U.S. patent application number 11/203137 was filed with the patent office on 2005-12-29 for antibody having a t-cell receptor-like specificity, yet higher affinity, and the use of same in the detection and treatment of cancer, viral infection and autoimmune disease.
This patent application is currently assigned to Technion Research & Development Foundation Ltd.. Invention is credited to Denkberg, Galit, Reiter, Yoram.
Application Number | 20050287141 11/203137 |
Document ID | / |
Family ID | 27732335 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050287141 |
Kind Code |
A1 |
Reiter, Yoram ; et
al. |
December 29, 2005 |
Antibody having a T-cell receptor-like specificity, yet higher
affinity, and the use of same in the detection and treatment of
cancer, viral infection and autoimmune disease
Abstract
An isolated molecule which comprises an antibody specifically
bindable with a binding affinity below 20 nanomolar, preferably
below 10 nanomolar, to a human major histocompatibility complex
(MHC) class I being complexed with a HLA-restricted antigen and
optionally further comprises an identifiable or therapeutic moiety
conjugated to the antibody.
Inventors: |
Reiter, Yoram; (Haifa,
IL) ; Denkberg, Galit; (Nofit, IL) |
Correspondence
Address: |
Martin Moynihan
c/o Anthony Castorina
Suite 207
2001 Jefferson Davis Highway
Arlington
VA
22202
US
|
Assignee: |
Technion Research & Development
Foundation Ltd.
Haifa
IL
|
Family ID: |
27732335 |
Appl. No.: |
11/203137 |
Filed: |
August 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11203137 |
Aug 15, 2005 |
|
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10073301 |
Feb 13, 2002 |
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Current U.S.
Class: |
424/144.1 ;
530/388.22 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 2039/505 20130101; C07K 16/2833 20130101; A61P 37/02 20180101;
A61K 47/6849 20170801; A61P 31/12 20180101; A61P 35/00 20180101;
C07K 2317/622 20130101; A61P 37/00 20180101; C07K 2317/34
20130101 |
Class at
Publication: |
424/144.1 ;
530/388.22 |
International
Class: |
A61K 039/395; C07K
016/28 |
Claims
What is claimed is:
1. An isolated molecule comprising an antibody specifically
bindable with a binding affinity below 20 nanomolar to a human
major histocompatibility complex (MHC) class I being complexed with
a HLA-restricted antigen.
2. The isolated molecule of claim 1, wherein said MHC class I
molecule is selected from the group consisting of HLA-A2, HLA-A1,
HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7,
HLA-B45 and HLA-Cw8.
3. The isolated molecule of claim 1, further comprising an
identifiable moiety being conjugated to said antibody.
4. The isolated molecule of claim 3, wherein said identifiable
moiety is selected from the group consisting of a member of a
binding pair and a label.
5. The isolated molecule of claim 3, wherein said member of said
binding pair is an antigen.
6. The isolated molecule of claim 3, wherein said label is selected
from the group consisting of a fluorescent protein and an
enzyme.
7. The isolated molecule of claim 1, further comprising a
therapeutic moiety being conjugated to said antibody.
8. The isolated molecule of claim 7, wherein said therapeutic
moiety is selected from the group consisting of a cytotoxic moiety,
a toxic moiety, a cytokine moiety and a bi-specific antibody
moiety.
9. The isolated molecule of claim 1, wherein said HLA-restricted
antigen is a tumor HLA-restricted antigen.
10. The isolated molecule of claim 1, wherein said HLA-restricted
antigen is a viral HLA-restricted antigen.
11. The isolated molecule of claim 1, wherein said HLA-restricted
antigen is an autoimmune HLA-restricted antigen.
12. A pharmaceutical composition comprising a therapeutically
effective amount of a molecule which comprises an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a HLA-restricted antigen, said molecule further
comprises a therapeutic moiety being conjugated to said
antibody.
13. The pharmaceutical composition of claim 12, further comprising
a pharmaceutically acceptable carrier.
14. The pharmaceutical composition of claim 12, wherein said MHC
class I molecule is selected from the group consisting of HLA-A2,
HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34,
HLA-B7, HLA-B45 and HLA-Cw8.
15. The pharmaceutical composition of claim 12, wherein said
therapeutic moiety is selected from the group consisting of a
cytotoxic moiety, a toxic moiety, a cytokine moiety and a
bi-specific antibody moiety.
16. The pharmaceutical composition of claim 12, wherein said
HLA-restricted antigen is a tumor HLA-restricted antigen.
17. The pharmaceutical composition of claim 12, wherein said
HLA-restricted antigen is a viral HLA-restricted antigen.
18. The pharmaceutical composition of claim 12, wherein said
HLA-restricted antigen is an autoimmune HLA-restricted antigen.
19. A diagnostic composition comprising a molecule which comprises
an antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a HLA-restricted antigen, said molecule
further comprises an identifiable moiety being conjugated to said
antibody.
20. The diagnostic composition of claim 19, wherein said MHC class
I molecule is selected from the group consisting of HLA-A2, HLA-A1,
HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7,
HLA-B45 and HLA-Cw8.
21. The diagnostic composition of claim 19, wherein said
identifiable moiety is selected from the group consisting of a
member of a binding pair and a label.
22. The diagnostic composition of claim 19, wherein said member of
said binding pair is an antigen.
23. The diagnostic composition of claim 19, wherein said label is
selected from the group consisting of a fluorescent protein and an
enzyme.
24. The diagnostic composition of claim 19, wherein said
HLA-restricted antigen is a tumor HLA-restricted antigen.
25. The diagnostic composition of claim 19, wherein said
HLA-restricted antigen is a viral HLA-restricted antigen.
26. The diagnostic composition of claim 19, wherein said
HLA-restricted antigen is an autoimmune HLA-restricted antigen.
27. An isolated molecule comprising a first polynucleotide encoding
an antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a HLA-restricted antigen.
28. The isolated molecule of claim 27, wherein said MHC class I
molecule is selected from the group consisting of HLA-A2, HLA-A1,
HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7,
HLA-B45 and HLA-Cw8.
29. The isolated molecule of claim 27, further comprising a second
polynucleotide being linked to said first polynucleotide and
encoding an identifiable moiety.
30. The isolated molecule of claim 29, wherein said identifiable
moiety is selected from the group consisting of a member of a
binding pair and a label.
31. The isolated molecule of claim 29, wherein said member of said
binding pair is an antigen.
32. The isolated molecule of claim 29, wherein said label is
selected from the group consisting of a fluorescent protein and an
enzyme.
33. The isolated molecule of claim 27, further comprising a second
polynucleotide being linked to said first polynucleotide and
encoding a therapeutic moiety.
34. The isolated molecule of claim 33, wherein said therapeutic
moiety is selected from the group consisting of a cytotoxic moiety,
a toxic moiety, a cytokine moiety and a bi-specific antibody
moiety.
35. The isolated molecule of claim 27, wherein said HLA-restricted
antigen is a tumor HLA-restricted antigen.
36. The isolated molecule of claim 27, wherein said HLA-restricted
antigen is a viral HLA-restricted antigen.
37. The isolated molecule of claim 27, wherein said HLA-restricted
antigen is an autoimmune HLA-restricted antigen.
38. A method of producing an antibody specifically bindable with a
binding affinity below 20 nanomolar to a human major
histocompatibility complex (MHC) class I being complexed with a
HLA-restricted antigen, the method comprising: immunizing a
genetically engineered non-human mammal having cells expressing
said human major histocompatibility complex (MHC) class I with a
soluble form of a MHC class I molecule being complexed with said
HLA-restricted antigen; isolating mRNA molecules from antibody
producing cells of said non-human mammal; producing a phage display
library displaying protein molecules encoded by said mRNA
molecules; and isolating at least one phage from said phage display
library, said at least one phage displaying said antibody
specifically bindable with said affinity below 10 nanomolar to said
human major histocompatibility complex (MHC) class I being
complexed with said HLA-restricted antigen.
39. The method of claim 38, wherein said non-human mammal is devoid
of self MHC class I molecules.
40. The method of claim 38, wherein said MHC class I molecule is
selected from the group consisting of HLA-A2, HLA-A1, HLA-A3,
HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and
HLA-Cw8.
41. The method of claim 38, wherein said HLA-restricted antigen is
a tumor HLA-restricted antigen.
42. The method of claim 38, wherein said HLA-restricted antigen is
a viral HLA-restricted antigen.
43. The method of claim 38, wherein said HLA-restricted antigen is
an autoimmune HLA-restricted antigen.
44. The method of claim 38, wherein said soluble form of a MHC
class I molecule is a single chain MHC class I polypeptide
including a functional human .beta.-2 microglobulin amino acid
sequence directly or indirectly covalently linked to a functional
human MHC class I heavy chain amino acid sequence.
45. A method of treating a cancer, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a molecule which comprises an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a tumor HLA-restricted antigen characterizing said
cancer, said molecule further comprises a therapeutic moiety being
conjugated to said antibody, said MHC class I molecule being
selected matching to the endogenous MHC class I of the subject.
46. The method of claim 45, wherein said MHC class I molecule is
selected from the group consisting of HLA-A2, HLA-A1, HLA-A3,
HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and
HLA-Cw8.
47. The method of claim 45, wherein said therapeutic moiety is
selected from the group consisting of a cytotoxic moiety, a toxic
moiety, a cytokine moiety and a bi-specific antibody moiety.
48. A method of treating a viral infection, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a molecule which comprises an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a viral HLA-restricted antigen characterizing a
virus causative of said viral infection, said molecule further
comprises a therapeutic moiety being conjugated to said antibody,
said MHC class I molecule being selected matching to the endogenous
MHC class I of the subject.
49. The method of claim 48, wherein said MHC class I molecule is
selected from the group consisting of HLA-A2, HLA-A1, HLA-A3,
HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and
HLA-Cw8.
50. The method of claim 48, wherein said therapeutic moiety is
selected from the group consisting of a cytotoxic moiety, a toxic
moiety, a cytokine moiety and a bi-specific antibody moiety.
51. A method of treating an autoimmune disease, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with an autoimmune HLA-restricted antigen, said
molecule further comprises a therapeutic moiety being conjugated to
said antibody, said MHC class I molecule being selected matching to
the endogenous MHC class I of the subject.
52. The method of claim 51, wherein said MHC class I molecule is
selected from the group consisting of HLA-A2, HLA-A1, HLA-A3,
HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and
HLA-Cw8.
53. The method of claim 51, wherein said therapeutic moiety is
selected from the group consisting of a cytotoxic moiety, a toxic
moiety, a cytokine moiety and a bi-specific antibody moiety.
54. A method of making an immunotoxin, the method comprising
ligating a first polynucleotide encoding an antibody specifically
bindable with a binding affinity below 20 nanomolar to a human
major histocompatibility complex (MHC) class I being complexed with
a HLA-restricted antigen in frame with a second polynucleotide
encoding a toxin moiety, so as to obtain a ligated polynucleotide
and expressing said ligated polynucleotide in an expression system
so as to obtain said immunotoxin.
55. The method of claim 54, wherein said HLA-restricted antigen is
selected from the group consisting of a tumor HLA-restricted
antigen, a viral HLA-restricted antigen and an autoimmune
HLA-restricted antigen.
56. A method of making an immunolabel, the method comprising
ligating a first polynucleotide encoding an antibody specifically
bindable with a binding affinity below 20 nanomolar to a human
major histocompatibility complex (MHC) class I being complexed with
a HLA-restricted antigen in frame with a second polynucleotide
encoding an identifiable moiety, so as to obtain a ligated
polynucleotide and expressing said ligated polynucleotide in an
expression system so as to obtain said immunolabel.
57. The method of claim 56, wherein said HLA-restricted antigen is
selected from the group consisting of a tumor HLA-restricted
antigen, a viral HLA-restricted antigen and an autoimmune
HLA-restricted antigen.
58. A method of making an immunolabel, the method comprising
ligating a first polynucleotide encoding an antibody specifically
bindable with a binding affinity below 20 nanomolar to a human
major histocompatibility complex (MHC) class I being complexed with
a HLA-restricted antigen in frame with a second polynucleotide
encoding an identifiable moiety, so as to obtain a ligated
polynucleotide and expressing said ligated polynucleotide in an
expression system so as to obtain said immunolabel.
