of this polyclonal IgG were
treated with sulfo-SMCC to add maleimide to free amino groups. This
conjugate was reacted with equimolar A77 F(ab'), yielding the
A77Xanti-Candida BsAb, which was purified from the uncoupled
fragments by chromatography on Superdex 200 (Pharmacia, Piscataway,
N.J.). The A77Xanti-Candida bound effector and target cells
consistent with the specificity of the component antibodies.
[0194] Candida albicans cells cultured overnight at 37.degree. C.
were harvested by centrifugation, washed three times with
phosphate-buffered saline (PBS), and fluorescently labeled by
incubation with FITC (Sigma, St. Louis, Mo.) at a concentration of
0.1 mg/ml in 0.1 M NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 buffer (pH
9.6) for 30 minutes at 4.degree. C. Fluorescent fungal cells washed
three times with PBS (5.times.10.sup.5) were incubated for 30
minutes at 37.degree. C. with 2.times.10.sup.5 isolated neutrophils
in the presence of 10 .mu.g/ml A77Xanti-Candida (A77 X
.alpha.Candida) BsAb; controls were incubated without BsAb.
Phagocytosis was quantitated by measuring FITC-fluorescence
intensities of cells on a flow cytometer using PE-conjugated CD11b
MoAb (Becton Dickinson, San Jose, Calif.) as a gate marker for
neutrophils, and phagocytosis was determined also in cytospin
preparations and evaluated by microscopy. Each experiment was
repeated three times.
[0195] Phagocytosis of Candida by neutrophils, the most important
effector cell population against pathogenic fungi such as Candida
and Aspergillus species (Pizzo, P., 1984, Cancer 54:2649;
Schaffner, A. et al., 1986, J. Clin. Invest. 78:511), and the
stimulation of phagocytosis by the bispecific antibody of the
invention, are shown in FIG. 21. In the presence of A77 X
anti-Candida, 37.3%.+-.7.5% of neutrophils were found to have
phagocytosed fingal particles; in the absence of this BsAb
4.2.+-.1.5% of neutrophils had ingested fungal particles (average
of three experiments). Thus the Fc.alpha.RI BsAb caused 8.9-fold
stimulation of neutrophil phagocytosis of the fungal pathogen as
demonstrated photographically in panels A and B of FIG. 21, and by
the shift in fluorescence intensity of the FITC-labeled fungal
cells to that of PE-labeled neutrophils, in panels C and D.
[0196] Fc.alpha.RI-directed BsAb can be used to combat a number of
infectious diseases, since most infectious agents (bacteria,
viruses, fungi etc.) express unique antigens on their surface and
several pathogen-specific antibodies have been described. This
technology can be applied to combat antibiotic resistant pathogens
such as methicillin resistant Staphylococcus aureus, or other
pathogenic bacterial species, and to pathogenic fingal species in
addition to Candida.
Example 11
BsAb-Mediated Cytolysis With Effector Cells Treated With Cytokines
and Growth Factors
[0197] FIG. 22 shows percent cytotoxicity of SKBR-3 breast tumor
target cells, as a function of concentration of A77X520C9 BsAb, by
neutrophil effector cells pre-incubated with G-CSF (Panel A) or
both G-CSF and IFN-.gamma. (Panel B). Neutrophils were
pre-incubated with these factors overnight at 37.degree. prior to
the cytotoxicity assay. In comparison with the data described in
Example 4, in which neutrophils were not pre-incubated with
cytokines, percent cytotoxicity was here found to be enhanced by
pre-incubation of effector cells with both G-CSF and IFN-.gamma.,
such that at 0.1 .mu.g/ml BsAb, greater than 10% of tumor cells
were specifically lysed compared to less than 10% seen in Example
1, and at 1.0 .mu.g/ml BsAb, greater than 40% cytotoxicity was
observed compared to approximately 24% to 32% in FIG. 7 in the
absence of these factors.
