U.S. patent application number 12/481248 was filed with the patent office on 2009-12-24 for autophilic antibodies and method of making the same.
This patent application is currently assigned to InNexus Biotechnology International Limited. Invention is credited to Heinz Kohler, Alton Charles Morgan, JR., Sybille Muller.
Application Number | 20090317379 12/481248 |
Document ID | / |
Family ID | 37308630 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317379 |
Kind Code |
A1 |
Kohler; Heinz ; et
al. |
December 24, 2009 |
AUTOPHILIC ANTIBODIES AND METHOD OF MAKING THE SAME
Abstract
Antibodies having noncovalent, autophilic properties are
disclosed. The autophilic antibodies are derived from antibodies
conjugated with an autophilic peptide. Such autophilic antibodies
can promote apoptosis of target cells and enhance therapeutic
efficacies in the treatment of patients with diseases or disorders
responsive to antibody therapy. Compositions containing the
antibodies, and methods of making and using the antibodies are also
disclosed.
Inventors: |
Kohler; Heinz; (Lexington,
KY) ; Muller; Sybille; (Lexington, KY) ;
Morgan, JR.; Alton Charles; (Scottsdale, AZ) |
Correspondence
Address: |
GIFFORD, KRASS, SPRINKLE,ANDERSON & CITKOWSKI, P.C
PO BOX 7021
TROY
MI
48007-7021
US
|
Assignee: |
InNexus Biotechnology International
Limited
Scottsdale
AZ
|
Family ID: |
37308630 |
Appl. No.: |
12/481248 |
Filed: |
June 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11119404 |
Apr 29, 2005 |
7569674 |
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12481248 |
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09865281 |
May 29, 2001 |
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11119404 |
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09070907 |
May 4, 1998 |
6238667 |
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09865281 |
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10652864 |
Aug 29, 2003 |
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11119404 |
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60059515 |
Sep 19, 1997 |
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60407421 |
Aug 30, 2002 |
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Current U.S.
Class: |
424/130.1 ;
424/178.1; 435/7.1; 530/300; 530/325; 530/326; 530/391.7 |
Current CPC
Class: |
A61K 47/6851 20170801;
C07K 16/2803 20130101; G01N 2510/00 20130101; C07K 16/32 20130101;
A61K 47/6803 20170801; A61P 25/28 20180101; A61K 47/6871 20170801;
A61P 35/00 20180101; C07K 16/3084 20130101; A61K 47/6849 20170801;
G01N 33/92 20130101; C07K 2317/73 20130101; C07K 16/40 20130101;
A61P 37/06 20180101; C07K 16/2896 20130101; A61K 41/0042 20130101;
A61K 2039/505 20130101; A61P 9/10 20180101; A61K 47/6811 20170801;
G01N 2800/323 20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/130.1 ;
424/178.1; 435/7.1; 530/300; 530/325; 530/326; 530/391.7 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; C07K 1/00 20060101
C07K001/00; C07K 14/00 20060101 C07K014/00; C07K 7/08 20060101
C07K007/08; C07K 19/00 20060101 C07K019/00 |
Claims
1. An autophilic antibody comprising an autophilic peptide
conjugated to an immunoglobulin component of a non-autophilic
antibody.
2. An autophilic antibody according to claim 1, wherein the
autophilic peptide comprises a conserved self-binding sequence.
3. An autophilic antibody according to claim 1, wherein the
autophilic peptide is a humanized peptide.
4. An autophilic antibody according to claim 1, wherein the
autophilic peptide is selected from the group consisting of T15
peptide, T15-scr2 peptide, R24 peptide, and R24-Charged
peptide.
5. An autophilic antibody according to claim 1, wherein the
autophilic peptide comprises SEQ ID NO.1.
6. An autophilic antibody according to claim 1, wherein the
autophilic peptide comprises SEQ ID NO.4.
7. An autophilic antibody according to claim 1, wherein the
autophilic peptide comprises SEQ ID NO.5.
8. An autophilic antibody according to claim 1, wherein the
autophilic peptide comprises SEQ ID NO.6.
9. An autophilic antibody according to claim 1 comprising one or
more functional peptides conjugated to the non-autophilic
antibody.
10. An autophilic antibody according to claim 9, wherein the one or
more functional peptides are conjugated to the non-autophilic
antibody at a site that is different from a site that the
autophilic peptide is conjugated to the non-autophilic
antibody.
11. An autophilic antibody according to claim 9, wherein the one or
more functional peptides comprises an MTS peptide.
12. An autophilic antibody according to claim 9, wherein the one or
more functional peptides comprises SEQ ID NO.2.
13. An autophilic antibody according to claim 9, wherein the one or
more functional peptides comprises SEQ ID NO.7.
14. An autophilic antibody according to claim 1, wherein the
non-autophilic antibody is selected from the group consisting of
5D10, S1C5, rituximab, anti-GM2, trastuzumab, anti-caspase
antibodies, humanized TEPC-15, humanized R24, and humanized
S107.
15. A method of producing an autophilic antibody according to claim
1 comprising attaching the autophilic peptide via photoactivation
crosslinking to an immunoglobulin component of the antibody.
16. A method according to claim 15, wherein the autophilic peptide
is cross-linked to a nucleotide affinity site of the immunoglobulin
component.
17. A method according to claim 15, wherein the autophilic peptide
is cross-linked to a heterocyclic compound affinity site of the
immunoglobulin component.
18. A method according to claim 15, wherein the autophilic peptide
is cross-linked to a carbohydrate site of the Fc portion of the
immunoglobulin component.
19. A method according to claim 15, wherein the autophilic peptide
is conjugated to an amino or sulfhydryl group of the immunoglobulin
component.
20. A method of producing an autophilic antibody according to claim
1 comprising expressing the autophilic antibody as a fusion protein
containing the autophilic peptide sequence and the non-autophilic
antibody.
21. A method of producing an autophilic antibody according to claim
9 comprising attaching the one or more functional peptides via
photoactivation crosslinking to an immunoglobulin component of the
antibody.
22. A method according to claim 21, wherein the one or more
functional peptides is cross-linked to a nucleotide affinity site
of the immunoglobulin component.
23. A method according to claim 21, wherein the one or more
functional peptides is cross-linked to a heterocyclic compound
affinity site of the immunoglobulin component.
24. A method according to claim 21, wherein the one or more
functional peptides is cross-linked to a carbohydrate site of the
Fc portion of the immunoglobulin component.
25. A method according to claim 21, wherein the one or more
functional peptides is conjugated to an amino or sulfhydryl group
of the immunoglobulin component.
26. A method according to claim 21, wherein the one or more
functional peptides is conjugated to the immunoglobulin component
at a site that is different from a site of conjugation of the
autophilic peptide.
27. A method of producing an autophilic antibody according to claim
9 comprising expressing the autophilic antibody as a fusion protein
containing the autophilic peptide sequence, the one or more
functional peptides, and the non-autophilic antibody.
28. A composition comprising one or more autophilic antibodies
according to claim 1 and a pharmaceutically acceptable carrier.
29. A composition comprising one or more autophilic antibodies
according to claim 9 and a pharmaceutically acceptable carrier.
30. A method of formulating an autophilic antibody composition
according to claim 28 to reduce or mitigate dimerization of the
autophilic antibody in solution comprising one or more steps
selected from the group consisting of: a. Maintaining a hypertonic
salt concentration; b. Adding detergents to the composition; and c.
Decreasing the isoelectric point of the antibody composition with
succinyl anhydride.
31. A method of formulating an autophilic antibody composition as
claimed in claim 30, wherein the detergents are SDS or anionic
detergents.
32. A method of formulating an autophilic antibody composition as
claimed in claim 30, wherein the salt concentration is equal to or
greater than 0.5M.
33. A method of selecting an antigen target in vitro for an
autophilic antibody according to claim 1 comprising administering
the autophilic antibody to one or more cell lines expressing
antigens, and assaying for apoptosis of the one or more cell
lines.
34. A method of identifying an autophilic antibody according to
claim 1 for use in humans comprising administering the autophilic
antibody to SCID or nude mice having human tumor xenografts.
35. A method of synthesizing a peptide sequence having enhanced
autophilic binding properties comprising providing a plurality of
peptides, each having one or more amino acid substitutions compared
to a template peptide, and comparing self-binding properties of the
peptides relative to those of the template peptide.
36. The method of claim 35, wherein the template peptide is
selected from the group consisting of T15 peptide, T15-scr2
peptide, R24 peptide, and R24-Charged peptide.
37. A method of treating a patient suffering from a disease or
disorder responsive to antibody therapy comprising administering to
the patient one or more autophilic antibodies according to claim 1,
or a composition comprising one or more autophilic antibodies
according to claim 1 and a pharmaceutically acceptable carrier, in
a therapeutically effective amount to alleviate symptoms of the
disease or disorder.
38. The method of claim 37, wherein the disease or disorder is a
malignancy, an auto-immune disorder, transplantation rejection,
Alzheimer's disease, a neuro-degenerative condition,
atheroschlerosis, or any other condition responsive to antibody
therapy.
39. The method of claim 37, wherein the autophilic antibody is
administered in one or more dose amounts substantially identical to
amounts used to administer non-autophilic antibodies.
40. The method of claim 37, wherein an initial dose is about 250 mg
per day and a later dose is about 100 mg per week.
41. The method of claim 37, wherein a maintenance dose is about 50
mg per week.
42. A method of potentiating apoptosis, complement fixation, or
cell-mediated killing of selected cells in a patient comprising
administering to the patient one or more autophilic antibodies
according to claim 1.
43. A method according to claim 42 further comprising administering
to the patient a second antibody directed to the autophilic peptide
of the autophilic antibody.
44. A method of restoring autophilic activity to an antibody that
has lost its autophilic activity comprising conjugating an
autophilic peptide to the antibody.
45. A method according to claim 44, wherein the autophilic peptide
is selected from the group consisting of T15 peptide, T15-scr2
peptide, R124 peptide, and 124-Charged peptide.
46. A method according to claim 44, wherein the autophilic peptide
is conjugated to the antibody via photoactivation crosslinking to
an immunoglobulin component of the antibody.
47. A method according to claim 44, wherein the autophilic peptide
is cross-linked to a nucleotide affinity site of the immunoglobulin
component.
48. A method according to claim 44, wherein the autophilic peptide
is cross-linked to a heterocyclic compound affinity site of the
immunoglobulin component.