59. The method of claim 58, wherein said HLA-restricted antigen is
selected from the group consisting of a tumor HLA-restricted
antigen, a viral HLA-restricted antigen and an autoimmune
HLA-restricted antigen.
60. A method of detecting the presence and/or level of antigen
presenting cells presenting a HLA-restricted antigen in a sample of
cells, the method comprising: interacting cells of said sample with
an antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a HLA-restricted antigen; and monitoring said
interaction, thereby detecting the presence and/or level of said
antigen presenting cells presenting said HLA-restricted
antigen.
61. The method of claim 60, wherein said HLA-restricted antigen is
selected from the group consisting of a tumor HLA-restricted
antigen, a viral HLA-restricted antigen and an autoimmune
HLA-restricted antigen.
62. An isolated nucleic acid comprising a polynucleotide as set
forth in SEQ ID NO:8.
63. An isolated nucleic acid comprising a polynucleotide encoding a
polypeptide as set forth in SEQ ID NO:9.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 10/073,301, filed on Feb. 13, 2002, the
content of which is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to an antibody having a T-cell
receptor specificity and higher affinity, conjugates of same with
identifiable and/or therapeutic moieties, method of making the
antibody and the conjugates, polynucleotides encoding the antibody
and conjugates and methods of using the conjugates in the detection
and treatment of cancer, viral infection and autoimmune
disease.
[0003] The expression of specific peptides in complex with major
histocompatibility complex (MHC) class I molecules on cells was
shown to be associated with cancer and autoimmune disorders (1-3)
and viral infections. In cancer, the discovery of these peptides
emerged from the now well-established observation that human tumor
cells often express antigens that are recognized by cytotoxic T
lymphocytes (CTLs) derived from patients (1-5).
[0004] Moreover, it has been demonstrated that the immune response
against the tumor is insufficient to cause tumor regression and
that tumor cells can develop effective mechanisms to escape such an
immune attack (6-9). Therefore, numerous approaches are being
developed in the field of tumor vaccination in an attempt to
augment the antitumor immune responses, including cancer peptide
vaccines, autologous cancer vaccines, and the cancer-dendritic cell
hybrid vaccine (7, 10, 11).
[0005] Because the specificity of the immune response is regulated
and dictated by these class I MHC-peptide complexes, it should be
possible to use these very specific and unique molecular
cell-surface markers as targets to eliminate the cancer cells,
while sparing the normal cells. A similar approach can be
undertaken to eradicate viral infected cells and cells presenting
targets for autoimmune attack. Thus, it would be very desirable to
devise new molecules in a soluble form that will mimic the fine,
unique specificity of the T-cell antigen receptor (TCR) to the
cancer/viral/autoimmune-associated MHC-peptide complexes.
[0006] One promising approach is to generate recombinant antibodies
that will bind the MHC-peptide complex expressed on the cancer
cells surface with the same specificity as the TCR. These unique
antibodies can subsequently be armed with an effector cytotoxic
moiety such as a radioisotope, a cytotoxic drug, or a toxin. For
example, antibodies that target cancer cells were genetically fused
to powerful toxins originating from both plants and bacteria, thus
generating molecules termed recombinant immunotoxins (12).
[0007] Antibodies with the MHC-restricted specificity of T cells
are rare and have been difficult to generate by conventional
hybridoma techniques because B cells are not educated to be
self-MHC-restricted (13-16). The advantages of antibody
phage-displayed technology makes it possible to also select large
antibody repertoires for unique and rare antibodies against very
defined epitopes. This has be demonstrated by the ability to
isolate by phage display a TCR-like restricted antibody to a murine
class I MHC H-2K.sup.k complexed with a viral epitope (17).
Evidently, this antibody, being directed at mouse MHC, is useless
in the treatment and diagnosis of humans. So far, attempts made by
the same group to develop a TCR-like restricted antibody to a human
class I MHC have failed. More recently an antibody was isolated
reactive with the melanoma antigen MAGE-A1 in a complex with
HLA-A1; however this antibody exhibited a low affinity and could be
used to detect the specific complexes on the surface of
antigen-presenting cells only when expressed in a multimeric form
on a phage and not as a soluble antibody (18).
[0008] There is thus a widely recognized need for, and it would be
highly advantageous to have, a TCR-like restricted antibody to a
human class I MHC devoid of the above limitations.
SUMMARY OF THE INVENTION
[0009] In recent years, many cancer-associated, viral and
autoimmune associated MHC-restricted peptides have been isolated
and because of their highly restricted fine specificity, they are
desirable targets for novel approaches in immunotherapy and
immunodiagnosis. Antibodies that are able to recognize
cancer-associated, viral and autoimmune associated MHC-peptide
complexes, with the same specificity as the T-cell antigen
receptor, would be valuable reagents for studying antigen
presentation by tumor cells, viral infected cells and autoimmune
related cells, for visualizing MHC-peptide complexes on such cells,
and eventually for developing new targeting agents for cancer,
viral and autoimmune immunotherapy and immunodiagnosis.
[0010] While reducing the present invention to practice, and in
order to generate exemplary molecules with such a unique, fine
specificity, HLA-A2 transgenic mice were immunized with a soluble
single-chain HLA-A2, complexed with a common antigenic T cell
HLA-A2-restricted epitope derived from the melanoma differentiation
antigen gp100. Using phage display, a high affinity recombinant
scFv antibody that exhibits a characteristic TCR-like binding
specificity to the gp100-derived epitope, yet unlike TCRs, it does
so with an affinity in the nanomolar range was isolated. The
TCR-like antibody recognizes the native MHC-peptide complex
expressed on the surface of antigen-presenting cells. Moreover,
when fused to a very potent cytotoxic effector molecule in the form
of a truncated bacterial toxin, it was able to specifically kill
antigen-presenting cells in a peptide-dependent manner and with
TCR-like specificity. These results demonstrate, for the first
time, the ability to isolate high-affinity human recombinant
antibodies with the antigen-specific, MHC-restricted specificity of
T cells directed toward human cancer T-cell epitopes. The selected
TCR-like antibodies are useful for monitoring and visualizing the
expression of specific MHC-peptide complexes on the surface of
tumor cells, other cells presenting antigens, and lymphoid tissues,
as well as for developing a new family of targeting agents for
immunotherapy.
[0011] Hence, according to one aspect of the present invention
there is provided an isolated molecule comprising an antibody
specifically bindable with a binding affinity below 20 nanomolar,
preferably below 10 nanomolar, to a human major histocompatibility
complex (MHC) class I being complexed with a HLA-restricted
antigen.
[0012] According to further features in preferred embodiments of
the invention described below, the isolated molecule further
comprising an identifiable moiety being conjugated to the
antibody.
[0013] According to still further features in the described
preferred embodiments the isolated molecule further comprising a
therapeutic moiety being conjugated to the antibody.
[0014] In one example, the antibody is a single chain antibody
having an amino acid sequence as set forth in SEQ ID NO:9.
[0015] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a HLA-restricted antigen, the molecule further
comprises a therapeutic moiety being conjugated to the antibody.
Preferably, the pharmaceutical composition further comprising a
pharmaceutically acceptable carrier.
[0016] According to yet another aspect of the present invention
there is provided a diagnostic composition comprising a molecule
which comprises an antibody specifically bindable with a binding
affinity below 20 nanomolar to a human major histocompatibility
complex (MHC) class I being complexed with a HLA-restricted
antigen, the molecule further comprises an identifiable moiety
being conjugated to the antibody.
[0017] According to still another aspect of the present invention
there is provided an isolated molecule comprising a first
polynucleotide encoding an antibody specifically bindable with a
binding affinity below 20 nanomolar to a human major
histocompatibility complex (MHC) class I being complexed with a
HLA-restricted antigen.
[0018] In one example, the first polynucleotide encodes a protein
having an amino acid sequence as set forth in SEQ ID NO:9. In a
specific example, the first polynucleotide has a nucleic acid
sequence as set forth in SEQ ID NO:8.
[0019] According to further features in preferred embodiments of
the invention described below, the isolated molecule further
comprising a second polynucleotide being linked to the first
polynucleotide and encoding a therapeutic moiety.
[0020] According to alternative features in preferred embodiments
of the invention described below, the isolated molecule of claim
24, further comprising a second polynucleotide being linked to the
first polynucleotide and encoding an identifiable moiety.
[0021] According to still further features in the described
preferred embodiments the identifiable moiety is selected from the
group consisting of a member of a binding pair and a label.
[0022] According to still further features in the described
preferred embodiments the member of the binding pair is an
antigen.
[0023] According to still further features in the described
preferred embodiments the label is selected from the group
consisting of a fluorescent protein and an enzyme.
[0024] According to an additional aspect of the present invention
there is provided a method of producing an antibody specifically
bindable with a binding affinity below 20 nanomolar to a human
major histocompatibility complex (MHC) class I being complexed with
a HLA-restricted antigen, the method comprising: immunizing a
genetically engineered non-human mammal having cells expressing the
human major histocompatibility complex (MHC) class I with a soluble
form of a MHC class I molecule being complexed with the
HLA-restricted antigen; isolating mRNA molecules from antibody
producing cells of the non-human mammal; producing a phage display
library displaying protein molecules encoded by the mRNA molecules;
and isolating at least one phage from the phage display library,
the at least one phage displaying the antibody specifically
bindable with the affinity below 10 nanomolar to the human major
histocompatibility complex (MHC) class I being complexed with the
HLA-restricted antigen.
[0025] According to still further features in the described
preferred embodiments the non-human mammal is devoid of self MHC
class I molecules.
[0026] According to still further features in the described
preferred embodiments the HLA-restricted antigen is a tumor
HLA-restricted antigen.
[0027] According to still further features in the described
preferred embodiments the HLA-restricted antigen is a viral
HLA-restricted antigen.
[0028] According to still further features in the described
preferred embodiments the HLA-restricted antigen is an autoimmune
HLA-restricted antigen.
[0029] According to still further features in the described
preferred embodiments the soluble form of a MHC class I molecule is
a single chain MHC class I polypeptide including a functional human
.beta.-2 microglobulin amino acid sequence directly or indirectly
covalently linked to a functional human MHC class I heavy chain
amino acid sequence.
[0030] According to still an additional aspect of the present
invention there is provided a method of treating a cancer, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a tumor HLA-restricted antigen characterizing
the cancer, the molecule further comprises a therapeutic moiety
being conjugated to the antibody, the MHC class I molecule being
selected matching to the endogenous MHC class I of the subject.
[0031] According to yet an additional aspect of the present
invention there is provided a method of treating a viral infection,
the method comprising administering to a subject in need thereof a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a viral HLA-restricted antigen characterizing
a virus causative of the viral infection, the molecule further
comprises a therapeutic moiety being conjugated to the antibody,
the MHC class I molecule being selected matching to the endogenous
MHC class I of the subject.
[0032] According to yet an additional aspect of the present
invention there is provided a method of treating an autoimmune
disease, the method comprising administering to a subject in need
thereof a therapeutically effective amount of a molecule which
comprises an antibody specifically bindable with a binding affinity
below 20 nanomolar to a human major histocompatibility complex
(MHC) class I being complexed with an autoimmune HLA-restricted
antigen, the molecule further comprises a therapeutic moiety being
conjugated to the antibody, the MHC class I molecule being selected
matching to the endogenous MHC class I of the subject.
[0033] According to further features in preferred embodiments of
the invention described below, the MHC class I molecule is selected
from the group consisting of HLA-A2, HLA-A1, HLA-A3, HLA-A24,
HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and
HLA-Cw8.
[0034] According to still further features in the described
preferred embodiments the therapeutic moiety is selected from the
group consisting of a cytotoxic moiety, a toxic moiety, a cytokine
moiety and a bi-specific antibody moiety.
[0035] According to another aspect of the present invention there
is provided a method of making an immunotoxin, the method
comprising ligating a first polynucleotide encoding an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a HLA-restricted antigen in frame with a second
polynucleotide encoding a toxin moiety, so as to obtain a ligated
polynucleotide and expressing the ligated polynucleotide in an
expression system so as to obtain the immunotoxin.