[0198] FIG. 23 shows the results of pre-treating effector monocytes
with IFN-.gamma. (Panel B) or TNF (Panel C), using BsAb A77XH425,
which combines the binding determinant for Fc.alpha.R with the
binding determinant for EGF-R. As described above, EGF receptors
are known to be overexpressed on carcinomas of the breast, skin,
head and neck and other tumor cells, so that BsAb A77XH425
comprises another embodiment of a multispecific composition for
treatment of breast and other tumors. As shown in FIG. 23,
untreated monocytes caused substantial breast cancer cell
cytotoxicity mediated by A77XH425 BsAb, at BsAb concentrations of
0.1 .mu.g/ml and 1.0 .mu.g/ml. Specifically, BsAb A77XH425 caused
greater than 60% cytolysis at 1.0 .mu.g/ml with untreated monocytes
and with TNF treated monocytes, and greater than 40% at 1.0
.mu.g/ml with IFN-.gamma.-treated monocytes.
[0199] Overall, these results demonstrate that each of the two BsAb
preparations, A77X520C9 and A77XH425, bind to breast tumor cells or
skin carcinoma cells, respectively, and mediate cytolysis of the
cancer cells by neutrophils or monocytes without additional
treatment of these effector cells by exogenous cytokines.
Example 12
The A77 Fc.alpha.R Binding Site is Different From That of
Fc.alpha.R for its Natural Ligand
[0200] In design of a therapeutic BsAb directed to Fc.alpha.RI,
optimal functionality as a therapeutic agent in subjects would be
achieved if binding of this entity were independent of competition
with endogenous molecular species, such as the natural ligand IgA.
Thus a therapeutic BsAb or multispecific binding composition could
be administered, and not be blocked or inhibited, or not be
substantially blocked or inhibited, by endogenous IgA.
[0201] FIG. 24 shows that A77 antibody bound to the full extent to
effector cells in the presence of IgA at 200 .mu.g/ml, compared to
control binding in the absence of IgA. Mean fluorescence intensity
was unaffected by the presence of IgA. These results demonstrate
that A77 mAb specifically binds an epitope on Fc.alpha.RI that is
different from the site for binding of IgA (Monteiro et al. 1992,
J. Immunol. 148: 1764-1770).
Example 13
Fc.alpha.RI Modulation by A77, and Absence of Modulation by
A77X520C9 and A77 F(ab').sub.2
[0202] Characterization of the effect of addition of A77 mAb to
cells on Fc.alpha.RI regulation was achieved by studies in which
modulation (decrease in receptor number) of Fc.alpha.RI from the
cell surface was examined as a function of concentration of A77
mAb.
[0203] As shown in FIG. 25, incubation of monocytes with various
concentrations of A77 for 18h at 37.degree. C. caused modulation at
10 nanograms /ml, which reached a plateau at 55% to 60% of control
(number of receptors in the absence of A77) at 1.0 to 10 .mu.g/ml.
In contrast, incubation of monocytes with antibody 520C9, which has
the same isotype as A77 and which specifically binds the HER2/neu
receptor that is not expressed on monocytes, had no effect on
monocyte modulation of Fc.alpha.R. Thus the A77 mAb functional
determinant is capable of causing internalization and modulation of
Fc.alpha.R from the surface. Further, ability of the BsAb to bind
HER2/neu by virtue of a binding determinant derived from 520C9 is
independent of the Fc.alpha.RI binding determinant. This result
shows that down modulation of Fc.alpha.R is achieved by incubation
of cells bearing this receptor with antibody A77. Such modulation
can be used for regulation of autoimmune disorders.
[0204] These findings are confirmed and contrasted with data for
the BsAb A77X520C9, as well as A77 F(ab').sub.2, which show no down
modulation of Fc.alpha.RI in monocytes, and little in PMN compared
to that of A77. The modulation of Fc.alpha.RI expression upon BsAb
or A77 mAb binding to monocytes or PMN was examined by flow
cytometry. FIG. 26 shows that 1 and 10 .mu.g/ml whole A77 mAb
induced about 40%-50% reduction of Fc.alpha.RI on PMN and monocytes
after overnight incubation at 37.degree. C. This modulation
activity did not require cross-linking of the bound A77 by an
anti-murine antibody. However, 1 and 10 .mu.g/ml of A77X520C9 BsAb
or A77 F(ab').sub.2 induced minimum or no modulation of Fc.alpha.RI
under the similar conditions, indicating that the Fc region of the
A77 mAb may be required to down modulate Fc.alpha.RI
expression.