49. A method according to claim 44, wherein the autophilic peptide
is cross-linked to a carbohydrate site of the Fc portion of the
immunoglobulin component.
50. A method according to claim 44, wherein the autophilic peptide
is conjugated to an amino or sulfhydryl group of the immunoglobulin
component.
51. A method according to claim 44 comprising expressing the
autophilic antibody as a fusion protein containing the autophilic
peptide sequence and the antibody.
52. A peptide comprising SEQ ID NO. 4.
53. A peptides comprising SEQ ID NO. 5.
54. A peptide comprising SEQ ID NO. 6.
55. A peptide comprising SEQ ID NO.7.
56. An antibody conjugate according to claim 1 comprising S1C5
antibody conjugated with T15 peptide.
57. An antibody conjugate according to claim 1 comprising 5D10
antibody conjugated with T15 peptide.
58. An antibody conjugate comprising anti-caspase 3 antibody
conjugated with MTS peptide.
59. An antibody conjugate according to claim 1 comprising anti-CD20
antibody conjugated with T 5 peptide.
60. An antibody conjugate according to claim 1 comprising rituximab
conjugated with T15 peptide.
61. An antibody conjugate according to claim 1 comprising 1F5
conjugated with T15 peptide.
62. An antibody conjugate according to claim 1 comprising
tositumomab conjugated with T15 peptide.
63. An antibody conjugate according to claim 1 comprising anti-GM2
conjugated with T15 peptide.
64. An antibody conjugate according to claim 1 comprising a
non-human antibody to GM2 glycolipid conjugated with an autophilic
peptide.
65. An antibody conjugate according to claim 1 comprising Herceptin
conjugated with an autophilic peptide.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/119,404, filed Apr. 29, 2005, which is a
continuation-in-part of U.S. patent application Ser. No.
10/652,864, filed Aug. 29, 2003, which claims priority from U.S.
Provisional Patent Application Ser. No. 60/407,421, filed Aug. 30,
2002. U.S. patent application Ser. No. 11/119,404 is also a
continuation-in-part of U.S. patent application Ser. No.
09/865,281, filed May 29, 2001, which is a continuation-in-part of
U.S. patent application Ser. No. 09/070,907, filed May 4, 1998, now
U.S. Pat. No. 6,238,667, which claims priority from U.S.
Provisional Patent Application Ser. No. 60/059,515, filed Sep. 19,
1997. The entire content of each application and patent is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies, compositions
containing antibodies, and methods of using, the antibodies and
compositions in the treatment of a variety of diseases, including
those diseases treatable with passive antibody therapy.
BACKGROUND OF THE INVENTION
[0003] Antibodies have emerged as a major therapeutic tool for the
treatment of chronic diseases such as cancer and autoimmune
disorders. One of the principal advantages of these biological
agents lies in their ability to target disease-causing cells or
molecules, while sparing healthy tissue and normal products of the
body. However, antibodies that exhibit desired specificities often
fail in pre-clinical and clinical evaluations because of
inefficient targeting and/or low therapeutic activity.
[0004] A rare class of antibodies, known as SuperAntibodies, exist
in nature. These are antibodies that exhibit one or more properties
not usually associated with antibodies (Kohler H., et al., 1998;
Kohler H., 2000). The defined class of SuperAntibodies comprises
catalytic, membrane-penetrating, and autophilic antibodies and
includes many antibodies exhibiting superior targeting and
therapeutic properties. One example of a naturally occurring
SuperAntibody is the murine TEPC-15 antibody. TEPC-15 is an
autophilic antibody which targets a normally cryptic determinant of
phosphorylcholine on apoptotic cells and atheroschlerotic lesions.
TEPC-15 antibodies have high therapeutic efficacy due to their
ability to form dimers or multimers (on cell or bacteria surfaces,
after binding to antigen), which enhances apoptosis. TEPC-15
antibodies are able to form dimers and multimers due to an
autophilic peptide sequence. (Kang, C-Y, et al., 1988)
[0005] It is known that a major mechanism by which therapeutic
antibodies attack their target cells is through the induction of
apoptosis. Apoptosis is triggered by crosslinking cellular
receptors that are part of the apoptosis signal pathway. For
example, crosslinking the B-cell antigen receptor by means of
antibodies induces apoptosis in B-cell tumors (Ghetie M., et al.,
1997). Crosslinking of cellular receptors also increases the
binding avidity of an antibody to its target antigen, and thus is
likely to increase all cell surface-dependent therapeutic
mechanisms, such as complement-mediated killing and
complement-dependent opsonization and phagocytosis,
antibody-dependent cellular cytotoxicity (ADCC), as well as
enhanced inhibition of cell growth or alterations in metabolic
pathways within cells through increased binding to and blockade of
cellular receptors when using antibodies targeted to cellular
receptors.
[0006] To enhance the therapeutic efficacy of known antibodies,
others have proposed the use of hybrid molecules for therapeutic
purposes wherein the hybrid molecules comprise two distinct domains
covalently linked. For instance, U.S. Pat. No. 6,482,586 (issued to
Arab et al.) proposes covalent hybrid compositions for use in
intracellular targeting. U.S. Pat. No. 6,406,693 (issued to Thorpe
et al.) proposes antibodies and conjugates for killing tumor
vascular endothelial cells by binding to aminophospholipid on the
luminal surface.
[0007] These are but a few of the approaches that have been used to
enhance therapeutic efficacy of monoclonal antibodies that, in
their native or "humanized" state, are not effective in killing
their targets or triggering a biological function affording
therapeutic efficacy.
[0008] There is a need for a method of enhancing the therapeutic
efficacy of antibodies which have desired specificities without the
use of toxic agents.
SUMMARY OF INVENTION
[0009] The present invention is directed to autophilic antibodies,
compositions containing autophilic antibodies, methods of making
autophilic antibodies, methods of restoring autophilic activity to
antibodies that have lost that activity, methods of assaying target
antigens for autophilic antibodies, methods of enhancing apoptosis,
complement fixation or cell-mediated killing using the autophilic
antibodies, and methods of using the autophilic antibodies and
compositions in the treatment of various diseases responsive to
antibody therapy. The diseases include those treatable with passive
antibodies, including atheroschlerosis, cancers, autoimmune
disorders, Alzheimer's disease and other neuro-degenerative
conditions, as well as artifacts of a functioning immune system
such as graft or transplant rejection.
[0010] The present invention relates to antibodies having
autophilic properties that mimic those of rare, naturally
occurring, autophilic antibodies. Autophilic antibodies according
to the present invention have the unusual property of spontaneously
binding to one another after first binding to their target antigen
(differential oligomerization).
[0011] The antibodies can comprise any antibody conjugated with an
autophilic peptide sequence. In some embodiments, the antibodies
are capable of binding an antigen, which, when bound, has a
therapeutic effect on a disease state or disorder. In some specific
embodiments, the antibodies comprise 5D10, S1C5, anti-caspase
antibodies, anti-CD20 antibodies such as rituximab, 1F5, and
tositumomab, anti-GM2 antibodies, humanized S107, trastuzumab,
humanized TEPC-15, and humanized 124.
[0012] The antibodies can be conjugated with any autophilic peptide
which allows the antibodies to dimerize or oligomerize once bound
to an antigen. The peptide can comprise any autophilic peptide
sequence. In specific embodiments, the peptide comprises the T15
peptide sequence, the T15-scr2 peptide sequence, the R24 peptide
sequence, the R24-charged peptide sequence, and optimized versions
thereof.
[0013] Autophilic antibodies can also be conjugated with one or
more other peptides to add additional functionality. In one
embodiment, the autophilic antibodies can be conjugated to an
autophilic peptide sequence and a transmembrane peptide sequence
which allows the autophilic antibodies to penetrate inside cells
and bind to intracellular targets. In specific embodiments, the
transmembrane peptide sequence comprises MTS peptide or
MTS-optimized peptide.
[0014] The invention also relates to compositions containing one or
more autophilic antibodies of the invention and pharmaceutically
acceptable carriers. The compositions can be administered to
patients in need of treatment with the autophilic antibodies of the
invention. The compositions can be optimized to prevent the
autophilic antibodies from forming spontaneous dimers before
administration.
[0015] The antibodies and compositions containing antibodies of the
invention can be administered in doses similar to, or lower than,
those practicable for non-autophilic antibodies.
[0016] The autophilic antibodies of the invention are preferably
formed by one of several methods, including chemically crosslinking
a peptide capable of self-binding to an antibody. In a specific
embodiment, the peptide is cross-linked to an antibody through
oxidation of an N-linked carbohydrate. Alternatively, the
autophilic peptide can be linked to an antibody through the
nucleotide binding site or to a tryptophane binding site, or
through less specific methods, such as through antibody epsilon
amino groups or sulfhydryl groups obtained through partial
reduction of the antibody.
[0017] The invention also relates to a method of optimizing
autophilic peptide sequences for use in forming autophilic
antibodies comprising optimizing a template-peptide.
[0018] The invention also relates to a method of restoring
autophilic properties to an antibody, such as a humanized antibody,
which has lost its autophilic properties, in whole or in part,
during the humanization process, by conjugating an autophilic
peptide to the antibody as described above.
[0019] The invention also contemplates a method for assaying target
antigens for autophilic antibodies, and a method of testing the
efficacy of autophilic antibodies using animal models.
[0020] The invention also relates to methods of enhancing
apoptosis, complement fixation, or cell-mediated killing using the
autophilic antibodies of the invention comprising administering the
antibodies of the invention.
[0021] The invention also relates to a method of treating a patient
suffering from a disorder, disease, or condition responsive to
passive antibody therapy comprising administering an autophilic
antibody of the invention to the patient.