[0036] According to another aspect of the present invention there
is provided a method of making an immunolabel, the method
comprising ligating a first polynucleotide encoding an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a HLA-restricted antigen in frame with a second
polynucleotide encoding an identifiable moiety, so as to obtain a
ligated polynucleotide and expressing the ligated polynucleotide in
an expression system so as to obtain the immunolabel.
[0037] According to another aspect of the present invention there
is provided a method of detecting the presence and/or level of
antigen presenting cells presenting a HLA-restricted antigen in a
sample of cells, the method comprising interacting cells of the
sample with an antibody specifically bindable with a binding
affinity below 20 nanomolar to a human major histocompatibility
complex (MHC) class I being complexed with a HLA-restricted
antigen; and monitoring the interaction, thereby detecting the
presence and/or level of the antigen presenting cells presenting
the HLA-restricted antigen.
[0038] Depending on the application, the HLA-restricted antigen is
selected from the group consisting of a tumor HLA-restricted
antigen, a viral HLA-restricted antigen and an autoimmune
HLA-restricted antigen.
[0039] The present invention successfully addresses the
shortcomings of the presently known configurations by providing an
antibody having a T-cell receptor specificity and high affinity to
its antigen, and the use thereof in immunotherapy and
immunodiagnosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0041] In the drawings:
[0042] FIGS. 1A-B show bar graphs demonstrating polyclonal phage
ELISA on recombinant scHLA-A2-peptide complexes. Plates were coated
with the indicated scMHC-peptide complex as described in the
Examples section that follows. Shown is the binding of the
polyclonal phage population of the initial library (L) or phages
eluted after each round of panning (I-IV). FIG. 1A--phages from the
pCANTAB scFv library; FIG. 1B--phages from the scFv-CBD library.
Binding specificity studies were performed by ELISA using
biotinylated scMHC-peptide complexes. ELISA plates (Falcon) were
coated overnight with BSA-biotin (1 .mu.g/well), washed, incubated
(1 hr, RT) with streptavidin (1 .mu.g/well), washed again
extensively and further incubated (1 hr, RT) with 0.5 .mu.g of
MHC/peptide complexes. Plates were blocked with PBS/2% milk (30
min, RT), incubated with phage clones (.about.10.sup.9 phages/well,
1 hr, RT) or 0.5-1 .mu.g of soluble scFv or scFv-PE38, and
afterwards washed with 1:1000 HRP-conjugated/anti-M13, anti-myc
antibody or anti-PE antibody, respectively. The HLA-A2-restricted
peptides used for specificity studies are gp100 (154): KTWGQYWQV
(SEQ ID NO:1); gp100 (209): IMDQVPFSV (SEQ ID NO:2); gp100 (280):
YLEPGPVTV (SEQ ID NO:3); MUC1: LLLTVLTVL (SEQ ID NO:4); HTLV-1
(TAX): LLFGYPVYV (SEQ ID NO:5); hTERT (540): ILAKFLHWL (SEQ ID
NO:6); hTERT (865): RLVDDFLLV (SEQ ID NO:7);
[0043] FIGS. 2A-B show bar graphs demonstrating differential
binding of monoclonal phage clones to scHLA-A2/gp100 complexes.
Monoclonal phage were tested for binding to immobilized scHLA-A2
complexed with the gp100-derived epitopes. FIG. 2A--G9-209M; FIG.
2B--G9-280V. The assays were performed similar to as described
under FIG. 1 above;
[0044] FIG. 3A shows the nucleic (SEQ ID NO:8) and amino (SEQ ID
NO:9) acid sequences of the antibody G1 scFv. CDRs are marked in
bold, the peptide linker connecting the VH and VL domains is
underlined;
[0045] FIGS. 3B-C show SDS-PAGE analysis of purified G1scFv and
G1scFv-PE38. The G1 scFv gene was rescued from the phage clone by
PCR and was subcloned into the phagemid vector pCANTAB6 via the
SfiI-NotI cloning sites. A Myc and hexahistidine tags were fused to
the C-terminus of the scFv gene. The scFv antibody was expressed in
BL21 .lambda.DE3 cells as previously described (29) and purified
from the periplasmic fraction by metal-ion affinity chromatography.
For the expression of the G1scFv-PE38 fusion protein, the scFv gene
was subcloned as an NcoI-NotI fragment into the plasmid pIB-NN,
which encodes the translocation and ADP-ribosylation domains of PE
(PE38). Expression in BL21 .lambda.DE3 cells, refolding from
inclusion bodies, and purification of G1scFv-PE38 was performed as
previously described (30);
[0046] FIG. 4 is a bar graph demonstrating the binding specificity
of G1 scFv-PE38. Immunoplates were coated with various indicated
scHLA-A2-peptide complexes as described and binding of the G1
scFv-PE38 to immobilized complexes was detected with anti-PE
antibodies;
[0047] FIGS. 5A-B show plots demonstrating the binding
characteristics of the TCR-like G1 scFv. 5A--Titration ELISA of
purified soluble G1 scFv. Wells were coated with the MHC-peptide
complexes as described in the Examples section that follows.
5B--Competitive binding analysis of the ability of purified G1
scFv-PE38 to inhibit the binding of 125I-labeled G1 scFv-PE38 to
the HLA-A2/G9-209M complex. The apparent binding affinity of the
recombinant scFv was determined as the concentration of competitor
(soluble purified G1scFv-PE38) required for 50% inhibition of the
binding of the .sup.125I-labeled tracer. Flexible ELISA plates were
coated with BSA-biotin and scMHC-peptide complexes (10 .mu.g in 100
.mu.l) were immobilized as previously described. The recombinant
G1scFv-PE38 was labeled with [.sup.125I] using the Bolton-Hunter
reagent. Labeled protein was added to wells as a tracer
(3-5.times.10.sup.5 CPM/well) in the presence of increasing
concentrations of cold G1scFv-PE38 as a competitor and incubated at
room temperature for 1 hr in PBS. The plates were washed thoroughly
with PBS and the bound radioactivity was determined by a gamma
counter. The apparent affinity of the G1scFv-PE38 was determined by
extrapolating the concentration of a competitor necessary to
achieve 50% inhibition of [.sup.125I]-labeled G1scFv-PE38 binding
to the immobilized scMHC-peptide complex. Non-specific binding was
determined by adding a 20-40-fold excess of unlabeled Fab;
[0048] FIGS. 6A-C show plots demonstrating the binding of G1 scFv
to APCs. RMAS-HHD or JY cells were loaded with the indicated
HLA-A2-restricted peptides. Peptide loaded cells were then
incubated with the soluble purified G1 scFv antibody. Detection of
binding was with FITC-labeled anti-Myc. RMAS-HHD cells loaded with
the G9-209 and G9-280 peptides and stained together with control
unloaded cells the anti-HLA antibody w6/32 (6A) or anti-HLA-A2
antibody BB7.2 (6B) to demonstrate the stabilization/expression of
HLA-A2 complexes on the surface of peptide loaded but not on
peptide-unloaded cells. Cells loaded with G9-209 or G9-280 peptides
were stained with G1 scFv and the differential staining is shown
(6C). The B cell line RMAS-HHD transfected with a single-chain
.beta.2M-HLA-A2 gene (26) or the EBV-transformed B-lymphoblast JY
cells (10.sup.6 cells) were washed twice with serum-free RPMI and
incubated overnight at 26.degree. C. or 37.degree. C.,
respectively, in medium containing 100 .mu.M of the peptide. The
APCs were subsequently incubated at 37.degree. C. for 2-3 hours to
stabilize cell surface expression of MHC-peptide complexes followed
by incubation with recombinant scFv (10-50 .mu.g/ml, 60-90 minutes,
4.degree. C.) in 100 .mu.l. The cells were then washed, incubated
with FITC-labeled anti-Myc antibody (30-45 minutes, 4.degree. C.),
and finally washed and analyzed by a FACStar flow cytometer (Becton
Dickinson). Melanoma cells were pulsed at 37.degree. C. with 1-10
.mu.M of peptide and then stained with the scFv as described
herein; and
[0049] FIGS. 7A-C show plots and a bar graph demonstrating the
cytotoxic activity of G1 scFv-PE38 on peptide-loaded APCs. RMAS-HHD
(7A) or JY (7B) cells were loaded with the HLA-A2-restricted
peptides as indicated, followed by incubation with increasing
concentrations of G1 scFv-PE38. Protein synthesis was determined by
incorporation of .sup.3H-Leucine into cellular proteins. In (7C)
excess (0.15-0.25 mg/ml) of the indicated scHLA-A2-peptide complex
was added to the wells before the addition of G1 scFv-PE38 (25-50
ng/ml). RMAS-HHD and JY APCs were loaded with the G9-209 peptide
and control peptides as described above. Peptide-loaded cells were
subsequently incubated with increasing concentrations of
G1scFv-PE38 and the inhibition of protein synthesis was determined
by measuring the uptake of .sup.3H-Leucine into cellular proteins,
as previously described (30). IC.sub.50 was determined as the
concentration of G1scFv-PE38 required to inhibit protein synthesis
by 50%. In competition assays, excesses of specific and
non-specific HLA-A2-peptide complexes (35-50 .mu.g/well) were added
to wells 15 minutes before the addition of G1scFv-PE38.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0050] The present invention is of an antibody having a T-cell
receptor specificity yet far higher affinity, conjugates of same
with identifiable and/or therapeutic moieties, so as to generate
immunotoxins and immunolabels, method of making the antibody and
the conjugates, polynucleotides encoding the antibody and the
conjugates and methods of using the conjugates in the detection and
treatment of cancer, viral infection and autoimmune disease.
[0051] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0052] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0053] The immune system is controlled and regulated by the T-cell
receptor (TCR), which specifically recognizes peptide/Major
histocompatibility complex (MHC) molecules.
[0054] The advent of the application of recombinant class I
MHC-peptide complexes and their tetrameric arrays now enables to
detect and study rare populations of antigen-specific T cells (25,
35, 36). However, fundamental questions in immunology in general,
and in tumor immunology in particular, regarding antigen
presentation are still open because of the lack of reagents that
will enable phenotypic analysis of antigen (MHC-peptide)
presentation, the other side of the coin to MHC-peptide-TCR
interactions. One way to generate such reagents is by making
TCR-like antibodies; however, only a few publications have reported
the generation of self-MHC-restricted antibodies by conventional
means such as the hybridoma technology (13-16). The major reason
for these past difficulties may be found in the molecular nature
and the resolved structures of MHC-peptide complexes. More
specifically, the peptides are deeply buried inside the MHC-binding
groove and therefore they are presented as extended mosaics of
peptide residues intermingled with the MHC residues. It has been
shown that no more than 100-300 .ANG..sup.2 of class I MHC-bound
peptide faces outwards and thus is available for direct
recognition, whereas antibodies recognizing protein molecules
engage about 800 .ANG..sup.2 of their ligand (17). Thus, when
generating TCR-like antibodies, these molecules will presumably
recognize the peptide but will also have to be dominated by the
MHC.
[0055] Until now, antibodies with TCR-like specificity have been
generated against murine MHC-peptide complexes employing various
strategies of immunization (17). Recently, a large human Fab
library was used to select for HLA-A1-MAGE-A1-specific binding
antibodies (18). One specific clone, G8, was selected which
exhibited TCR-like specificity but revealed a relatively low
affinity of 250 nM.
[0056] While reducing the present invention to practice, the
ability to select from an immune repertoire of murine scFv
fragments a high affinity antibody directed toward a human T-cell
epitope was demonstrated.