[0205] From the data of FIG. 26 showing modulation by A77 mAb but
not by BsAb or A77 F(ab').sub.2, it can be concluded that BsAb
binding to monocytes and PMN does not lead to cross-linking and
subsequent down modulation of Fc.alpha.RI in the absence of target
antigen or cells. Therefore, the Fc.alpha.RI-directed BsAb can be
used to "arm" the effector cells in a subject or a patient, without
activation by receptor cross-linking, thus avoiding undesired
systemic side effects. Such effector cells armed with multispecific
antibodies of the invention can be activated locally only upon
cross-linking of Fc.alpha.RI by binding a target antigen, for
example, an antigen on a tumor cell or on a pathogen. Similar
arming of monocytes by Fc.gamma.RI-directed BsAb has been shown
(Valone, F. H. et al. 1995. J. Clin. Oncol. 13:2281-2292), however,
for Fc.gamma.RI-directed BsAb arming to be effective, pretreatment
of the subject with G-GSF or IFN-.gamma. is required to engage the
PMN effector population in vivo (Repp, R. et al. 1991. Blood.
78:995; Weber, J. S. et al. 1996. Proc. of ASCO. 15:354. (Abstr.)
Fc.alpha.Rl-directed BsAb described here could engage effector
cells such as monocytes, PMN and macrophages without cytokine
pretreatment, and without down-modulation of the cognate receptor
on the leukocytes.
Example 14
Cloning and Sequencing A77 Variable Region Genes
[0206] A77 RNA was prepared from A77 Fc.alpha.R specific antibody
producing hybridoma cells, and 33 .mu.g of total RNA was obtained
from approximately 4.times.10.sup.7 A77 cells using the RNAeasy
Total RNA kit (Qiagen). RT-PCR was then done on 200 ng of the total
RNA preparation using the GeneAmp Thermostable rTth Reverse
Transcriptase RNA PCR kit (Perkin Elmer). Ig V region cDNAs were
made using primers CG1FOR, 5'-GGAAGCTTAGACAGATGGGGGTGTCGTTTTG, SEQ
ID NO: 1 (encoding amino acids 115-122 of the murine IgG1 heavy
chain CH1 domain and a HindIII site) and
CK2FOR,5'-GGAAGCTTGAAGATGGATACAGTTGGTGCAGC, SEQ ID NO: 2 (encoding
amino acids 111-118 of the murine .kappa. light chain constant
domain and a HindIII site).
[0207] The V.sub.H and V.sub.78 cDNAs were amplified by PCR using
the cDNA primers along with SH1BACKBAM,
5'-GACTGGATCCATGGRATGGAGCTGGRTCWTBHTC- TT, SEQ ID NO: 3 (encoding a
consensus sequence of amino acids -20 to -12 of some V.sub.H signal
peptides and a BamHI site) and VK1BACKBAM,
5'-GACTGGATCCGACATTCAGCTGACCCAGTCTCCA, SEQ ID NO: 4 (encoding amino
acids -4 to -1 of the signal peptide and residues 1 to 4 of some
murine V.sub.78 domains and a BamHI site). The single-letter code
for combinations of nucleotides, known to those of skill in the
art, is given on p.174 of the 1996-1997 New England Biolabs catalog
(32 Tozer Rd., Beverly, Mass.).
[0208] Amplified V.sub.H and V.sub..kappa. DNA were purified using
Wizard PCR Prep kit (Promega), cloned into pUC 19, and sequenced by
the dideoxy method. DNA sequencing was carried out by National
Biosciences, Inc. At least 5 pUC19 clones of each V.sub..kappa. and
V.sub.H were sequenced to obtain consensus sequences of these
genes, which are shown in FIGS. 27 (SEQ ID NO: 5) and 28 (SEQ ID
NO: 7). The predicted amino acid sequences are shown in FIGS. 27
(SEQ ID NO: 6) and 28 (SEQ ID NO: 8) for V.sub..kappa. and V.sub.H
respectively.
[0209] These sequences are used to obtain recombinant humanized A77
Fc.alpha.R-binding determinants, to produce single-chain antibodies
and single-chain BsAbs, for engineering determinants with greater
affinity using recombinant methods, for modeling studies to develop
mimetic drugs using rational drug design, and for additional
applications described in the instant invention.
Equivalents
[0210] Those skilled in the 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.
[0211] The contents of all patents and publications referred to
herein are hereby incorporated by reference.
* * * * *