BRIEF DESCRIPTION OF DRAWINGS
[0022] In drawings which are intended to illustrate embodiments of
the invention:
[0023] FIG. 1 is a graph depicting improved binding of anti-CD20
antibodies conjugated with T15 peptide to DHL-4 cells at high
concentrations of antibody;
[0024] FIG. 2 is a graph depicting improved binding of anti-CD20
antibodies conjugated with T15 peptide at low concentrations of
antibody;
[0025] FIG. 3 is a graph depicting enhanced binding of anti-CD20
antibodies conjugated with T15 peptide;
[0026] FIG. 4 is a graph depicting enhanced induction of apoptosis
of tumor cells with mouse anti-CD20 conjugated with T15
peptide;
[0027] FIG. 5 is a graph depicting enhanced apoptosis of tumor
cells using anti-GM2 antibody conjugated with T15 peptide;
[0028] FIG. 6 is a graph comparing the efficacy of autophilic
peptide conjugation to an affinity site on an antibody (nucleotide)
versus a non-affinity site (CHO--carbohydrate) using anti-GM2;
[0029] FIG. 7 is a graph comparing the internalization of MTS
conjugated antibodies and non-MTS conjugated antibodies using
anti-caspase 3 antibodies;
[0030] FIG. 8 is a graph comparing the binding of Herceptin (upper
panel) and the autophilic peptide conjugated form of Herceptin
(lower panel) to small cell lung cancer cells
[0031] FIG. 9 is a graph comparing the binding of anti-GM2 antibody
and T15 conjugated anti-GM2 antibody to ganglioside GM2;
[0032] FIG. 10 is a graph illustrating the self-binding activity of
anti-GM2 antibody and T15 conjugated anti-GM2 antibody;
[0033] FIG. 11 is a graph demonstrating binding specificity of T15
conjugated anti-GM2 antibody to different gangliosides;
[0034] FIG. 12 is a graph depicting differences in cell surface
binding of anti-GM2 antibody and T15 conjugated anti-GM2 antibody
to Jurkat cells;
[0035] FIG. 13 is a graph depicting the effect of anti-GM2 antibody
and T15 conjugated anti-GM2 antibody on Jurkat cell growth; and
[0036] FIG. 14 is a graph depicting the effect of chemotherapeutic
drug (actinomycin D) on cell death in the presence and absence of
MTS-conjugated (Sab) antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Throughout the following description, specific details are
set forth in order to provide a more thorough understanding of the
invention. However, the invention may be practiced without these
particulars. In other instances, well known elements have not been
shown or described in detail to avoid unnecessarily obscuring the
invention. Accordingly, the specification and drawings are to be
regarded in an illustrative, rather than a restrictive, sense.
[0038] The present invention relates to non-covalent, autophilic
antibodies having enhanced therapeutic potencies. Such antibodies
are referred to as "autophilic" antibodies. Autophilic antibodies
belong to the class of SuperAntibodies--antibodies that exhibit one
or more properties not usually associated with antibodies (Kohler
H., et al., 1998; Kohler H., 2000). The defined class of
SuperAntibodies comprises catalytic, membrane-penetrating, and
autophilic antibodies and includes many antibodies exhibiting
superior targeting and therapeutic properties.
[0039] The autophilic antibodies of the present invention comprise
antibodies conjugated with a peptide having an autophilic sequence.
The autophilic antibodies of the invention can comprise any
antibody. In some embodiments, the antibodies bind to targets
implicated in a disease or disorder, where binding of the target
has a therapeutic effect on the disease or disorder. The target
antigens can include cell-surface antigens, including
trans-membrane receptors. In specific embodiments, the antibodies
comprise the monoclonal antibody 5D10, which binds human B-cell
receptors, the monoclonal antibody S1C5, which binds murine B-cell
receptors, anti-CD20 antibodies such as rituximab, which binds CD20
on normal and malignant pre-B and mature B lymphocytes, mouse
monoclonal antibody 1F5, which is specific for CD-20 on human
B-cell lymphomas 5D10 and 3H1, and tositumab which also binds CD20
on B lymphocytes, anti-GM7, which binds human ganglioside GM2
lymphocytes, trastuzumab, which binds the protein HERS that is
produced by breast cells, anti-caspase antibodies, which recognize
the caspase proteins involved in apoptosis, humanized TEPC-15
antibodies, which are capable of binding oxidized low density
lipoproteins (oxLDL) and can prevent uptake of oxidized LDL by
macrophages, humanized T15-idiotype positive antibodies, which bind
phosphocholine, and humanized R24 antibodies which recognize the
human (3D3 ganglioside on melanoma cell surfaces.
[0040] The autophilic antibodies of the invention are conjugated
with an autophilic peptide component. The autophilic peptide can
comprise any autophilic peptide sequence. The autophilic peptide
can also comprise optimized sequences which may include sequences
with enhanced functionality, such as ones which act as linkers to
enhance display and cross-linking activity of antibodies, or
residues which enhance solubility of autophilic sequences. In all
situations, the autophilic sequences are complementary and are able
to bind to themselves.
[0041] In a specific embodiment, the autophilic peptide comprises
the autophilic T15 peptide, which originally comprised regions of
CDR2 and FR3 of the murine germline-encoded S107/TEPC15 antibody.
The T15 peptide comprises amino acid sequence:
ASRNKANDYTTDYSASVKGPRFIVSR (SEQ ID NO.: 1) (Kang C--Y, et al.,
1988). Its autophilic property has been shown to be
antigen-independent. Therefore, attachment of the peptide to any
monomeric antibodies can impart autophilic and increased avidity
properties to the antibodies (Y. Zhao, and H. Kohler, 2002). In
other specific embodiments, the autophilic peptide can comprise a
humanized T15 peptide sequence, for increased or optimized binding
and effectiveness of antibodies.
[0042] In other specific embodiments, the autophilic peptide can
also comprise the peptide T15-scr2 comprising the sequence
NH-SKAVSRFNAKGIRYSETNVDTYAS-COOH (SEQ ID NO. 4), the peptide R24
comprising the sequence NH-GAAVAYISSGGSSINYA-COOH (SEQ ID NO. 5),
the peptide RC4-Charged comprising the sequence
NII-GKAVAYISSGGSSINYAE-COOH (SEQ ID NO. 6), and any modifications
to the peptides which optimize or enhance the binding and
therapeutic effectiveness of antibodies.
[0043] The autophilic antibody conjugates of the invention can also
comprise one or more other bioactive or functional peptides which
confer additional functionality on the antibody conjugates. For
example, the antibody conjugate can comprise an antibody that bears
a T15 autophilic peptide and an MTS membrane translocation peptide
(Y. Zhao et al., 2003; Y. Lin et al., 1995). In a specific
embodiment, the MTS translocation peptide can have the amino acid
sequence KGEGAAVLLPVLLAAPG (SEQ ID NO. 2). In another embodiment,
the translocation peptide can be an optimized MTS peptide,
MTS-optimized, comprising the sequence WKGESAAVILPVLIASPG (SEQ ID
NO. 7). The T15 peptide provides autophilicity to the conjugate,
and the MTS sequence allows the antibody to penetrate into cells.
Such a conjugate can target, for example, cancer cells for
radio-immunotherapy, when its antibody region targets a primarily
intracellular, tumor-associated antigen, such as carcinio-embryonic
antigen (CEA) (See, e.g., U.S. Pat. No. 6,238,667 which is hereby
incorporated by reference). The autophilic conjugate, upon
administration, targets CEA-bearing, colon carcinoma cells, is
internalized by translocation of the antibody mediated by the MTS
peptide, and is enabled to bind to the more prevalent intracellular
form of CEA. Crosslinking of CEA antibody with, for instance, a
therapeutic isotope such as .sup.131I will be retained in a cell
longer than unmodified, labeled antibody and will deliver a higher
radioactive dose to the tumor. In addition, such therapeutic
isotopes as .sup.125I, which release beta particles of short path
length and are not normally considered useful for therapy, can,
when delivered intracellularly in closer proximity to the nucleus,
be efficacious against certain targets, especially those of
lymphoid origin and accessible in the blood and lymph tissues.
Other categories of secondary, bioactive or functional peptides
include peptides capable of binding to receptors, and peptide
mimetics, capable of binding to a distinctive antigen or epitope of
the same antigen, targeted by the primary antigen combining
site.
[0044] Autophilic antibodies conjugated with one or more other
functional peptides may also be useful for targeting intracellular
antigens. Such antigens could include tumour associated antigens
and viral proteins. For example, an autophilic antibody specific
for viral proteins which is conjugated with a self-binding peptide
and a MTS peptide can also be used to bind to intracellular viral
proteins and prevent production of viruses. The antibody could be
internalized through the MTS peptide, and would be optimized to
bind intracellular viral proteins (Zhao, Y., et al. 2003). Many
other functional peptides may also be conjugated to the autophilic
antibodies to increase functionality.
[0045] The invention also relates to compositions containing the
autophilic antibodies of the invention and a pharmaceutically
acceptable carrier. The conjugate autophilic antibodies can bind
non-covalently with other autophilic antibodies when bound to their
target antigen(s). However, premature formation of dimers or
multimers of the antibodies may lead to difficulties in
manufacturing, such as during purification and concentration, as
well as drawbacks in administration, which may lead to side
effects. As such, compositions containing the autophilic
antibody-peptide conjugates of the invention are formulated to
reduce this dimerizing potential and maximize monomericity while in
solution and before administration. For example, it has been found
that solution dimerization can be reduced or mitigated by using a
hypertonic composition. In some embodiments, salt concentrations of
0.5M or more, low levels of SDS or other various detergents such as
those of an anionic nature (see U.S. Pat. No. 5,151,266 which is
hereby incorporated by reference), or modifications of the antibody
to decrease its isoelectric point, for example through the use of
succinyl anhydride (see U.S. Pat. No. 5,322,678, which is hereby
incorporated by reference), can be used to formulate
compositions.
[0046] According to the principles of the present invention, an
autophilic antibody or a composition containing an autophilic
antibody is preferably administered in one or more dosage amounts
substantially identical to, or lower than, those practicable for
unmodified antibodies. Thus, in the treatment of a lymphoma or a
breast cancer, an autophilic antibody of the invention can be
administered in one or more dose amounts substantially identical
to, or less than, the doses used for RITLTXAN.TM. (rittiximab) or
HERCEPTIN.TM. (trastuzumab). For example, treatment with
HERCEPTIN.TM. (a humanized monoclonal anti-HER2/neu antibody) in a
patient with HER2.sup.+ breast cancer employs an antibody
concentration of about 10 mg/ml. Intravenous infusion over 90
minutes provides a total dose of 250 mg on day 0. Beginning at day
7, 100 mg is administered weekly for a total of 10 doses. The
dosing regimen is reduced gradually from 250 mg to 100 mg to a
maintenance dose of 50 mg. Similar or lower dosage regimens to that
for HERCEPTIN.TM. can be employed with autophilic antibodies, with
any adjustments being well within the capabilities of a skilled
practitioner.