[0057] According to an aspect of the present invention there is
provided a method of producing an antibody specifically bindable
with a binding affinity below 20 nanomolar to a human major
histocompatibility complex (MHC) class I which is complexed with a
HLA-restricted antigen. The method according to this aspect of the
invention is effected by (i) immunizing a genetically engineered
non-human mammal having cells expressing the human major
histocompatibility complex (MHC) class I, with a soluble form of a
MHC class I molecule being complexed with the HLA-restricted
antigen; (ii) isolating mRNA molecules from antibody producing
cells, such as splenocytes, of the non-human mammal; (iii)
producing a phage display library displaying protein molecules
encoded by the mRNA molecules; and (iv) isolating at least one
phage from the phage display library, the at least one phage
displaying the antibody specifically bindable with the affinity
below 10 nanomolar to the human major histocompatibility complex
(MHC) class I being complexed with the HLA-restricted antigen. The
genetic material of the phage isolate is then used to prepare a
single chain antibody or other forms of antibodies as is further
described herein below. The genetic material of the phage isolate
is then used to prepare a single chain antibody or other forms of
antibodies as is further described hereinbelow and which are
conjugated to identifiable or therapeutic moieties. Preferably, the
non-human mammal is devoid of self MHC class I molecules. Still
preferably, the soluble form of a MHC class I molecule is a single
chain MHC class I polypeptide including a functional human .beta.-2
microglobulin amino acid sequence directly or indirectly covalently
linked to a functional human MHC class I heavy chain amino acid
sequence.
[0058] Hence, according to another aspect of the present invention
there is provided an isolated molecule comprising an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I complexed
with a HLA-restricted antigen. Such an antibody has a T-cell
receptor specificity, yet far higher affinity. In one, non-limiting
example, the antibody is a single chain antibody having an amino
acid sequence as set forth in SEQ ID NO:9, encoded, for example, by
the polynucleotide as set forth in SEQ ID NO:8.
[0059] Once a polynucleotide encoding an antibody having a T-cell
receptor specificity as herein described is cloned, it can be
modified in one of many ways in order to produce a spectrum of
related-products.
[0060] In one example, the polynucleotide encoding an antibody
having a T-cell receptor specificity is ligated with a second
polynucleotide encoding an identifiable moiety, so as to produce an
antibody having a T-cell receptor specificity conjugated to the
identifiable moiety, an immunolabel. Such a conjugate or
immunolabel can be used in a method of detecting the presence
and/or level of antigen presenting cells presenting a
HLA-restricted antigen in a sample of cells and serve for diagnosis
of cancer, viral infection or autoimmune disease. As used herein,
the phrase "antigen presenting cell" includes all cells expressing
MHC, class I molecules on their surface, and which are capable of
presenting HLA-restricted antigens. An antigen presenting cell can
be a cancer cell, a cell of the immune system, or any other cell
expressing MHC, class I molecules on its surface.
[0061] Hence, according to another aspect of the present invention
there is provided a method of making an immunolabel, the method
comprising ligating a first polynucleotide encoding an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a HLA-restricted antigen in frame with a second
polynucleotide encoding an identifiable moiety, so as to obtain a
ligated polynucleotide and expressing the ligated polynucleotide in
an expression system so as to obtain the immunolabel.
[0062] And, according to yet another aspect of the present
invention there is provided a method of detecting the presence
and/or level of antigen presenting cells presenting a
HLA-restricted antigen in a sample of cells, the method comprising
interacting cells of said sample with an antibody specifically
bindable with a binding affinity below 20 nanomolar to a human
major histocompatibility complex (MHC) class I being complexed with
a HLA-restricted antigen; and monitoring said interaction, thereby
detecting the presence and/or level of said antigen presenting
cells presenting said HLA-restricted antigen.
[0063] Depending on the application, the HLA-restricted antigen can
be a tumor HLA-restricted antigen, a viral HLA-restricted antigen
and an autoimmune HLA-restricted antigen, examples of which are
provided hereinbelow.
[0064] According to yet another aspect of the present invention
there is provided a diagnostic composition comprising a molecule
which comprises an antibody specifically bindable with a binding
affinity below 20 nanomolar to a human major histocompatibility
complex (MHC) class I being complexed with a HLA-restricted
antigen, the molecule further comprises an identifiable moiety
being conjugated to the antibody.
[0065] The identifiable moiety can be a member of a binding pair,
which is identifiable via its interaction with an additional member
of the binding pair, and a label which is directly visualized. In
one example, the member of the binding pair is an antigen which is
identified by a corresponding labeled antibody. In one example, the
label is a fluorescent protein or an enzyme producing a
colorimetric reaction.
[0066] In another example, the polynucleotide encoding an antibody
having a T-cell receptor specificity is ligated with a second
polynucleotide encoding a therapeutic moiety, so as to produce an
antibody having a T-cell receptor specificity conjugated to the
therapeutic moiety. Such a conjugate or immunotoxin can be used in
a method of treating cancer, viral infection or autoimmune
disease.
[0067] Hence, according to another aspect of the present invention
there is provided a method of making an immunotoxin, the method
comprising ligating a first polynucleotide encoding an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a HLA-restricted antigen in frame with a second
polynucleotide encoding a toxin moiety, so as to obtain a ligated
polynucleotide and expressing said ligated polynucleotide in an
expression system so as to obtain said immunotoxin.
[0068] The immunotoxin can be used in any one of the following
therapeutic protocols:
[0069] (i) A method of treating a cancer, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a molecule which comprises an antibody
specifically bindable with a binding affinity below 20 nanomolar to
a human major histocompatibility complex (MHC) class I being
complexed with a tumor HLA-restricted antigen characterizing the
cancer, the molecule further comprises a therapeutic moiety being
conjugated to the antibody, the MHC class I molecule being selected
matching to the endogenous MHC class I of the subject.
[0070] (ii) A method of treating a viral infection, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a viral HLA-restricted antigen characterizing
a virus causative of the viral infection, the molecule further
comprises a therapeutic moiety being conjugated to the antibody,
the MHC class I molecule being selected matching to the endogenous
MHC class I of the subject.
[0071] (iii) A method of treating an autoimmune disease, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with an autoimmune HLA-restricted antigen, said
molecule further comprises a therapeutic moiety being conjugated to
said antibody, said MHC class I molecule being selected matching to
the endogenous MHC class I of the subject.
[0072] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising a
therapeutically effective amount of a molecule which comprises an
antibody specifically bindable with a binding affinity below 20
nanomolar to a human major histocompatibility complex (MHC) class I
being complexed with a HLA-restricted antigen, the molecule further
comprises a therapeutic moiety being conjugated to the antibody.
Preferably, the pharmaceutical composition further comprising a
pharmaceutically acceptable carrier.
[0073] The therapeutic moiety can be, for example, a cytotoxic
moiety, a toxic moiety, a cytokine moiety and a bi-specific
antibody moiety, examples of which are provided hereinbelow.
[0074] In all applications the MHC class I can be, for example,
HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33,
HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8.
[0075] The following sections provide specific examples and
alternatives for each of the various aspects of the invention
described herein. These examples and alternatives should not be
regarded as limiting in any way, as the invention can be practiced
in similar, yet somewhat different ways. These examples, however,
teach one of ordinary skills in the art how to practice various
alternatives and embodiments of the invention.
[0076] Antibody:
[0077] The term "antibody" as used to describe this invention
includes intact molecules as well as functional fragments thereof,
such as Fab, F(ab').sub.2, Fv and scFv that are capable of
specific, high affinity binding to a human major histocompatibility
complex (MHC) class I complexed with a HLA-restricted antigen.
These functional antibody fragments are defined as follows: (i)
Fab, the fragment which contains a monovalent antigen-binding
fragment of an antibody molecule, can be produced by digestion of
whole antibody with the enzyme papain to yield an intact light
chain and a portion of one heavy chain; (ii) Fab', the fragment of
an antibody molecule that can be obtained by treating whole
antibody with pepsin, followed by reduction, to yield an intact
light chain and a portion of the heavy chain; two Fab' fragments
are obtained per antibody molecule; (iii) F(ab').sub.2, the
fragment of the antibody that can be obtained by treating whole
antibody with the enzyme pepsin without subsequent reduction;
F(ab').sub.2 is a dimer of two Fab' fragments held together by two
disulfide bonds; (iv) Fv, defined as a genetically engineered
fragment containing the variable region of the light chain and the
variable region of the heavy chain expressed as two chains; and (c)
scFv or "single chain antibody" ("SCA"), a genetically engineered
molecule containing the variable region of the light chain and the
variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain
molecule.
[0078] Methods of making these fragments are known in the art. (See
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0079] Antibody fragments according to the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli or mammalian cells (e.g. Chinese hamster ovary cell
culture or other protein expression systems) of DNA encoding the
fragment.
[0080] Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references
contained therein, which patents are hereby incorporated by
reference in their entirety. See also Porter, R. R., Biochem. J.,
73: 119-126, 1959. Other methods of cleaving antibodies, such as
separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0081] Fv fragments comprise an association of V.sub.H and V.sub.L
chains. This association may be noncovalent, as described in Inbar
et al., Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively,
the variable chains can be linked by an intermolecular disulfide
bond or cross-linked by chemicals such as glutaraldehyde.
Preferably, the Fv fragments comprise V.sub.H and V.sub.L chains
connected by a peptide linker. These single-chain antigen binding
proteins (sFv) are prepared by constructing a structural gene
comprising DNA sequences encoding the V.sub.H and V.sub.L domains
connected by an oligonucleotide. The structural gene is inserted
into an expression vector, which is subsequently introduced into a
host cell such as E. coli. The recombinant host cells synthesize a
single polypeptide chain with a linker peptide bridging the two V
domains. Methods for producing sFvs are described, for example, by
Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science
242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993;
and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby
incorporated by reference in its entirety.
[0082] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry, Methods, 2: 106-10,
1991.
[0083] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence.
[0084] The humanized antibody optimally also will comprise at least
a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin [Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta,
Curr. Op. Struct. Biol., 2:593-596 (1992)].
[0085] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0086] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016; and in the following scientific publications:
Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al.,
Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994);
Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger,
Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern.
Rev. Immunol. 13 65-93 (1995).
[0087] It will be appreciated that once the CDRs of an antibody are
identified, using conventional genetic engineering techniques can
be used to devise expressible polynucleotides encoding any of the
forms or fragments of antibodies described herein.
[0088] A Human Major Histocompatibility Complex (MHC) Class I:
[0089] The major histocompatibility complex (MHC) is a complex of
antigens encoded by a group of linked loci, which are collectively
termed H-2 in the mouse and HLA in humans. The two principal
classes of the MHC antigens, class I and class II, each comprise a
set of cell surface glycoproteins which play a role in determining
tissue type and transplant compatibility. In transplantation
reactions, cytotoxic T-cells (CTLs) respond mainly against foreign
class I glycoproteins, while helper T-cells respond mainly against
foreign class II glycoproteins.
[0090] Major histocompatibility complex (MHC) class I molecules are
expressed on the surface of nearly all cells. These molecules
function in presenting peptides which are mainly derived from
endogenously synthesized proteins to CD8+ T cells via an
interaction with the .alpha..beta. T-cell receptor. The class I MHC
molecule is a heterodimer composed of a 46-kDa heavy chain which is
non-covalently associated with the 12-kDa light chain .beta.-2
microglobulin. In humans, there are several MHC haplotypes, such
as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31,
HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, their sequences can
be found at the kabbat data base, at http://immuno.bme.nwu.edu/,
which is incorporated herein by reference.
[0091] Peptides that Bind to Class I MHC Molecules; HLA-Restricted
Antigens:
[0092] Class I, MHC-restricted peptides (also referred to herein
interchangeably as HLA-restricted antigens, HLA-restricted
peptides, MHC-restricted antigens) which are typically 8-10-amino
acid-long, bind to the heavy chain .alpha.1-.alpha.2 groove via two
or three anchor residues that interact with corresponding binding
pockets in the MHC molecule. The .beta.-2 microglobulin chain plays
an important role in MHC class I intracellular transport, peptide
binding, and conformational stability. For most class I molecules,
the formation of a heterodimer consisting of the MHC class I heavy
chain, peptide (self or antigenic) and .beta.-2 microglobulin is
required for biosynthetic maturation and cell-surface
expression.
[0093] Research studies performed on peptide binding to class I MHC
molecules enable to define specific MHC motifs functional in
displaying peptides derived from viral, tumor and self antigens
that are potentially immunogenic and might elicit specific response
from cytotoxic T lymphocytes (CTLs).
[0094] As used herein the term "peptide" refers to native peptides
(either degradation products or synthetically synthesized peptides)
and further to peptidomimetics, such as peptoids and semipeptoids
which are peptide analogs, which may have, for example,
modifications rendering the peptides more stable while in a body,
or more immunogenic. Such modifications include, but are not
limited to, cyclization, N terminus modification, C terminus
modification, peptide bond modification, including, but not limited
to, CH.sub.2--NH, CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH,
CH.sub.2--O, CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH or
CF.dbd.CH, backbone modification and residue modification. Methods
for preparing peptidomimetic compounds are well known in the art
and are specified in Quantitative Drug Design, C. A. Ramsden Gd.,
Chapter 17.2, F. Choplin Pergamon Press (1992), which is
incorporated by reference as if fully set forth herein. Further
detail in this respect are provided hereinunder.
[0095] As used herein in the specification and in the claims
section below the term "amino acid" is understood to include the 20
naturally occurring amino acids; those amino acids often modified
post-translationally in vivo, including for example hydroxyproline,
phosphoserine and phosphothreonine; and other unusual amino acids
including, but not limited to, 2-aminoadipic acid, hydroxylysine,
isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore,
the term "amino acid" includes both D- and L-amino acids. Further
elaboration of the possible amino acids usable according to the
present invention and examples of non-natural amino acids useful in
MHC-I HLA-A2 recognizable peptide antigens are given
hereinunder.
[0096] Based on accumulated experimental data, it is nowadays
possible to predict which of the peptides of a protein will bind to
MHC, class I. The HLA-A2 MHC class I has been so far characterized
better than other HLA haplotypes, yet predictive and/or sporadic
data is available for all other haplotypes.
[0097] With respect to HLA-A2 binding peptides, assume the
following positions (P1-P9) in a 9-mer peptide:
[0098] P1-P2-P3-P4-P5-P6-P7-P8-P9
[0099] The P2 and P2 positions include the anchor residues which
are the main residues participating in binding to MHC molecules.
Amino acid resides engaging positions P2 and P9 are hydrophilic
aliphatic non-charged natural amino (examples being Ala, Val, Leu,
Ile, Gln, Thr, Ser, Cys, preferably Val and Leu) or of a
non-natural hydrophilic aliphatic non-charged amino acid (examples
being norleucine (Nle), norvaline (Nva), .alpha.-aminobutyric
acid). Positions P1 and P3 are also known to include amino acid
residues which participate or assist in binding to MHC molecules,
however, these positions can include any amino acids, natural or
non-natural. The other positions are engaged by amino acid residues
which typically do not participate in binding, rather these amino
acids are presented to the immune cells. Further details relating
to the binding of peptides to MHC molecules can be found in Parker,
K. C., Bednarek, M. A., Coligan, J. E., Scheme for ranking
potential HLA-A2 binding peptides based on independent binding of
individual peptide side-chains. J Immunol.152, 163-175, 1994., see
Table V, in particular. Hence, scoring of HLA-A2.1 binding peptides
can be performed using the HLA Peptide Binding Predictions software
approachable through a worldwide web interface at
http://www.bimas.dcrt.nih.gov/molbio/hla_bind/index.html- . This
software is based on accumulated data and scores every possible
peptide in an analyzed protein for possible binding to MHC HLA-A2.1
according to the contribution of every amino acid in the peptide.
Theoretical binding scores represent calculated half-life of the
HLA-A2.1-peptide complex.
[0100] Hydrophilic aliphatic natural amino acids at P2 and P9 can
be substituted by synthetic amino acids, preferably Nleu, Nval
and/or .alpha.-aminobutyric acid. P9 can be also substituted by
aliphatic amino acids of the general formula
--HN(CH.sub.2).sub.nCOOH, wherein n=3-5, as well as by branched
derivatives thereof, such as, but not limited to, 1
[0101] wherein R is, for example, methyl, ethyl or propyl, located
at any one or more of the n carbons. The amino terminal residue
(position P1) can be substituted by positively charged aliphatic
carboxylic acids, such as, but not limited to,
H.sub.2N(CH.sub.2).sub.nCOOH, wherein n=2-4 and
H.sub.2N--C(NH)--NH(CH.sub.2).sub.nCOOH, wherein n=2-3, as well as
by hydroxy Lysine, N-methyl Lysine or ornithine (Orn).
Additionally, the amino terminal residue can be substituted by
enlarged aromatic residues, such as, but not limited to,
H.sub.2N--(C.sub.6H.sub.6)--CH.sub.2--COOH, p-aminophenyl alanine,
H.sub.2N--F(NH)--NH--(C.sub.6H.sub.6)--CH.sub.2--C- OOH,
p-guanidinophenyl alanine or pyridinoalanine (Pal). These latter
residues may form hydrogen bonding with the OH.sup.- moieties of
the Tyrosine residues at the MHC-1 N-terminal binding pocket, as
well as to create, at the same time aromatic-aromatic
interactions.
[0102] Derivatization of amino acid residues at positions P4-P8,
should these residues have a side-chain, such as, OH, SH or
NH.sub.2, like Ser, Tyr, Lys, Cys or Orn, can be by alkyl, aryl,
alkanoyl or aroyl. In addition, OH groups at these positions may
also be derivatized by phosphorylation and/or glycosylation. These
derivatizations have been shown in some cases to enhance the
binding to the T cell receptor.
[0103] Longer derivatives in which the second anchor amino acid is
at position P10 may include at P9 most L amino acids. In some cases
shorter derivatives are also applicable, in which the C terminal
acid serves as the second anchor residue.
[0104] Cyclic amino acid derivatives can engage position P4-P8,
preferably positions P6 and P7. Cyclization can be obtained through
amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn,
di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at
various positions in the chain (--CO--NH or --NH--CO bonds).
Backbone to backbone cyclization can also be obtained through
incorporation of modified amino acids of the formulas
H--N((CH.sub.2)n--COOH)--C(R)H--COOH or H--N((CH.sub.2).sub.n--C-
OOH)--C(R)H--NH.sub.2, wherein n=1-4, and further wherein R is any
natural or non-natural side chain of an amino acid.
[0105] Cyclization via formation of S--S bonds through
incorporation of two Cys residues is also possible. Additional
side-chain to side chain cyclization can be obtained via formation
of an interaction bond of the formula
--(--CH.sub.2--).sub.n--S--CH.sub.2--C--, wherein n=1 or 2, which
is possible, for example, through incorporation of Cys or homoCys
and reaction of its free SH group with, e.g., bromoacetylated Lys,
Orn, Dab or Dap.
[0106] Peptide bonds (--CO--NH--) within the peptide may be
substituted by N-methylated bonds (--N(CH.sub.3)--CO--), ester
bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH.sub.2--), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R
is any alkyl, e.g., methyl, carba bonds (--CH.sub.2--NH--),
hydroxyethylene bonds (--CH(OH)--CH.sub.2--), thioamide bonds
(--CS--NH--), olefinic double bonds (--CH.dbd.CH--), retro amide
bonds (--NH--CO--), peptide derivatives (--N(R)--CH.sub.2--CO--),
wherein R is the "normal" side chain, naturally presented on the
carbon atom.
[0107] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
Preferably, but not in all cases necessary, these modifications
should exclude anchor amino acids.
[0108] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as TIC,
naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0109] Tumor HLA-Restricted Antigens:
[0110] The references recited in the following Table provide
examples of human MHC class I, tumor HLA-restricted peptides
derived from tumor associated antigens (TAA) or protein markers
associated with various cancers. Additional tumor HLA-restricted
peptides derived from tumor associated antigens (TAA) can be found
in http://www.bmi-heidelberg.com/s- yfpeithi/
1 Cancer TAA/Marker HLA Reference Transitional cell Uroplakin II
HLA-A2 WO 00/06723 carcinoma Transitional cell Uroplakin Ia HLA-A2
WO 00/06723 carcinoma Carcinoma of the prostate specific HLA-A2 WO
00/06723 prostate antigen Carcinoma of the prostate specific HLA-A2
WO 00/06723 prostate membrane antigen Carcinoma of the prostate
acid HLA-A2 WO 00/06723 prostate phosphatase Breast cancer BA-46
HLA-A2 WO 00/06723 Breast cancer Muc-1 HLA-A2 WO 00/06723 Melanoma
Gp100 HLA-A2 Reference 54 Melanoma MART1 HLA-A2 Reference 54 All
tumors Telomerase HLA-A2 Reference 54 Leukemia TAX HLA-A2 Reference
54 Carcinomas NY-ESO HLA-A2 Reference 54 Melanoma MAGE-A1 HLA-A2
Reference 54 Melanoma MAGE-A3 HLA-A24 Reference 54 Carcinomas HER2
HLA-A2 Reference 54 Melanoma Beta-catenine HLA-A24 Reference 54
Melanoma Tyrosinase HLA-DRB1 Reference 54 Leukemia Bcr-abl HLA-A2
Reference 54 Head and neck Caspase 8 HLA-B35 Reference 54
[0111] Viral HLA-Restricted Antigens:
[0112] The references recited in the following Table provide
examples of human MHC class I, viral HLA-restricted peptides
derived from viral antigens associated with various cancers.
2 Disease Viral antigen HLA Reference AIDS (HTLV-1) HIV-1 RT
476-484 HLA-A2 http://www.bmi- heidelberg.com/syfpeithi/ Influenza
G I L G F V F T L HLA-A2 http://www.bmi- (SEQ ID NO: 10)
heidelberg.com/syfpeithi/ CMV disease CMV HLA-A2 http://www.bmi-
heidelberg.com/syfpeithi- / Burkitts Lymphoma TAX HLA-A2
http://www.bmi- heidelberg.com/syfpeithi/ Hepatitis C HCV HLA-A2
http://www.bmi- heidelberg.com/syfpeithi/ Hepatitis B HBV pre-S
protein 85-66 HLA-A2 http://www.bmi- S T N R Q S
heidelberg.com/syfpeith- i/ G R Q (SEQ ID NO: 11) HTLV-1 Leukemia
HTLV-1 tax 11-19 HLA-A2 http://www.bmi- heidelberg.com/syfpeithi/
Hepatitis HBV surface HLA-A2 http://www.bmi- antigen 185-194
heidelberg.com/syfpeithi/
[0113] Autoimmune HLA-Restricted Antigens:
[0114] The website http://www.bmi-heidelberg.com/syfpeithi/
provides examples of human MHC class I, autoimmune HLA-restricted
peptides derived from autoimmune antigens.
[0115] Soluble MHC Class I Molecules:
[0116] Recombinant MHC class I and class II complexes which are
soluble and which can be produced in large quantities are described
in, for example, references 23, 24 and 41-53 and further in U.S.
patent application Ser. No. 09/534,966 and PCT/IL01/00260
(published as WO 01/72768), all of which are incorporated herein by
reference. Soluble MHC class I molecules are available or can be
produced for any of the MHC haplotypes, such as, for example,
HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33,
HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, following, for example the
teachings of PCT/IL01/00260, as their sequences are known and can
be found at the kabbat data base, at http://immuno.bme.nwu.edu/,
the contents of the site is incorporated herein by reference. Such
soluble MHC class I molecules can be loaded with suitable
HLA-restricted antigens and used for vaccination of Non-human
mammal having cells expressing the human major histocompatibility
complex (MHC) class I as is further detailed hereinbelow.
[0117] Non-Human Mammal having Cells Expressing the Human Major
Histocompatibility Complex (MHC) Class I:
[0118] Non-human mammal having cells expressing a human major
histocompatibility complex (MHC) class I and devoid of self major
histocompatibility complex (MHC) class I can be produced using (i)
the sequence information provided in the kabbat data base, at
http://immuno.bme.nwu.edu/, which is incorporated herein by
reference and pertaining to human MHC haplotypes, such as, for
example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31,
HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, (ii) conventional
constructs preparation techniques, as described in, for example,
"Molecular Cloning: A laboratory Manual" Sambrook et al., (1989);
"Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.