[0047] The present invention also relates to a method of producing
the autophilic antibody conjugates. The antibody conjugates can be
produced by chemical or genetic engineering techniques. For
instance, a peptide component of an autophilic antibody can be
attached to the immunoglobulin component via its variable domain
structures using azido-tryptophan or azido-purine photoactivation
crosslinking. In this approach, the peptide attaches to the
variable domain at a location that does not interfere with antigen
recognition. This method can incorporate two peptide moieties into
a single immunoglobulin molecule. See, for example U.S. Pat. No.
6,238,667, U.S. Reissue Pat. No. RE38,008, U.S. Pat. No. 5,635,180,
and U.S. Pat. No. 5,106,951, the disclosures of which are
incorporated herein by reference.
[0048] The peptides can be photo-crosslinked to a heterocyclic
compound affinity site (such as a tryptophane affinity site) or a
nucleotide affinity site of antibodies to produce the autophilic
antibodies of the invention. Alternatively, the peptides can be
crosslinked to a carbohydrate site of the Fc portion or to an amino
or sulfhydryl group of an antibody. In an alternative embodiment,
the autophilic antibody can be conveniently expressed as a fusion
protein of the autophilic peptide and whole immunoglobulin, or
fragment thereof.
[0049] The present invention also contemplates a method of
producing an autophilic conjugate of the invention in which a
template peptide has been modified to enhance the crosslinking
potential of the autophilic antibodies as described above. In one
embodiment of the invention, such functionally enhanced peptides
are determined by producing a series of synthetic peptides with
substitutions at each amino acid position within the template
sequence and then testing this library of peptides for autophilic
binding or for binding to the original peptide sequence. Those
peptides with superior binding to the original sequence are then
conjugated to immunoglobulins and the resultant conjugates are
tested for potency, specificity, and the unwanted ability to induce
aggregation. In one specific embodiment, the T15 peptide sequence
is altered and modified sequences are selected for enhanced
function.
[0050] In other embodiments of the invention, the self-binding
potential of a peptide can be enhanced by increasing
complementarity of the sequence, such as described in U.S. Pat. No.
4,863,857 to Blalock et al., which is incorporated herein by
reference. The self-binding potential of a peptide can also be
enhanced by humanizing a self-binding peptide sequence which is
derived from non-human animals. Humanizing a peptide sequence
involves optimizing the sequence for expression or functionality in
humans. Examples and methods of humanizing peptides and proteins
have been previously described (Roque-Navarro et al., 2003; Caldas
et al., 2003; Leger et al., 1997; Isaacs and Waldmann, 1994; Miles
et al. 1989; Veeraraghavan et al., 2004; Dean et al., 2004;
Halkenberg et al., 2003; Gonzales et al., 2004; and H. Schellekens,
2002).
[0051] An assay method is also contemplated that penrits
pre-selection of target antigens most suitable as targets for the
autophilic antibodies of the present invention. Such method entails
the in vitro assay of apoptosis with multiple antigen-positive
target cell lines, and if possible, fresh isolates of
antigen-positive cells. A non-modified antibody is incubated with a
secondary (anti-immunoglobulin) antibody to enhance the potential
for cross-linking. Cells may be enumerated by pre-labeling, such as
with .sup.51Cr or .sup.131I-UDR, or by FACS analysis using
indicators of apoptosis. Positive results in this assay predict a
positive outcome using an autophilic conjugate. However, negative
results in the assay do not necessarily mean that subsequent
conjugation with an autophilic peptide will not improve one or more
antibody effector properties.
[0052] Autophilic antibodies of the present invention have a higher
potential for forming dimers in vitro under laboratory conditions,
such as in solution with PEG. This laboratory characteristic
correlates with a crosslinking ability upon binding to a
cell-surface target and higher therapeutic potency through such
mechanisms as triggering apoptosis. This characteristic can be used
to identify natural SuperAntibodies and to screen for proper
conjugation of self-binding peptides to a non-autophilic
antibody.
[0053] A method of enhancing apoptosis, complement fixation,
effector cell-mediated killing of targets, or preventing the
development of, or enhancement of, a disease state, is also
disclosed employing an autophilic conjugate of the invention or a
composition comprising an autophilic conjugate of the invention. In
one embodiment, an autophilic conjugate of the invention, or a
composition containing an autophilic conjugate of the invention, is
administered to a subject. Once administered, the antibodies bind
to target cells and enhance apoptosis, complement fixation,
effector cell-mediated killing of targets, or prevent target
antigens or cells from stimulating the development of, or further
enhancing, a disease state. In a further embodiment, allowing time
for the autophilic conjugate to bind to target cells and enhance
apoptosis, complement fixation, effector cell-mediated killing of
targets, or prevent target antigens or cells from further enhancing
a disease state, and for the autophilic conjugate to be cleared
from normal tissues, a second anti-autophilic peptide antibody can
be administered. For example, if an autophilic conjugate contains a
non-native autophilic peptide, such as the murine T15 sequence, an
anti-T15 peptide antibody would be administered, which would only
recognize and bind to antibodies conjugated with the T15 sequence.
This allows binding to and enhancement of apoptosis of
pre-localized SuperAntibodies.
[0054] A further method of enhancing apoptosis, complement
fixation, or effector cell-mediated killing of targets is
contemplated, which employs administering an autophilic conjugate
of the invention in which a template autophilic peptide has been
modified to enhance the crosslinking potential of the autophilic
antibodies as described above.
[0055] In another aspect of the invention, a method of potentiating
apoptosis of targeted cells of a patient comprises administering a
first autophilic antibody-peptide conjugate, or a composition
containing an autophilic antibody-peptide conjugate, and a second
antibody, or composition containing the second antibody, that
recognizes the autophilic peptide domain of the conjugate. In this
embodiment, the antibody-peptide conjugate recognizes an antigen on
a target cell. Owing to its homodimerization property, the
antibody-peptide conjugate can bind more avidly to the target than
the corresponding antibody lacking the autophilic peptide domain.
This is likely due to the ability to crosslink antigen at the
surface of target cells. Moreover, whenever the autophilic
antibodies bind to two or more antigens, with those antigens being
brought in close proximity and crosslinked, due to the autophilic
property of the antibodies, an apoptosis signal within the cell can
be triggered. In those instances when the peptide domain of the
conjugate presents an exposed epitope, a second antibody, specific
for the autophilic peptide, can be administered, bind to the
modified antibody, and enhance the process of crosslinking and even
cause temporary clearance of the target antigen. As an example, if
the target antigen is a receptor, clearance from the cell surface,
endocytosis, and degradation will subsequently require synthesis of
new receptor protein, meaning that the biological function of the
receptor will be more effectively inhibited for a longer period
than using either a simple blocking antibody or small molecule
inhibitor. Alternatively, the second antibody can bear a radiolabel
or other potentially therapeutic substance, so that when
administered, it can attack the targeted cells. The key to use of
this second antibody is the antibody's specificity. The autophilic
peptide is present on only a small number of immunoglobulins, or if
it is a peptide derived from another organism, or if it is
modified, will not be present on any immunoglobulins in a patient.
Thus, antibody specific to the autophilic peptide will have the
requisite selectivity to be used in vivo.
[0056] In another aspect of the invention, a patient who suffers
from a disease or condition responsive to antibody therapy is
administered at least one autophilic antibody of the invention in
an amount effective to alleviate symptoms of the disease or
condition. A disease or condition contemplated for treatment by an
antibody of the invention can be a malignancy, neoplasm, cancer,
auto-immune disorder, Alzheimer's disease or other
neuro-degenerative condition, graft or transplantation rejection,
atherosclerosis, or any other disease or condition responsive to
antibody therapy.
[0057] The following examples are presented to illustrate certain
aspects of the invention, and are not intended to limit the scope
of the invention.
EXAMPLES
Example 1
Conjugation of T15 Peptide to Two Mabs Specific for B-Cell
Receptor
[0058] Cell Line and Antibodies
[0059] The human B-cell tumor line (Su-DHL4) and murine B-cell
tumor line (38C13) are grown in RPMI 1640 medium (supplemented with
10% fetal bovine serum, 2 .mu.mol/L glutamine, 10 .mu.mol/L HEPES,
50 U/mL penicillin, and 50 .mu.g/mL streptomycin, 50 .mu.mol/L
2-mercaptoethanol) at 37.degree. C. under 5% carbon dioxide. Two
mAb 5D10 and S1C5, specific for the human or murine BCR,
respectively, were used in this study. The antibodies are purified
from the culture supernatant by protein G and protein A affinity
chromatography.
[0060] Synthesis of Antibody-Peptide Conjugate.
[0061] T15H peptide (ASRNKANDYTTDYSASVKGRFIVSR) (SEQ ID NO. 1), a
VH-derived peptide from an autophilic antibody-T15, was synthesized
by Genemed Synthesis (San Francisco, Calif., U.S.A.). Antibodies
were dialyzed against PBS (pH 6.0) and 1/10 volume of 200 .mu.mol/L
sodium periodate was added and incubated at 4.degree. C. for 30
minutes in the dark. The reaction was stopped by adding glycerol to
30 .mu.mol/L, and the sample was dialyzed at 4.degree. C. for 30
minutes against PBS (pH 7.0). One hundred times molecular excess of
T15H or scrambled peptide was added to the antibodies and incubated
at 37.degree. C. for 1 hour. L-Lysine was added and incubated at
37.degree. C. for 30 minutes to block the remained aldehyde group.
The same oxidation reaction steps (except adding the peptides) were
applied to antibodies used as controls. After the blocking step,
the antibody conjugates were dialyzed against PBS (pH 7.2)
overnight.
[0062] Ig Capture ELISA.
[0063] Four .mu.g/mL of S1C5-T15H was coated to Costar vinyl assay
plates (Costar, Cambridge, Mass.). After blocking with 3.degree. %
BSA solution, 8 .mu.g/mL of photobiotinylated S1C5-T15H,
S1C5-scrambled peptide conjugate, and control S1C5 were added to
the first wells, and 1:1 dilution was performed. The antibodies
were incubated for 2 hours at room temperature. After washing with
PBS buffer, Avidin-HRP (Sigma, St. Louis, Mo.) was added as a
1:2500 dilution. The binding antibodies were visualized by adding
substrate o-phenylenediamine.
[0064] Size Exclusion Chromatography.
[0065] Antibody conjugate was chromatographed on a 75 mL Sephacryl
300HR column (Pharmacia, Peapack, N.J.). 1:10 diluted PBS (pH 7.2)
was chosen as elution buffer. Fractions (0.5 mL/each) were
collected and aliquots (100 .mu.L) were assayed on antihuman IgG
capture ELISA. The ELISA reading (OD 490 nm) is plotted against
elution volume.