M., ed. (1994); Ausubel et al., "Current Protocols in Molecular
Biology", John Wiley and Sons, Baltimore, Md. (1989); Perbal, "A
Practical Guide to Molecular Cloning", John Wiley & Sons, New
York (1988); Watson et al., "Recombinant DNA", Scientific American
Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory
Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New
York (1998); and (iii) conventional gene knock-in/knock-out
techniques as set forth, for example, in U.S. Pat. Nos. 5,487,992,
5,464,764, 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846,
5,221,778, 5,175,385, 5,175,384, 5,175,383, 4,736,866; in
International Publications WO 94/23049, WO93/14200, WO 94/06908 and
WO 94/28123; as well as in Burke and Olson, Methods in Enzymology,
194:251-270, 1991; Capecchi, Science 244:1288-1292, 1989; Davies et
al., Nucleic Acids Research, 20 (11) 2693-2698, 1992; Dickinson et
al., Human Molecular Genetics, 2(8): 1299-1302, 1993; Duff and
Lincoln, "Insertion of a pathogenic mutation into a yeast
artificial chromosome containing the human APP gene and expression
in ES cells", Research Advances in Alzheimer's Disease and Related
Disorders, 1995; Huxley et al., Genomics, 9:742-750 1991;
Jakobovits et al., Nature, 362:255-261, 1993; Lamb et al., Nature
Genetics, 5: 22-29, 1993; Pearson and Choi, Proc. Natl. Acad. Sci.
USA, 1993. 90:10578-82; Rothstein, Methods in Enzymology,
194:281-301, 1991; Schedl et al., Nature, 362: 258-261, 1993;
Strauss et al., Science, 259:1904-1907, 1993, all of which are
incorporated herein by reference.
[0119] Of particular interest is the paper by Pascolo et al.,
published in J. Exp. Med. 185: 2043-2051, 1997, which describe the
preparation of mice expressing the human HLA-A2.1, H-2Db and HHD
MHC class I molecules and devoid of mice MHC class I
altogether.
[0120] Identifiable Moieties:
[0121] In some aspects thereof, the present invention employ
conjugates of an antibody and an identifiable moiety. To this end,
in one example, first and second polynucleotides encoding the
antibody and the identifiable moiety, respectively, are ligated in
frame, so as to encode an immunolabel. The following table provide
examples of sequences of identifiable moieties.
3 Amino Acid sequence Nucleic Acid sequence (Genebank (Genebank
Identifiable Moiety Accession No.) Accession No.) Green Fluorescent
protein AAL33912 AF435427 Alkaline phosphatase AAK73766 AY042185
Peroxidase NP_568674 NM_124071 Histidine tag AAK09208 AF329457 Myc
tag AF329457 AF329457 Biotin lygase tag NP_561589 NC_003366 orange
fluorescent protein AAL33917 AF435432 Beta galactosidase NM_125776
NM_125776 Fluorescein isothiocyanate AAF22695 AF098239 Streptavidin
S11540 S11540
[0122] Therapeutic Moieties:
[0123] In some aspects thereof, the present invention employ
conjugates of an antibody and a therapeutic moiety. To this end, in
one example, first and second polynucleotides encoding the antibody
and the therapeutic moiety, respectively, are ligated in frame, so
as to encode an immunotoxin. The following table provide examples
of sequences of therapeutic moieties.
4 AminoAcid sequence Nucleic Acid sequence (Genebank (Genebank
Therapeutic Moiety Accession No.) Accession No.) Pseudomonas
exotoxin AAB25018 S53109 Diphtheria toxin E00489 E00489 interleukin
2 CAA00227 A02159 CD3 P07766 X03884 CD16 AAK54251 AF372455
interleukin 4 P20096 ICRT4 HLA-A2 P01892 K02883 interleukin 10
P22301 M57627 Ricin A toxin 225988 A23903
[0124] Chemical Conjugates:
[0125] Many methods are known in the art to conjugate or fuse
(couple) molecules of different types, including peptides. These
methods can be used according to the present invention to couple an
antibody another moiety, such as a therapeutic moiety or an
identifiable moiety, to thereby provide an immunotoxin or
immunolabel.
[0126] Two isolated peptides can be conjugated or fused using any
conjugation method known to one skilled in the art. A peptide can
be conjugated to an antibody of interest, using a
3-(2-pyridyldithio)propion- ic acid Nhydroxysuccinimide ester (also
called N-succinimidyl 3-(2pyridyldithio) propionate) ("SDPD")
(Sigma, Cat. No. P-3415), a glutaraldehyde conjugation procedure or
a carbodiimide conjugation procedure.
[0127] SPDP Conjugation:
[0128] Any SPDP conjugation method known to those skilled in the
art can be used. For example, in one illustrative embodiment, a
modification of the method of Cumber et al. (1985, Methods of
Enzymology 112: 207-224) as described below, is used.
[0129] A peptide, such as an identifiable or therapeutic moiety,
(1.7 mg/ml) is mixed with a 10-fold excess of SPDP (50 mM in
ethanol) and the antibody is mixed with a 25-fold excess of SPDP in
20 mM sodium phosphate, 0.10 M NaCl pH 7.2 and each of the
reactions incubated, e.g., for 3 hours at room temperature. The
reactions are then dialyzed against PBS.
[0130] The peptide is reduced, e.g., with 50 mM DTT for 1 hour at
room temperature. The reduced peptide is desalted by equilibration
on G-25 column (up to 5% sample/column volume) with 50 mM
KH.sub.2PO.sub.4 pH 6.5. The reduced peptide is combined with the
SPDP-antibody in a molar ratio of 1:10 antibody:peptide and
incubated at 4.degree. C. overnight to form a peptide-antibody
conjugate.
[0131] Glutaraldehyde Conjugation:
[0132] Conjugation of a peptide (e.g., an identifiable or
therapeutic moiety) with an antibody can be accomplished by methods
known to those skilled in the art using glutaraldehyde. For
example, in one illustrative embodiment, the method of conjugation
by G. T. Hermanson (1996, "Antibody Modification and Conjugation,
in Bioconjugate Techniques, Academic Press, San Diego) described
below, is used.
[0133] The antibody and the peptide (1.1 mg/ml) are mixed at a
10-fold excess with 0.05% glutaraldehyde in 0.1 M phosphate, 0.15 M
NaCl pH 6.8, and allowed to react for 2 hours at room temperature.
0.01 M lysine can be added to block excess sites. After-the
reaction, the excess glutaraldehyde is removed using a G-25 column
equilibrated with PBS (10% v/v sample/column volumes)
[0134] Carbodiimide Conjugation:
[0135] Conjugation of a peptide with an antibody can be
accomplished by methods known to those skilled in the art using a
dehydrating agent such as a carbodiimide. Most preferably the
carbodiimide is used in the presence of 4-dimethyl aminopyridine.
As is well known to those skilled in the art, carbodiimide
conjugation can be used to form a covalent bond between a carboxyl
group of peptide and an hydroxyl group of an antibody (resulting in
the formation of an ester bond), or an amino group of an antibody
(resulting in the formation of an amide bond) or a sulfhydryl group
of an antibody (resulting in the formation of a thioester
bond).
[0136] Likewise, carbodiimide coupling can be used to form
analogous covalent bonds between a carbon group of an antibody and
an hydroxyl, amino or sulfhydryl group of the peptide. See,
generally, J. March, Advanced Organic Chemistry: Reaction's,
Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985.
By means of illustration, and not limitation, the peptide is
conjugated to an antibody via a covalent bond using a carbodiimide,
such as dicyclohexylcarbodiimide. See generally, the methods of
conjugation by B. Neises et al. (1978, Angew Chem., Int. Ed. Engl.
17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); E. P.
Boden et al. (1986, J. Org. Chem. 50:2394) and L. J. Mathias (1979,
Synthesis 561).
[0137] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0138] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0139] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Materials and Experimental Methods
[0140] Production of Biotinylated scMHC/Peptide Complexes:
[0141] Single-chain MHC/peptide complexes were produced by in vitro
refolding of inclusion bodies produced in E. coli, as described
previously (23, 24, U.S. patent application Ser. No. 09/534,966 and
PCT/IL01/00260 (published as WO 01/72768). Biotinylation was
performed using the BirA enzyme (Avidity, Denver, Colo.) as
previously described (25).
[0142] Mice Immunization:
[0143] D.sup.b-/.sup.- X .beta.2 microglobulin (.beta.2m) null
mice, transgenic for a recombinant HLA-A2.1/D.sup.b-.beta.2
microglobulin single chain (HHD mice) (26) were immunize with an
emulsion containing purified protein-derived peptide of tuberculin
(PPD) covalently coupled with HLA-A2/G9-209 complexes, as described
previously (17). Briefly, mice were initially immunized subdermally
and subsequently subcutaneously for two-week intervals for a period
of 3-5 months with 20-30 .mu.g/mice of the antigenic mixture in
incomplete Freund's adjuvant. Spleens were collected two weeks
after the last immunization.
[0144] Library Construction and Selection of Phage-Antibodies on
Biotinylated Complexes:
[0145] Total RNA was isolated from immunized mice and an antibody
scFv library was constructed by room temperature-PCR from the mRNA
as described (27). The scFv repertoire was cloned as an SfiI-NotI
fragment into the pCANTAB5E or pCC-CBD phagemid vectors (28). The
complexity of both libraries was 1.times.10.sup.8 independent
clones. For panning, biotinylated scHLA-A2/G9-209M complexes (20
.mu.g) were incubated with streptavidin-coated magnetic beads
(2.times.10.sup.8), washed, and incubated with 10.sup.11 cfu of the
libraries (1 hour at room temperature). Starting with the 2.sup.nd
round, panning was performed in the presence of an excess (5 .mu.g)
of scHLA-A2/G9-280V complexes. Beads were washed extensively 10-12
times with 2% MPBS+0.1% TWEEN20. Bound phages were eluted by using
1 ml of Triethylamine (100 mM, pH 12) for 5 minutes at room
temperature followed by neutralization with 0.1 ml of 1 M Tris-HCl,
pH 7.4. Eluted phages were expanded in exponentially growing E.
coli TG1 cells that were later superinfected with M13KO7 helper
phage as described (28).
[0146] Expression and Purification of Soluble Recombinant scFv and
scFv-P38 Fusion Protein:
[0147] The G1 scFv gene was rescued from the phage clone by PCR and
was subcloned into the phagemid vector pCANTAB6 by using the
SfiI-NotI cloning sites. A Myc and hexahistidine tags were fused to
the C-terminus of the scFv gene. The scFv antibody was expressed in
BL21 .lambda.DE3 cells as previously described (29) and purified
from the periplasmic fraction by metal-ion affinity chromatography.
For the expression of the G1scFv-PE38 fusion protein, the scFv gene
was subcloned as an NcoI-NotI fragment into the plasmid pIB-NN,
which encodes the translocation and ADP-ribosylation domains of PE
(PE38). Expression in BL21 .lambda.DE3 cells, refolding from
inclusion bodies, and purification of G1scFv-PE38 was performed as
previously described (30).
[0148] ELISA with Phage Clones and Purified scFv or scFv-PE38:
[0149] Binding specificity studies were performed by ELISA using
biotinylated scMHC-peptide complexes. Briefly, ELISA plates
(Falcon) were coated overnight with BSA-biotin (1 .mu.g/well),
washed, incubated (1 hour, room temperature) with streptavidin (1
.mu.g/well), again washed extensively and further incubated (1
hour, room temperature) with 0.5 .mu.g of MHC/peptide complexes.
Plates were blocked with PBS/2% milk (30 minutes, room
temperature), incubated with phage clones (about 10.sup.9
phages/well, 1 hour, room temperature) or 0.5-1 .mu.g of soluble
scFv or scFv-PE38, and afterwards washed with 1:1000
HRP-conjugated/anti-M13, anti-myc antibody or anti-PE antibody,
respectively. The HLA-A2-restricted peptides used for specificity
studies are gp100 (154): KTWGQYWQV (SEQ ID NO:1); gp100 (209):
IMDQVPFSV (SEQ ID NO:2); gp100 (280): YLEPGPVTV (SEQ ID NO:3);
MUC1: LLLTVLTVL (SEQ ID NO:4); HTLV-1 (TAX): LLFGYPVYV (SEQ ID
NO:5); hTEroom temperature (540): ILAKFLHWL (SEQ ID NO:6); hTEroom
temperature (865): RLVDDFLLV (SEQ ID NO:7).