[0066] Viability Assay for Antibody-Treated Cells.
[0067] The lymphoma cells were grown in 96-well tissue culture
wells in 1-mL medium. 2 .mu.g of antibodies or antibody-peptide
conjugates were added and incubated for various times as described
herein. Ten .mu.L aliquots from the cell suspension were used to
determine viability by using trypan blue exclusion.
[0068] FACS Assay of the B-Cell Lymphoma.
[0069] The Su-DHL4 and 38C13 cells were fixed with 1%
paraformaldehyde. 1.times.10.sup.6 cells were suspended in 50 .mu.L
of staining buffer (Hank's balanced salt solution, containing 0.1%
NaN3, 1.0% BSA), then 1.5 .mu.g of photobiotinylated S1C5-T15H
conjugates was added and incubated for 30 minutes on ice. Control
antibodies and antibody-scrambled T15 peptide conjugates served as
controls. The cells were washed twice with staining buffer before
Avidin-FITC (Sigma) was added to the cells for 30 minutes on ice.
Then the cells were washed twice with staining buffer, re-suspended
in 200 .mu.L PBS and analyzed by flow cytometry.
[0070] Hoechst-Merocyanin 540 Staining to Detect Apoptosis.
[0071] 1.times.10.sup.6 of lymphoma cells were placed into 24-well
tissue culture wells. Four pig of antibodies or antibody-peptide
conjugates were added and incubated for various times as described
herein. 1.times.10.sup.6 cells were removed from the culture,
re-suspended in 900 .mu.L cold PBS (pH 7.2). One hundred .mu.L of
Hoechst 33342 (50 .mu.g/mL; Molecular Probe, Eugene, Oreg., U.S.A.)
was added, the cells were incubated at 37.degree. C. for 30 minutes
in the dark. The cells were centrifuged and re-suspended in 100
.mu.L PBS. Then, 4 .mu.L of MC540 solution (Molecular Probe) was
added, and a 20-minute incubation was performed at room temperature
in the dark. The cells were pelleted, re-suspended in 1 mL cold PBS
(pH 7.2), and analyzed by flow cytometry.
[0072] Results
[0073] Characterization of Autophilic Antibodies.
[0074] The T15H (24-mer) peptide was crosslinked to two murine mAb
(S1C5 and 5D 10), using carbohydrate periodate conjugation. The mAb
S1C5 (IgG1) is specific for the tumor idiotype of the mouse 38C13
B-cell line and the 5D10 antibody for the human Su-DHL4 B-cell
tumor. Both antibodies recognize unique idiotypes of the BCR IgM on
the B-cell tumors. Autophilic Behavior can Easily be Demonstrated
by ELISA.
[0075] The autophilic effect was studied with the T15H
peptide-crosslinked mAb S1C15. The T15H-crosslinked S1C5 binds to
insolubilized S1C5-T15H detected by biotin-avidin ELISA. Control
S1C5 does not bind significantly to S1C5-T15H or S1C5 crosslinked
with a scrambled peptide. Similar self-binding of T15H
peptide-crosslinked mAb 5D10 to insolubilized T15H-5D10 was also
observed. The specificity of the peptide mediated autophilic effect
was tested using the 24-mer peptide T151H itself as an inhibitor.
Only the T15H peptide inhibited S1C5-T15H and 5D10-T15H
self-binding while the control-scrambled peptide did not inhibit
it. These results are similar to the previously published
inhibition data with the naturally occurring autophilic T15/S107
antibody (not shown).
[0076] T15H-Antibody Conjugates Form an Equilibrium of Monomer and
Dimer in Solution.
[0077] The non-covalent nature of the self-aggregation of
T15H-linked antibodies raises the question of its physical state in
solution. To address this issue, the molecular species of
T15H-linked monoclonal antibodies were analyzed using gel
electrophoresis and sizing gel filtration. The electrophoretic
mobility of control and T15H peptide conjugated to S1C5 and 5D 10
under reducing and non-reducing conditions show no differences,
indicating the absence of chemical bonds between the antibody
chains. The molecular species of the peptide-conjugated antibodies
(5D0-T15H) was further analyzed by size exclusion chromatography.
The elution profile indicated two immunoglobulin species of
different sizes. The larger first peak eluted in the position of an
antibody dimer. The second smaller peak eluted in the position of
non-conjugated 5-D10 antibody. The appearance of two peaks
resembled monomer and dimer antibodies and could indicate that
either a fraction of antibodies was not modified to polymerize, or
that the modification was complete and the antibody establishes an
equilibrium of dimers and monomers. To test the latter possibility,
material from both peaks were subjected to a second gel filtration
on the same column. Reruns of both peaks yielded again two peaks at
the same position as in the first chromatography (Zhao and Kohler,
2002). These data show that the T15H peptide-linked antibodies
exist in solution as two distinct molecular species in equilibrium
as monomer and dimer.
[0078] Enhanced Binding of Autophilic Antibodies to Tumors.
[0079] The binding of the peptide-conjugated antibodies against
their respective tumor targets was compared with that of the
control antibodies in indirect fluorescence activated cell sorting
(FACS). As control, antibodies linked with a scrambled peptide were
included. The fluorescence intensity of the T15H-S1C5 on 38C13
cells is compared with that by the control S1C5 and the scrambled
peptide S1C5. The difference in mean fluorescence channels between
S1C5-T15H and controls was greater than 10-fold. Similarly, the
FACS analysis of autophilic 5D 10-T15H on Su-DHL4 cells shows
enhancement of binding over binding of control 5D10 and control
peptide-crosslinked 5D 10. In both tumor systems, the conjugation
of the T15H peptide to tumor-specific antibody enhanced the FACS
signals over control antibodies used at the same concentration
(Zhao, Lou, et al., 2002). The enhancement of fluorescence can be
explained with the increase of targeting antibodies caused by
self-aggregation and lattice formation on the surface of the tumor
cells.
[0080] Inhibition of Tumor Growth.
[0081] Antibodies binding to the BCR induce crosslinking of the
BCR, which, in turn, inhibits cell proliferation and produces a
death signal. Furthermore, chemically dimerized antibodies directed
against a B-cell tumor induce hyper-crosslinking of the BCR
followed by inhibition of cell division and apoptosis of the tumor.
To see if similar enhancement of the antitumor effects of
dimerizing antibody were induced by our noncovalent, dimerizing
T15H-linked antibodies, the two B cell tumors were cultured in the
absence or presence of control and T15H-linked antibodies.
Co-culture of both tumors, 38C13 and Su-DHL4, with their respective
T15H-linked antibodies inhibited the cell growth significantly
better compared with the control antibodies. To test the tumor
target specificity of autophilic antibodies in growth inhibition,
criss-cross experiments were performed with the 38C13 and Su-DHL-4
cell lines. Inhibition of 38C13 cell growth with S1C5-T15H was
statistically greater than mismatched 5D10-T15H. Similar results on
the specificity of autophilic antibodies were obtained with the
Su-DHL4 cells (Zhao, Lou, et al., 2002).
[0082] Induction of Apoptosis.
[0083] As suggested by earlier studies, the antitumor effect of
antibodies directed against the BCR of B-cell lymphomas in vitro
and in vivo might be caused by the induction of apoptosis. Aliquots
of tumor cells (38C13 and Su-DI-L-4) cultured in the presence of
control or T15H-linked antibodies were analyzed for apoptosis using
a double stain FACS protocol. 38C13 and Su-DHL4 cells underwent a
moderate amount of apoptosis without antibodies over a 6,
respectively 18-hour culture. This apoptosis was enhanced when the
respective antibody was added. However, when the T15H-linked
antibodies were added, the accumulated number of apoptotic 38C13
cells was almost doubled, and apoptosis of Su-DHL4 cells was more
than doubled during the entire culture (Zhao, Lou, et al.,
2002).
[0084] The biologic advantage of the autophilic property is
exemplified with the S107/T15 anti-phosphorylcholine antibody. This
autophilic antibody is several times more potent in protecting
immune-deficient mice against infection with pneumococci pneumoniae
than non-autophilic antibodies with the same antigen specificity
and affinity.
[0085] As shown here, the autophilic antibody function can be
transferred to other antibodies by chemically crosslinking a
peptide derived from the T15 VH germline sequence. The modified
antibody mimics the autophilic property of the T15/S107 antibody,
producing a autophilic antibody with increased avidity and enhanced
targeting. Enhancing the binding of autophilic engineered
antibodies to the BCR of B-cell tumor increases the strength of the
death signals leading to profound inhibition of cell proliferation
in culture. Even though the doubling of apoptosis is demonstrated
here, other mechanisms of growth inhibition can be involved.
[0086] Crosslinking the BCR of the mature murine B-cell lymphoma
A20 can protect against mediated apoptosis. This anti-apoptotic
activity of engagement of the BCR by crosslinking antibodies is
highly restricted to the time window of CD95 stimulation and is not
dependent upon protein synthesis. The finding that BCR
hypercrosslinking per se is pro-apoptotic is not at variance with
reports on the anti-apoptotic activity of the BCR engagement,
because it can be a result of the use of less mature B-cell lines
in our study, to different strength of delivered signals by
homodimerizing antibodies, or to Fas-independent apoptosis.
[0087] The use of two BCR idiotope-specific antibodies against
different tumors offered the opportunity to test the biologic
effect of targeting receptors other than the idiotope specific BCR.
In criss-cross experiments with autophilic antibodies binding in
FACS analysis and inhibition of growth in vitro show a significant
enhancement only with the autophilic matched antibody. In this
context, it is interesting to speculate whether enhanced tumor
targeting would also augment cellular effector functions.
[0088] In an earlier study using chemically homodimerized
antibodies, the Fe domain was not involved in the augmentation of
growth inhibition and tumor cells lacking Fc receptors were
susceptible to the antigrowth activity of homodimers. Thus, the
anti-tumor effect induced by dimerizing antibodies would not be
restricted to lymphoid tumors such as non-Hodgkin's B-cell
lymphoma, where anti-tumor effects require the participation of
Fc-receptor-bearing effector cells.
[0089] The described approach of transferring the naturally
occurring autophilic property to other antibodies thereby enhancing
their anti-tumor effect outlines a general method to improve the
therapeutic efficacy of antibodies in passive immunotherapy.
Example 2
Internalization of Antibodies Conjugated with MTS Peptide
[0090] Cell Line and Antibodies
[0091] Human Jurkat T cells were grown in RPMI 1640 supplemented
with 10% fetal bovine serum and antibiotic (penicillin,
streptomycin and amphotericin). Rabbit polyclonal anti-active
caspase-3 antibody (#9661 S) and anti cleaved-fodrin, i.e. alpha II
spectrins (#2121S) were purchased from Cell Signaling, Inc
(Beverly, Mass.). Monoclonal (rabbit) anti-active caspase-3
antibody (#C92-605) was purchased from BD PharMingen (San Diego,
Calif.). Mouse monoclonal antibody 3H1 (anti-CEA) was purified from
cell-culture supernatant by protein G affinity chromatography.
Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were
purchased from Santa Cruz Biotechnologies, Inc. ApoAlert Caspase-3
Fluorescent Assay kit was purchased from Clonetech Laboratories
(Palo Alto, Calif.). The Cell Death Detection ELISA was purchased
from Roche Applied Science (Indianapolis, Ind.). Synthesis of MTS
Peptide-Antibody Conjugate MTS peptide (KGEGAAVLLPVLLAAPG) (SEQ ID
NO. 2) was a signal peptide-based membrane translocation sequence,
and synthesized by Genemed Synthesis (San Francisco, Calif.).
Antibodies were dialyzed against PBS (pH 6.0) buffer, oxidized by
adding 1/10 volume of 200 mmol/L NaIO.sub.4 and incubating at
4.degree. C. for 30 min in the dark. Adding glycerol to a final
concentration of 30 mM terminated the oxidation step. Samples were
subsequently dialyzed at 4.degree. C. for 1 h against 1.times.PBS
(pH6.0) buffer. The MTS peptide (50 times molar excess) was added
to couple the antibodies and the samples were incubated at
37.degree. C. for 1 h and the resulting antibody-peptide conjugate
was dialyzed against 1.times.PBS (pH 7.4).
[0092] Effect of MTS-Conjugated Antibody on Cell Growth
[0093] Jurkat cells (2.5.times.10.sup.5) were seeded into 96-well
culture plate. After incubation with 0.5 .mu.g MTS-antibody
conjugates for 6, 12, 18 and 24 hour, aliquots were removed and
viability was determined by trypan blue exclusion.
[0094] Study of Antibody Internalization by ELISA
[0095] Jurkat cells, grown in 1-ml medium in a 6-well culture
plate, were incubated with 2 .mu.g of unconjugated or MTS
conjugated antibodies for 0, 1, 3, 6, 12 and 18 h. The cells were
centrifuged and the culture supernatant was then transferred to a
new tube. The cell pellet was washed twice with PBS (pH 7.4) before
being homogenized by Pellet Pestle Motor (Kontes, Vineland, N.J.)
for 30 sec. All of the cell homogenate and an equal volume of the
culture (10 .mu.l) supernatant were added to sheep anti-rabbit IgG
coated ELISA plate (Falcon, Oxnard, Calif.) and incubated for 2 h
at room temperature. After washing step, HRP-labeled goat
anti-rabbit light chain antibody was added, and visualized using
o-phenylenediamine.
[0096] DNA Fragmentation
[0097] Jurkat cells were pre-treated with antibodies or a caspase-3
inhibitor (DEVD-fmk) for 1 h, centrifuged, and incubated with fresh
medium containing actinomycin ID alone (1 .mu.g/ml) for 4 h. After
treatment, Jurkat cells were collected, washed, and resuspended in
700 .mu.l of HL buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2%
Triton X-100.sup.4,11 for 15 min at room temperature. DNA was
extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and
precipitated 24 h at 20.degree. C. with 0.1 volume of 5 M NaCl and
1 volumes of isopropanol. The DNA was washed, dried, and
resuspended in TE pH 8.0. The DNA was resolved by electrophoresis
on a 1.5% agarose gel and visualized by UV fluorescence after
staining with ethidium bromide. DNA fragmentation was also
determined using the Cell Death Detection ELISA according to the
manufacturer's instructions.
[0098] Preparation of Total Cell Lysate
[0099] Jurkat cells were treated as described in the DNA
fragmentation section. After treatment, cells were collected and
washed with PBS (pH 7.4) twice, then suspended in 300 .mu.l of
CLAPS buffer (50 mM PIPES, pH 6.5, 2 mM EDTA, 0.1% CHAPS). The
samples were sonicated for 10 sec and centrifuged at 14,000 rpm for
15 min at 4.degree. C. The supernatant was transferred to a new
tube and referred as total cell lysate.
[0100] Caspase-3-like Cleavage Activity Assay
[0101] Jurkat cells were treated as described in the DNA
fragmentation section. Equal amounts of protein of the total cell
lysate was applied for caspase-3 activity assay using ApoAlert
Caspase-3 Fluorescent Assay Kit according to the manufacturer's
instruction. Fluorescence was measured with a Spectra MAX GEMINI
Reader (Molecular Devices, Sunnyvale, Calif.).
[0102] Western Blot Analysis
[0103] Jurkat total cell lysates (10 ug) were separated on a 10%
SDS-PAGE gel to detect immunoreactive protein against cleaved
spectrin. Ponceau staining was used to monitor the uniformity of
protein transfer onto the nitrocellulose membrane. The membrane was
washed with distilled water to remove excess stain and blocked in
Blotto (5% milk, 10 mm Tris-HCl [pH 8.0], 150 mM NaCl and 0.05%
Tween 20) for 2 h at room temperature. Before adding the secondary
antibody, the membrane was washed twice with TBST (10 mM Tris-HCl
with 150 mM NaCl and 0.05% Tween 20), and then incubated with
horseradish peroxidase-conjugated secondary antibodies. The blot
was washed extensively and reactivity was visualized by enhanced
chemiluminescence (AmershamBiotech, Piscataway, N.J.).
[0104] Statistical Analysis
[0105] Statistical analysis was performed using the student t-test
(for a pair-wise comparison) and one-way ANOVA followed by
Newman-Keuls posttest. Data are reported as means.+-.SE.
[0106] Results
[0107] As shown in FIG. 7, an MTS conjugated anti-active caspase 3
antibody is internalized more rapidly than unmodified antibody.
When cells were exposed to the chemotherapeutic drug, actinomycin
D, apoptosis was triggered and the cells died (see FIG. 14).
However, if cells were exposed at the same time to the MTS
conjugated antibody (transMab), most of the toxicity of the
chemotherapeutic drug was inhibited.
Example 3
Enhancing Binding and Apoptosis Using Peptide-Conjugated Anti-CD20
Antibodies
[0108] Materials and Methods
[0109] Cell Line and Antibodies
[0110] The human B-cell tumor lines SU-DHL-4 and Raj were grown in
RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2
mmol/L glutamine, 10 .mu.mol/L Hepes, 50 U/mL penicillin, 50
.mu.g/mL streptomycin, and 50 .mu.mol/L 2-mercaptoethanol at
37.degree. C. under 5% CO.sub.2. Mouse monoclonal antibodies 1F5
IgG2a (ATTC #HB-9645) specific for human B-cell lymphomas 5D10 and
3H1 (Zhao, Lou, et al., 2002.) were purified from cell culture
supernatant by protein G or protein A affinity chromatography.
[0111] Synthesis of Antibody-Peptide Conjugate
[0112] T15 peptide (ASRNKANDYTTDYSASVKGRFIVSR) (SEQ ID NO. 1), a
VH-derived peptide from a self-binding antibody-T15, was
synthesized by Genemed Synthesis, California. The antibodies were
dialyzed against phosphate-buffered saline (PBS. pH 6.0) buffer;
incubated at 4.degree. C. for 30 minutes in the dark. The oxidation
was stopped by adding glycerol (30 .mu.mol, final concentration),
and dialysis was performed at 4.degree. C. for 30 minutes against
PBS (pH 7.0) buffer. One hundred times molecular excess of T15
peptide was coupled to the antibody 1F5 by incubation at 37.degree.
C. for 1 hour; 1-lysine was used to block the unreacted aldehyde by
incubation at 37.degree. C. for 30 minutes. After the blocking
step, the antibody conjugates were dialyzed against PBS (pH 7.2)
overnight.
[0113] 8-azido-adenosine-biotin was synthesized and used to
affinity cross-link biotin to antibodies. The 8-azidoadenosine
dialdehyde was prepared as previously published (U.S. Pat. No.
5,800,991 for "Nucleotide or nucleoside photoaffinity compound
modified antibodies, methods for their manufacture and use thereof
as diagnostics and therapeutics," issued to Haley et al., 1998,
which is incorporated herein by reference)
[0114] Self-Binding Enzyme-Linked Immunosorbant Assay
[0115] Four micrograms per milliliter of 1F5-T15 was used to coat
Costar vinyl assay plates (Costar, Cambridge, Mass., U.S.A.). After
blocking with 1% (BSA) solution. 8 ug/mL photobiotinylated (see
U.S. Pat. No. 5,800,991 discussed above) 1F5-T15 naked 1F5 and
control antibody (5D10) were added, diluted to 1:1, and incubated
for 2 hours at room temperature. After washing with PBS buffer,
avidin-HRP (Sigma, St. Louis, Mo., U.S.A.) was added, and
enzyme-linked immunosorbent assay color was developed with
o-phenylenediamine.
[0116] FACS Assay of the B-Cell Lymphoma
[0117] SU-DHL-4 cells were fixed using 1% paraformaldehyde, and
1.times.06 cells were suspended in 50 .mu.L staining buffer (Hanks,
containing 0.1% NaN.sub.3 and 1.0% BSA); 1.5 .mu.g
photobiotinylated 1F5-T15 conjugates (see U.S. Pat. No. 5,800,991
discussed above), naked 1F5, and control antibodies were added and
incubated for 30 minutes on ice. The cells were washed twice with
staining buffer, and then avidin-FITC was added for 30 minutes on
ice. After washing twice with staining buffer, the cells were
resuspended in 200 .mu.L PBS for FACS analysis.
[0118] Hoechst-Merocyanin 540 Staining to Detect Apoptosis
[0119] After 1.times.10.sup.6 lymphoma cells were placed into
24-well tissue culture wells, 4 .mu.g antibodies and
antibody-peptide conjugates were added. After 24 hours of
incubation, 1.times.10.sup.6 cells were removed from the culture
pellet and resuspended in 900 mL cold PBS (pH 7.2), and 100 .mu.L
Hoechst (Pierce, Rockford, Ill., U.S.A.) 33342 (50 .mu.g/mL) was
added and incubated at 37.degree. C. for 30 minutes in the dark.