[0150] Flow Cytometry:
[0151] The B cell line RMAS-HHD transfected with a single-chain
.beta.2M-HLA-A2 gene (26) or the EBV-transformed B-lymphoblast JY
cells (10.sup.6 cells) were washed twice with serum-free RPMI and
incubated overnight at 26.degree. C. or 37.degree. C.,
respectively, in medium containing 100 .mu.M of the peptide. The
APCs were subsequently incubated at 37.degree. C. for 2-3 hours to
stabilize cell surface expression of MHC-peptide complexes,
followed by incubation with recombinant scFv (10-50 .mu.g/ml, 60-90
minutes, 4.degree. C.) in 100 .mu.l. The cells were then washed,
incubated with FITC-labeled anti-Myc antibody (30-45 minutes,
4.degree. C.), and finally washed and analyzed by a FACStar flow
cytometer (Becton Dickinson).
[0152] Competition Binding Assays:
[0153] Flexible ELISA plates were coated with BSA-biotin and
scMHC-peptide complexes (10 .mu.g in 100 .mu.l) were immobilized
thereto. The recombinant G1scFv-PE38 was labeled with [.sup.125I]
using the Bolton-Hunter reagent. Labeled protein was added to wells
as a tracer (3-5.times.10.sup.5 CPM/well) in the presence of
increasing concentrations of the cold G1scFv-PE38 as a competitor
and incubated at room temperature for 1 hour in PBS. The plates
were washed thoroughly with PBS and the bound radioactivity was
determined by a gamma counter. The apparent affinity of the
G1scFv-PE38 was determined by extrapolating the concentration of a
competitor necessary to achieve 50% inhibition of
[.sup.125I]-labeled G1scFv-PE38 binding to the immobilized
scMHC-peptide complex. Non-specific binding was determined by
adding a 20-40-fold excess of unlabeled Fab.
[0154] Cytotoxicity Assays:
[0155] RMAS-HHD and JY APCs were loaded with the G9-209 peptide and
control peptides as previously described. Peptide-loaded cells were
subsequently incubated with increasing concentrations of
G1scFv-PE38 and the inhibition of protein synthesis was determined
by measuring the uptake of .sup.3H-Leucine into cellular proteins,
as previously described (30). IC.sub.50 was determined as the
concentration of G1scFv-PE38 required to inhibit protein synthesis
by 50%. In competition assays, excesses of specific and
non-specific HLA-A2-peptide complexes (35-50 .mu.g/well) were added
to wells 15 minutes before the addition of G1scFv-PE38.
Experimental Results
[0156] Generation of Recombinant Single-Chain MHC-Peptide Complexes
with the Melanoma gp100-Derived Peptide G9-209M:
[0157] Gp100 is a melanocyte lineage-specific membrane glycoprotein
consisting of 661 amino acids that is expressed in most melanoma
cells (19-22). This protein is recognized by many
HLA-A2-restricted, melanoma-reactive, tumor
infiltrating-lymphocytes (TILs) that have been isolated from
melanoma patients (19-22). Several T cell HLA-A2-restricted
epitopes have been identified in gp100; they have been improved in
MHC anchor positions for enhanced immunogenicity without altering
T-cell specificity (31). The G9-209M (IMDQVPFSV, SEQ ID NO:2)
peptide is one of a three major immunogenic epitopes (19-22).
Recombinant MHC-peptide complexes that present the G9-209M peptide
were generated by using a single-chain MHC (scMHC) construct
expressed in E. coli that has been described previously (23, 24,
U.S. patent application Ser. No. 09/534,966 and PCT/IL01/00260
(published as WO 01/72768). The scMHC-peptide complexes are
produced by in vitro refolding of inclusion bodies in the presence
of the G9-209M or other HLA-A2-restricted peptides, followed by a
purification protocol employing ion-exchange chromatography. The
refolded gp100-derived and control scHLA-A2-peptide complexes were
very pure, homogenous, and monomeric, as determined by analysis on
SDS-PAGE and gel filtration chromatography. The G9-209M-containing
scHLA-A2 complexes have been previously shown to be functional, by
their ability to stimulate specific CTL lines and clones and stain
G9-209M-specific T cells in the form of tetramers (23, 24).
[0158] Construction of an Antibody scFv Phage Library and Selection
of a Phage that Binds HLA-A2/G9-209M Complexes with TCR-Like
Specificity:
[0159] For immunization purposes PPD was coupled to the purified
complex and the D.sup.b-/.sup.- X .beta.2 microglobulin (.beta.2m)
null mice transgenic for a recombinant HLA-A2.1/D.sup.b-.beta.2
microglobulin single chain (HHD mice) (26) was immunized therewith.
These mice combine classical HLA transgenesis with selective
destruction of murine class I H-2. Hence, unlike the classical HLA
transgenics, these mice showed only HLA-A2.1-restricted responses
with muli-epitope proteins such as intact viruses. Moreover, it is
presume that these mice are a useful tool for immunization with
HLA-A2-peptide complexes because they should be largely tolerant to
HLA-A2 as a B-cell immunogen and thus may favor the generation of
an antibody response directed against the MHC-restricted epitope
when in complex with HLA-A2 (the specific tumor-associated
peptide). PPD was used for conjugation because it is a highly
reactive T cell immunogen (17).
[0160] Total spleen mRNA was isolated from immunized mice and
reverse transcribed to cDNA. Specific sets of degenerated primers
were used to PCR-amplify the cDNA segments corresponding to the
immunoglobulin heavy and light chain variable domains (27). The VH
and VL PCR pools were assembled into a scFv repertoire by a PCR
overlap extension reaction and subsequently cloned into the
pCANTAB5E phagemid vector or to the phagemid vector pCC-Gal6(Fv) in
which the scFv is expressed as an in frame fusion protein with a
cellulose-binding domain (CBD) (28). The resulting libraries were
transduced into E. coli TG1 cells by electroporation and expressed
as fusion with the minor phage coat protein pIII after rescue with
a helper phage. The library complexity consisted of
1.times.10.sup.8 independent clones using both types of
vectors.
[0161] The library was subjected to 3-4 panning cycles followed by
elution of bound phages and reamplification in E. coli. To enhance
the efficiency of selection biotinylated scMHC-peptide complexes
were used. A BirA sequence tag for site-specific biotinylation was
engineered at the C-terminus of the HLA-A2 gene as previously
described (25). Several selection strategies were employed, the
most successful of which resulted in the isolation of specific
binders consisting of panning protocols with a negative depletion
step starting from the .sub.2nd round of panning. The specific
HLA-A2/G9-209M biotinylated complexes were immobilized onto
streptavidin-coated magnetic beads and the library was incubated
with the immobilized complex in the presence of a large excess of
HLA-2 complexes that displayed a different gp100-derived epitope,
the G9-280V peptide. When this strategy is used G9-209M-specific
phage will bind to streptavidin-biotin-immobilized complexes that
are captured by a magnetic force, whereas pan-MHC binders that are
not specific to the G9-209M peptide in the complex will bind to the
non-specific complex in the solution and thus can be separated and
removed from the specific phage. As shown in Table 1 below, a
progressive, marked enrichment for phages that bind the immobilized
complexes was observed after 3-4 rounds of panning, two of which
were performed with the negative depletion strategy.
5TABLE 1 Phage selection on scHLA-A2/G9-209M complexes Library
Cycle Input Output Enrichment scFv 1 1 .times. 10.sup.12 1 .times.
10.sup.4 -- 2 5 .times. 10.sup.11 1 .times. 10.sup.5 10 3 5 .times.
10.sup.11 1 .times. 10.sup.9 10,000 scFv-CBD 1 5 .times. 10.sup.9 1
.times. 10.sup.4 -- 2 5 .times. 10.sup.11 1 .times. 10.sup.5 10 3 5
.times. 10.sup.11 1 .times. 10.sup.8 1,000 A 4.sup.th round of
selection resulted with similar enrichments as observed in round
3.
[0162] Polyclonal phage ELISA was performed to determine phage
specificity on biotinylated recombinant scMHC-peptide complexes
immobilized to BSA-Biotin-streptavidin-coated immunoplates. The
BSA-biotin-streptavidin spacer enables the correct presentation of
the complexes, which can be distorted by direct binding to plastic.
Phage analyzed already after the 2.sup.nd and more dramatically,
after the 3.sup.rd round of panning revealed a unique specificity
pattern only directed toward the specific G9-209M-containing HLA-A2
complexes (FIGS. 1A-B). No binding was observed with control HLA-A2
complexes that display the gp100-derived epitope, G9-280V or the
telomerase-derived epitope 654.
[0163] Individual monoclonal phage clones were isolated from the
population of phages from the last round of panning (no further
enrichment observed after a 4.sup.th round) and re-screened for
specificity by phage ELISA (FIGS. 2A-B). Of the 93 clones tested,
85 (91%) reacted with the HLA-A2/G9-209M complex (FIG. 2A).
Seventy-seven out of the 85 reactive clones (90%) reacted
specifically with the specific HLA-A2/G9-209M complex but not with
the control G9-280V-containing complex (FIG. 2B). Only a small
percentage of the clones (5/93; 5%) did not exhibit peptide
specificity (FIG. 2B). Thus, the panning procedure yielded a
successful enrichment of phage antibodies with TCR-like specificity
toward the HLA-A2/G9-209M complex. Fingerprint analysis by means of
multicutter restriction enzyme digestion revealed that 50 positive,
HLA-A2/G9-209M-specific clones had a similar digestion pattern,
indicating that all are similar (data not shown). Similar results
were obtained with the two libraries. Since they were constructed
from the same genetic material (the same pool of mRNA), only phage
clones derived from the pCANTAB5E scFv library were further
characterized.
[0164] DNA sequencing of V.sub.H and V.sub.L variable domains from
10 clones revealed that all were identical (data not shown),
suggesting that they were all derived from a single productive
antibody VH/VL combinatorial event.
[0165] Characterization of the Soluble Recombinant scFv Antibody
with TCR-Like Specificity:
[0166] DNA sequencing revealed that the antibody VH sequence
belongs to the mouse heavy chains subgroup III (D) and the VL
sequence to mouse kappa light chains group IV (according to
Kabbat). The nucleotide sequence (SEQ ID NO:8) and deduced amino
acid sequence (SEQ ID NO:9) are shown in FIG. 3A. To further
characterize the binding specificity and the biological properties
of the selected scFv antibody, termed G1, two expression systems
were used; for the first, the scFv was subcloned into the phagemid
vector pCANTAB6 in which a myc and a hexahistidine tag is fused to
the C-terminus of the scFv gene. The second was a T7
promoter-driven expression system in which the scFv gene is fused
to a truncated form of Pseudomonas Exotoxin A (PE38) to generate a
scFv-immunotoxin (12). This truncated form of PE contains the
translocation and ADP-ribosylation domains of whole PE but lacks
the cell-binding domain, which is replaced by the scFv fragment
fused at the N-terminus of the truncated toxin. The G1 scFv was
produced in E. coli BL21 (.lambda.DE3) cells by secretion and was
purified from periplasmic fractions by metal affinity
chromatography using the hexahistidine tag fused to the C-terminus
(FIG. 3B). The G1 scFv-PE38 was expressed in BL21 cells and upon
induction with IPTG, large amounts of recombinant protein
accumulated as intracellular inclusion bodies. SDS-PAGE showed that
inclusion bodies from cultures expressing G1 scFv-PE38 contained
more than 90% recombinant protein. Using established renaturation
protocols, G1 scFv-PE38 was refolded from solubilized inclusion
bodies in a redox-shuffling refolding buffer and was thereafter
purified by ion-exchange chromatography on Q-Sepharose and MonoQ
columns, and later by size-exclusion chromatography. A highly
purified G1 scFv-PE38 fusion protein with the expected size of 63
kDa was obtained as analyzed by SDS-PAGE under non reducing
conditions (FIG. 3C). The molecular profile of the G1scFv and
G1scFv-immunotoxin was analyzed by size-exclusion chromatography
and revealed a single protein peak in a monomeric form with an
expected molecular mass of 26 and 63 kDa, respectively (data not
shown). The yield of the refolded G1 scFv-immunotoxin was about 2%,
thus, 2 mg of highly pure protein could be routinely obtained from
the refolding of 100 mg of protein derived from inclusion bodies
containing 80-90% of recombinant protein. This yield is similar to
previously reported scFv-immunotoxins that expressed well and were
produced using a similar expression and refolding system (30). The
yield of the G1 scFv was 3 mg from a 1-liter bacterial culture.