The cells were centrifuged and resuspended in 100 .mu.L PBS; 4
.mu.L MC540 dilution solution was added and the cells were
incubated for 20 minutes at room temperature in the dark. The cells
were pelleted, resuspended in 1 mL PBS, and analyzed by flow
cytometry.
[0120] Inhibition of Cell Growth in Culture
[0121] One.times.10.sup.5 tumor cells were seeded in complete
culture medium. At days 1, 2, and 3 of culture, aliquots were
removed and viable cells were counted using dye exclusion (trypan
blue).
[0122] Results
[0123] Mouse monoclonal antibodies 1F5 IgG2a were conjugated with
self-binding peptide as in Example 1. An average of 1.8 peptides
were found by competitive analysis. The parental antibody was
compared to the conjugated form for binding by flow cytometry. As
shown in FIG. 3 the binding was increased for the conjugated
antibody (Mab-ap) when assessed with a limiting dilution of
antibody. This was characterized by a shift in the binding
fluorescence to a higher intensity. When compared over a series of
dilutions, conjugated antibody required almost one-tenth the
concentration of antibody to achieve the same level of intensity as
parental antibody (FIG. 2). As shown in FIG. 1, increasing the
amount of conjugated antibody caused a reduction in fluorescence
intensity, presumably due to internalization, a property of SAT
technology that can be used to enhance potency of immunoconjugates
of drugs, toxins and short path length radiotherapeutic isotopes.
Furthermore, when tested for the ability to trigger apoptosis, the
conjugated form (Sab) was much more active than native antibody,
with most cells dead by 3 days, compared to only a small fraction
with the native antibody (see FIG. 4).
Example 4
Enhanced Binding and Apoptosis with Anti-GM2 Antibodies
[0124] Materials and Methods
[0125] Cell Lines and Antibody
[0126] Human T-cell leukemia Jurkat cells were grown in RPMI 1640
supplemented with 10% fetal bovine serum and antibiotic
(penicillin, streptomycin and amphotericin). Chimeric hamster
anti-GM2 antibody (ch-.alpha.-GM2) was obtained from Corixa
Corporation (Seattle, Wash.). After chimerization, the resulting
antibody lost its ability to induce apoptosis in ganglioside GM2
expressing target cells.
Synthesis of Antibody-Peptide Conjugate
[0127] Both the T15 peptide (GAAASRNKANDYTTEYSASVKGRFIVSR) (SEQ ID
NO. 8), a VH-derived peptide from a self-binding antibody-T15
(Kaveri et al, 1991), and a scrambled peptide (T15-scr) (SEQ. ID.
NO, 3), which was randomly generated using the T15 amino acid
sequence, were synthesized by Genemed Synthesis (South San
Francisco, Calif.). The scrambled peptide was used as a control.
Antibodies were dialyzed against PBS (pH 6.0), then 1/10 volume of
200 .mu.M NaIO4 was added and incubated at 4.degree. C. for 30 min
in the dark. The reaction was stopped by adding glycerol to a final
concentration of 30 .mu.M, and the samples were dialyzed at
4.degree. C. for 30 min against PBS (pH 6.0). Fifty (50) times
molecular excess of T15 or scrambled peptide was added to the
antibodies and incubated at 37.degree. C. for 1 h. L-Lysine was
added and incubated at 37.degree. C. for 30 min to block the
remaining reactive aldehyde group. After the blocking step, the
antibody-conjugates were dialyzed against PBS (pH 7.2) at 4.degree.
C. overnight, then stored at 4.degree. C. until used.
[0128] Direct Binding ELISA
[0129] GM2 ganglioside was dissolved in methanol and 0.5 .mu.g was
coated per well in 96 well polystyrene plates (Costar, Cambridge,
Mass.) and allowed to dry overnight. The wells were blocked with 1%
BSA for 2 h at room temperature and 400 .mu.g of anti-GM2
antibodies, diluted in 1% BSA, were added in the first well and
then serially diluted 1:1. After incubation for 1 h, the wells were
washed 5.times.. and HRP-conjugated anti-human IgG (Sigma, St.
Louis, Mo.) was added at a 1:1000 dilution and incubated for 1.5 h.
After washing three times, the bound antibodies were visualized
using substrate o-phenylenediamine and read at OD 492 using a
spectrophotometer.
[0130] Specific Binding ELISA
[0131] Gangliosides GM2, GM1, GM3 were dissolved in DMSO in 0.5
.mu.g and coated in 96 well polystyrene plate (Costar, Cambridge,
Mass.) dried over night. The wells were blocked with 1% BSA for 2 h
at room temperature, 400 .mu.g of ch-.alpha.-GM2 antibodies
(anti-GM2-T15) were added in the first well and then serially
diluted 1:1. After incubation for 1 h, the wells were washed
5.times. and HRP-conjugated anti-human IgG (Sigma, St. Louis, Mo.)
was added and incubated for 1.5 h. After washing three times, the
bound antibodies were visualized using substrate o-phenylenediamine
and assayed as described previously.
[0132] Antibody Self-Binding ELISA
[0133] 2 .mu.g/ml of naked ch-.alpha.-GM2 (anti-GM2) or
ch-.alpha.-GM2-T15 (anti-GM2-T15) were coated onto Costar vinyl
assay plates. After blocking with 3% BSA solution, 0.5 .mu.g/well
of photobiotinylated anti-GM2-T15 was added. The antibodies were
then incubated for 2 h at room temperature. After washing three
times, avidin-HRP (Sigma) was added at a 1:1000 dilution and
incubated for 1 hour. The bound antibodies were visualized by
adding substrate o-phenylenediamine and assayed as described
previously.
[0134] Cell Surface Binding Detected by FACS
[0135] Two.times.10.sup.5 Jurkat cells per well were seeded in a
6-well plate and incubated overnight, then cells were collected and
washed twice with P/B/G/A buffer (0.5% BSA, 5% Goat Serum in PBS).
Cells were then resuspended in 100 .mu.l P/B/G/A buffer containing
5 .mu.g/ml anti-GM2 antibodies for 30 min. After washing with
P/B/G/A buffer, FITC-conjugated anti-Human IgG (Sigma, 1:1000
dilution in 100 .mu.l P/B/G/A) was added and incubated on ice for
30 min. After washing with P/B/G/A buffer, cells were resuspended
in 400 .mu.l P/B/G/A containing 10 .mu.g/ml propidium iodide (as
viability probe) and analyzed by flow cytometry.
[0136] Apoptosis Detected by Annexin V Staining
[0137] 2.times.10.sup.5 Jurkat cells were seeded per well in a
6-well plate. After 6 h, cells were incubated with 20 .mu.g/ml of
the anti-GM2 or anti-GM2-T15 antibodies for 12 hr. Following the
incubation, a small portion of cells (50 .mu.l) was saved and
assayed for viability, while the remainder of the cells were
harvested and washed with cold PBS. Cells were then resuspended in
100 .mu.l annexin staining buffer (5 .mu.l Alex fluor 488 was added
into 95 .mu.l 1.times.annexin binding buffer, and Sytox was added
at a dilution of 1:1000. After incubation at room temp for 15 min,
400 .mu.l of 1xannexin binding buffer was then added, and samples
were analyzed by FACS.
[0138] Viability Assay for Antibody-Treated Cells
[0139] A small portion of the cell samples saved from the annexin
experiment was used for viability assay. 10-.mu.l aliquots from the
cell suspension were taken to determine viability using trypan blue
exclusion assay.
[0140] Statistical Analysis.
[0141] Statistical analysis was performed using one-way ANOVA
followed by Newman-Keuls post test. Data are reported as
means+SD.
Results
[0142] Self-Binding Peptide Enhanced Antibody Binding to its
Specific Ganglioside.
[0143] Following antibody-peptide conjugation, the binding capacity
of the T15-conjugated ch-.alpha.-GM2 antibody (anti-GM2-T15) was
determined using a direct binding ELISA. As seen in FIG. 9, both
ch-.alpha.-GM2 antibody (anti-GM2) and anti-GM2-T15 antibody showed
a dose-dependent increase in binding to ganglioside GM2. The
anti-GM2-T15 antibody demonstrated a higher binding capacity
compared with the naked anti-GM2 at all the doses tested,
confirming that the self-binding T15 peptide had increased the
antigen binding capacity of the ch-.alpha.-GM2 antibody at a given
antibody concentration.
[0144] Antibody Self-Binding Behavior Demonstrated by ELISA
[0145] Next, it was investigated by ELISA whether the increase in
binding to ganglioside GM2 by the T15 peptide-linked antibody was
due to its self-binding feature. As seen in FIG. 10, the
anti-GM2-T15 antibody demonstrated a greater dose-dependent
increase in binding to the peptide-conjugated anti-GM2-T15 antibody
coated on the wells, whereas it did not show significant binding to
the non-peptide conjugated anti-GM2 antibody. These data
demonstrate that the anti-GM2-T15 antibody can bind to itself or
homodimerize through the Fc-conjugated, autophilic peptide
moiety.
[0146] Conjugation does not Change the Specificity of the
ch-.alpha.-GM2 Antibody.
[0147] To assess whether conjugation of the T15 peptide might alter
the cognate binding specificity of the antibody, a direct
antigen-binding ELISA was used to determine the binding specificity
of the anti-GM2-T15 conjugated antibody. As shown in FIG. 11, the
anti-GM2-T15 antibody demonstrated a specific, dose-dependent
increase in binding to ganglioside GM2, whereas no binding above
background levels to gangliosides GM1 or GM3 was detected. This
result confirms that addition of the self-binding T15 peptide did
not alter nor reduce the specificity of the ch-.alpha.-GM2
antibody.
[0148] Enhanced Surface Binding of Anti-GM2 Antibody to Target
Tumor Cells
[0149] The human T-cell leukemic cell line Jurkat is known to
express ganglioside GM2 (Suzuki et al, 1987). The ability of the
peptide-conjugated anti-GM2-T15 antibody to bind to native
ganglioside GM2 expressed on the surface of Jurkat cells was
compared to that of the non-conjugated anti-GM2 antibody by flow
cytometry. As shown in FIG. 12, the ch-.alpha.-GM2 antibody
(anti-GM2) demonstrated a GM2 specific binding signal 3 times
greater than background levels, whereas the binding demonstrated by
the T15-conjugated anti-GM2 antibody was 2-fold higher than that of
the non-peptide conjugated antibody. This result suggests that the
enhanced binding demonstrated by the peptide-conjugated Ab is due
to self-aggregation of this antibody.
[0150] Inhibition of Tumor Growth
[0151] Antibodies binding to the B cell receptor have been shown to
induce crosslinking of the BCR, which, in turn, inhibits cell
proliferation (Ward et al, 1988) and produces a death signal
(Hasbold et al, 1990; Wallen-Olunan et al, 1993). Furthermore,
chemically dimerized antibodies directed against a B-cell tumor
induce hyper-crosslinking of the BCR followed by inhibition of cell
division and induction of apoptosis of the tumor cells (Ghetie et
al, 1994; Ghetie et al, 1997). To determine whether the
T15-conjugated anti-GM2 antibody induced a similar
anti-proliferative effect, 2.times.10.sup.5 Jurkat cells were
cultured in the presence or absence of anti-GM2 or control
antibodies for 12 h, and then the number of viable cells remaining
were counted. As summarized in FIG. 13, no antibody or control
human IgG antibody (HuIgG) treatment had no effect on cell growth
or viability, whereas there was some effect with the anti-GM2
antibody. However, the T15-linked antibody demonstrated a marked
inhibition of Jurkat cell growth, as cell numbers were reduced
>2-fold compared to naked anti-GM2 antibody treated cells, and
more than 4 fold versus the control IgG treatment. As a comparison
and positive control, Actinomycin D demonstrated the ability to
induce apoptosis, at levels slightly higher than the
SuperAntibody.
[0152] Induction of Apoptosis
[0153] In order to determine whether the anti-tumor effect of
antibodies directed against cell surface expressed gangliosides
might be due to the induction of apoptosis, we took the cell
samples used in the cell growth study and analyzed them for
apoptosis induction by measuring annexin V staining. The results
are summarized in Table I.
TABLE-US-00001 TABLE I Apoptosis Analysis Using Annexin V Staining
Antibody Jurkat* No treatment 7.7 .+-. 1.55 HuIgG 7.2 .+-. 1.94
Anti-GM2 14.8 .+-. 7.55 Anti-GM2-T15scr 13.0 .+-. 4.60 Anti-GM2-T15
54.2 .+-. 23.4 Actinomycin D 81.9 .+-. 10.2 *Data were summarized
from four sets of experiments.
[0154] Treatment of Jurkat cells with the ch-.alpha.-GM2 antibody
(anti-GM2) or the ch-.alpha.-GM2 antibody conjugated with a
scrambled, control peptide (anti-GM2-T15scr) did not induce
apoptosis significantly over levels induced by treatment with
control human IgG, as a modest 2-fold increase was observed.
However, Jurkat cells treated with the anti-GM2-T15 conjugated
underwent a significant amount of apoptosis, nearly 8-fold over
background and more than 4-fold higher than that induced by the
non-conjugated antibody or the control-conjugated antibody. These
results confirmed the activity and specificity of T15-conjugated
antibody.
Example 5
Generation of Autophilic Peptide Sequences T15-scr, T15-scr2, R24
and R124-Charged
[0155] Peptides were synthesized as in Example 5. The sequences are
given in Table II.
TABLE-US-00002 TABLE II Sequences for Autophillic Binding Peptides
Name Sequence (NH2 to COOH) T15 ASRNKANDYTTDYSASVKGRFIVSR (SEQ ID
NO. 1) T15scr or T15s SYSASRFRKNGSIRAVEATTDVNSAYAK (SEQ ID NO. 3)
T15scr2 SKAVSRFNAKGIRYSETNVDTYAS (SEQ ID NO. 4) R24
GAAVAYISSGGSSINYA (SEQ ID NO. 5) R24-Charged GKAVAYISSGGSSINYAE
(SEQ ID NO. 6)
[0156] The peptide derived from R24 is difficult to solubilize
except in DMSO or alcohol. Using such solubilizers can not only
denature the antibody but also makes it difficult to conjugate to
hydrophilic regions of the antibody. To overcome this solubility
problem the addition and changes of sequence to charged amino
acids, as shown in Table II were undertaken. The resultant modified
peptide (R124--Charged) was soluble in aqueous buffer, was able to
be conjugated to the tryptophane or nucleotide binding sites and
preserved self-binding as well as induced apoptosis when conjugated
to anti-GM2 antibody. The same amino acids present in the T15
sequence were randomly re-arranged and used to construct a further
synthetic peptide; this scrambled sequence (T15scr or T15s), had no
self-binding and when conjugated to anti-GM2 antibody did not
induce apoptosis (see Example 5, Table I). In like manner, a
second, randomly selected sequence, derived from the amino acids of
the T15 sequence, was used to generate a synthetic peptide
(T15scr2). Unlike the first scrambled sequence, this peptide
demonstrated self-binding and when conjugated to anti-GM2 antibody,
induced apoptosis in levels higher than the original T15 sequence.
Thus, self-binding behavior can be generated, using the same amino
acids from the original T15 sequence but arranged in a different
order from the original T15. A peptide library generated using
these same amino acids, combined with a screen for self-binding
could be used to identify other self-binding sequences.
Example 6
Method of Conjugating Autophilic Peptides to Antibodies (Comparison
of Various Immunoglobulin Conjugation Sites)
[0157] The T15 peptide sequence was conjugated to anti-GM2 antibody
via the nucleotide binding site, tryptophane affinity sites, and
through periodate oxidation, the carbohydrate on the Fe region. As
shown in FIG. 6, when tested for the ability to trigger apoptosis,
the nucleotide site conjugation (GM2-N-3-ATP-T15/biotin) generated
a higher level of apoptosis, than the carbohydrate linkage
(Anti-GM2-T15). This was in spite of the fact that carbohydrate
linkage installed 8-10 peptides per antibody and nucleotide linkage
only 2 peptides per antibody. Affinity site conjugation was the
best method of conjugation of peptides. Conjugation to
epsilon-amino acids of antibody, via hetero-bifunctional
cross-linking agents, gave an inactive conjugate (not shown).
Example 7
Restoration of Apototic Activity
[0158] A parental antibody to GM2 glycolipid, derived from a
non-human hybridoma, was tested for the ability to trigger
apoptosis against human cancers including non-small cell lung
cancer (FIG. 5). The parental antibody demonstrated a high level of
apoptosis and linking of cancer cells. The antibody was also
effective in inhibiting growth of cancers in nude mouse models (not
shown). To remove the potential for immunogenicity in humans, the
antibody was "humanized" via cloning the heavy and light chain
CDR's into the context of a human IgG1. Despite retention of
affinity and specificity (not shown), the humanized antibody
demonstrated much reduced ability to trigger apoptosis. In
contrast, the humanized antibody, conjugated to a self-binding
peptide (Sab), demonstrated high levels of apoptosis, similar to
that of the parental antibody.
[0159] A further example is of a murine antibody, R24 which targets
the GD3 ganglioside on human melanoma cells. When naturally
expressed, this antibody has self-binding and therapeutic activity
in patients, but as a humanized antibody it loses avidity,
self-binding and therapeutic activity (Chapman et al., 1994).
Restoration of therapeutic activity of the humanized R124 antibody
can also be achieved by conjugation of a self-binding peptide to
the antibody.
[0160] The humanized versions of antibody TEPC-15 and T15/S107 will
also benefit from conjugation with a self-binding peptide to
restore or enhance self-binding and therapeutic activity.
Example 8
Enhanced Binding and Tumor Recognition by Herceptin
SuperAntibody
[0161] Herceptin (monoclonal antibody to HER2/neu), has been
approved by the FDA for treatment of breast cancer. The antigen is
expressed in approximately 30% of breast cancers but in only about
half of those patients is the level of expression sufficient to
trigger therapeutic effects. In fact, patients are normally
pre-screened in a diagnostic test to determine their suitability
for treatment. HER2/neu is also expressed on other cancers, such as
non-small cell lung cancer but typically in only low levels, making
this type of cancer unsuitable for treatment. We conjugated an
autophilic peptide to Herceptin and tested for ability to bind
non-small cell lung cancer. As shown in FIG. 8 (top panel),
Herceptin reacts very weekly to this cancer; only 0.5% of cells are
positive compared to an irrelevant antibody. In contrast, the same
cancer can be better detected with the autophilic peptide
conjugated form (i.e. SuperAntibody form) of Herceptin; over 57%
are positive compared to irrelevant antibody (bottom panel). In
separate tests, a SuperAntibody form of Herceptin also inhibited
growth better than the parent antibody and could trigger apoptosis
unlike the parent.
[0162] As will be apparent to those skilled in the art in the light
of the foregoing disclosure, many alterations and modifications are
possible in the practice of this invention without departing from
the spirit or scope thereof. Accordingly, the scope of the
invention is to be construed in accordance with the substance
defined by the following claims.
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Sequence CWU 1
1
7125PRTmouse 1Ala Ser Ala Ala Leu Ala Ala Ala Thr Thr Thr Ala Thr
Ser Ala Ser1 5 10 15Val Leu Gly Ala Pro Ile Val Ser Ala 20
25217PRTmouse 2Leu Gly Gly Gly Ala Ala Val Leu Leu Pro Val Leu Leu
Ala Ala Pro1 5 10 15Gly328PRTArtificial SequenceT15 peptide -
scrambled 3Ser Thr Ser Ala Ser Ala Pro Ala Leu Ala Gly Ser Ile Ala
Ala Val1 5 10 15Gly Ala Thr Thr Ala Val Ala Ser Ala Thr Ala Leu 20
25424PRTArtificial SequenceT15 peptide - scrambled 4Ser Leu Ala Val
Ser Ala Pro Ala Ala Leu Gly Ile Ala Thr Ser Gly1 5 10 15Thr Ala Val
Ala Thr Thr Ala Ser 20517PRTArtificial SequenceR24 peptide 5Gly Ala
Ala Val Ala Thr Ile Ser Ser Gly Gly Ser Ser Ile Ala Thr1 5 10
15Ala618PRTArtificial SequenceR24 - charged peptide 6Gly Leu Ala
Val Ala Thr Ile Ser Ser Gly Gly Ser Ser Ile Ala Thr1 5 10 15Ala
Gly718PRTArtificial SequenceMembrane transporter sequence optimized
peptide 7Thr Leu Gly Gly Ser Ala Ala Val Ile Leu Pro Val Leu Ile
Ala Ser1 5 10 15Pro Gly
* * * * *