[0167] The binding specificity of the soluble purified G1 scFv
antibody and G1 scFv-PE38 fusion protein was determined by ELISA
assays on biotinylated MHC-peptide complexes immobilized to wells
through BSA-biotin-streptavidin to ensure correct folding of the
complexes, which can be distorted by direct binding to plastic. The
correct folding of the bound complexes and their stability during
the binding assays were determined by their ability to react with
the conformational, specific monoclonal antibody w6/32, which binds
HLA complexes only when folded correctly and when it contains
peptide (data not shown). When we used the soluble-purified G1 scFv
or G1scFv-PE38 protein, the ELISA assays revealed a very specific
recognition pattern corresponding to the hallmarks of
MHC-restricted T-cell specificity (FIG. 4). The G1 scFv selected to
bind the G9-209M-containing HLA-A2 complex reacted only with the
specific complex and not with complexes displaying the G9-280 and
G9-154 gp100-derived MHC-peptide complexes nor to other control
complexes containing HLA-A2-restricted telomerase-derived epitopes
540 and 865 (32), a MUC1-derived peptide (33), or the
HTLV-1-derived TAX peptide (34) (FIG. 4). In these assays the
binding was detected with an anti-PE38 antibody. Similar results
were obtained when using the unfused G1 scFv antibody where
detection was performed with anti-Myc tag antibody (data not
shown). Thus, this antigen-specific scFv fragment exhibits binding
characteristics and the fine specificity of a TCR-like molecule.
The G1 scFv or G1 scFv-PE38 did not recognize the peptide alone nor
empty HLA-A2 molecules (which are difficult to produce because they
are unstable in the absence of a peptide), neither streptavidin nor
other protein antigens (data not shown).
[0168] Next, the binding properties of the TCR-like soluble
purified G1 scFv-PE38 were determined using a saturation ELISA
assay in which biotinylated complexes were bound to
BSA-biotin-streptavidine-coated plates to which increasing amounts
of G1 scFv-PE38 were added. The binding of G1scFv-PE38 to the
specific gp100-derived HLA-A2/G9-209M complex was dose-dependent
and saturable (FIG. 5A). Extrapolating the 50% binding signal
revealed that this antibody possessed high affinity, with a binding
affinity in the nanomolar range. To determine the apparent binding
affinity of the TCR-like scFv fragments to its cognate MHC-peptide
complex, a competition binding assay was performed in which the
binding of .sup.125I-labeled G1scFv-PE38 was competed with
increasing concentrations of unlabeled protein. These binding
assays revealed an apparent binding affinity in the low nanomolar
range of 5 nM (FIG. 5B). Importantly, these results underscore a
previous success in isolating a high affinity scFv antibody with
TCR-like specificity from the phage-displayed antibody repertoire
of immunized HLA-A2 transgenic mice.
[0169] Binding of G1 scFv to APCs Displaying the gp100-Derived
Epitope:
[0170] To demonstrate that the isolated soluble G1 scFv can bind
the specific MHC-peptide complex, not only in its recombinant
soluble form but also in the native form, as expressed on the cell
surface, two APC systems were utilized. One consisted of the murine
TAP2-deficient RMA-S cells that were transfected with the human
HLA-A2 gene in a single-chain format (26) (HLA-A2.1/Db-.beta.2m
single chain) (RMA-S-HHD cells). The gp100-derived peptide and
control peptides were loaded on the RMA-S-HHD cells and the ability
of the selected G1 scFv antibody to bind to peptide-loaded cells
was monitored by FACS (FIGS. 6A-C). Peptide-induced MHC
stabilization of the TAP2 mutant RMA-S-HHD cells was determined by
analyzing the reactivity of the conformational anti HLA antibody
w6/32 and the anti-HLA-A2 MAb BB7.2 with peptide loaded and
unloaded cells (FIGS. 6A and B). The G1 scFv, which recognized the
G9-209M-containing HLA-A2 complex, reacted only with RMA-S-HHD
cells loaded with the G9-209M peptide but not with cells loaded
with the G9-280 peptide (FIG. 6C) or control cells not loaded with
peptide. The G1 scFv did not bind to cells loaded with other
HLA-A2-restricted control peptides such as TAX, MUC1 or
telomerase-derived peptides used for the specificity analysis (see
FIG. 4).
[0171] A second type of APCs was also used, namely the
EBV-transformed B lymphoblast JY cells, which express HLA-A2; these
cells were incubated with the gp100-derived or control peptides.
They are TAP+, and consequently, the displaying of the exogenously
supplied peptide is facilitated by peptide exchange. Using this
strategy, a similar binding specificity with the G1 scFv antibody
was observed (data not shown). These results demonstrate that the
scFv antibody can specifically recognize its corresponding native
HLA-A2 complex in situ on the surface of cells.
[0172] Cytotoxic Activity of G1scFv-PE38 Toward APCs:
[0173] To determine the ability of the G1 scFv antibody to serve as
a targeting moiety for T cell-like specific elimination of
antigen-presenting cells, a G1scFv-PE38 molecule was constructed in
which the very potent truncated form of Pseudomonas exotoxin A is
fused to the C-terminus of the scFv gene and its ability to kill
peptide-loaded APCs was tested. RMAS-HHD or JY cells were loaded
with the gp100-derived epitopes G9-209M and G9-280V as well as with
other control HLA-A2-restricted peptides. FACS analysis with
anti-HLA-A2 antibody revealed a similar expression pattern of
HLA-A2 molecules with G9-209M, G9-280V, and other control
peptide-loaded cells (FIG. 6B). As shown in FIG. 7A, cytotoxicity
by G1scFv-PE38 was observed only on RMAS-HHD cells loaded with the
G9-209 peptide with an IC.sub.50 of 10-20 ng/ml. No cytotoxic
activity was observed on RMAS-HHD cells that were loaded with the
gp100-derived G9-280V epitope or with other control
HLA-A2-restricted peptides or cells that were not loaded with
peptide. G9-209M-loaded RMAS-HHD cells were not killed with an
irrelevant immunotoxin in which an anti human Lewis Y scFv antibody
is fused to PE38 [B3(Fv)-PE38] (FIG. 7A). In the EBV-transformed JY
cells, which express normal TAP, the display of the
exogenously-supplied peptide is facilitated by peptide exchange.
Using this strategy, similar specific activity was observed in
which G1scFv-PE38 kills only cells loaded with the G9-209M peptide
(FIG. 7B). Additional proof for specificity was demonstrated in
competition experiments in which excess specific and control
soluble scHLA-A2-peptide complex was present in solution, in order
to compete for binding and inhibit cytotoxicity by G1 scFv-PE38. An
example of this type of assay is shown in FIG. 7C, in which excess
soluble G9-209M-containing HLA-A2 but not the G9-280V/HLA-A2
complex competed and inhibited the cytotoxic activity of G1
scFv-PE38 toward G9-209M-loaded JY cells. These results further
demonstrate the fine and unique specificity of the G1scFv antibody
and its ability to serve as a targeting moiety to deliver a
cytotoxic effector molecule with antigen (peptide)-specific,
MHC-restricted specificity of T cells directed toward a human tumor
T-cell epitope.
Discussion of the Results
[0174] In this example, the ability to select from an immune
repertoire of murine scFv fragments a high affinity antibody
(referred to herein as G1scFv) directed toward a human T-cell
epitope derived from a cancer antigen, the melanoma-associated
antigen gp100, was demonstrated.
[0175] G1scFv exhibits a very specific and special binding pattern;
it can bind in a peptide-specific manner to HLA-A2 complexes.
Hence, this is a recombinant antibody with T-cell antigen
receptor-like specificity. In contrast to the inherent low affinity
of TCRs, this molecule displays the high affinity binding
characteristics of antibodies, while retaining TCR specificity.
[0176] This example strikingly demonstrates the power of the phage
display approach and its ability to select especially fine
specificities from a large repertoire of different antibodies.
[0177] The ability to select high-affinity TCR-like antibodies,
despite the fact that such peptide-specific binders are thought to
be rare and hence difficult to isolate, may result from the
following considerations.
[0178] One is the mode of immunization and selection which included
immunization of transgenic animal combined with the power of
various selection strategies employed by phage display. It is
believed that using HLA transgenic mice, such as HLA-A2 transgenic
mice, is an advantage because they are usually tolerant to HLA
complexes unless a new foreign peptide is presented on the complex.
The ability to isolate TCR-like antibody molecules may represent a
situation in which lymphocytes that were tolerant to HLA-A2 are now
exposed to new epitopes contributed, in the example provided
herein, by the melanoma gp100-derived peptide presented on HLA-A2.
The panning procedure that combined an excess of non-specific
complex in solution significantly contributed to the selection
process and allowed to isolate a rare antibody clone (one out of
10.sup.8).
[0179] Another important issue relates to the state of the antigen
used in the selection process. The conformation of the antigen has
to be as "natural" as possible, especially when produced in a
recombinant form. As described in references 23 and 24 and in U.S.
patent application Ser. No. 09/534,966 and PCT/IL01/00260
(published as WO 01/72768), it was found that in vitro refolding
from inclusion bodies produced in E. coli of a single-chain MHC
molecule complexed with various peptides yields large amounts of
correctly folded and functional protein. The fact that the
exemplary antibody G1scFv was isolated from a relatively small
library of about 10.sup.8 clones, yet is highly specific with an
affinity in the nanomolar range, strongly indicates that the HLA-A2
transgenic mice that was used for immunization indeed developed
high-affinity antibodies to the HLA-A2/G9-209 complexes. The
observation that only a single anti-HLA-A2/G9-209 antibody was
isolated may reflect that only one such specificity exists or that
other specificities were not generated during the immune response
because such a response could not be easily generated and tolerated
by the HLA-A2 transgenic mice. Quite astonishing is the fact that
similar results were reported in the past for a murine MHC-peptide
system where, using phage display, a recombinant TCR-like antibody
directed toward a class I murine H-2K.sup.k molecule in complex
with the influenza hemagglutinin peptide Ha.sub.255-262 was
isolated (17). Similar to the results presented here, of the 50
clones tested, seven reacted specifically with the
H-2K.sup.k/Ha.sub.255-262 complexes only, and not with other
H-2K.sup.k/peptide complexes. Interestingly, the DNA sequences of
these specific clones were determined and found to be identical
(17). These anti-H-2K.sup.k/Ha.sub.255-262 complexes antibodies,
however, cannot be used to monitor antigen presentation and or kill
antigen presenting cells of human origin.
[0180] Despite the fact that antibodies having a T-cell antigen
receptor-like specificity are rare, the phage display approach can
be applied to isolate recombinant antibodies with TCR-like
specificity to a variety of MHC-peptide complexes related to
various pathological conditions such as cancer, viral infections,
and autoimmune diseases.
[0181] Recombinant antibodies with TCR-like specificity represent a
new, valuable tool for future research in two major areas of tumor
immunology. First, these antibodies may now be used to detect and
directly visualize the presence of specific T-cell epitopes or
MHC-peptide complexes by standard methods of flow cytometry and
immuno-histochemistry. They should be very useful for the study and
analysis of antigen presentation in cancer by determining the
expression of specific tumor-related MHC-peptide complexes on the
surface of tumor cells, metastasis, antigen-presenting cells, and
lymphoid cells. Moreover, such antibodies can be used to analyze
immunotheraphy-based approaches by determining the alterations in
MHC-peptide complex expression on antigen-presenting cells before,
during, and after vaccination protocols with peptides or with APCs
loaded with tumor cell extracts or dendritic-tumor cell hybrid
vaccinations (7-11). Thus, questions relating to how and where
certain events occur during antigen presentation may be directly
addressed, for the first time, and the expression of T-cell
epitopes on the antigen-presenting cell may be visualized and
quantitated.
[0182] Second, antibodies with such exquisitely fine specificity
directed toward a very specific and unique human tumor antigen
present new opportunities for use as targeting moieties for various
antibody-based immunotherapeutic approaches. This includes using
such antibodies to construct recombinant immunotoxins (12), fusion
with cytokine molecules (37) or for bi-specific antibody therapy
(38). The open question with respect to these applications relates
to the low density of the specific epitope on the target cell's
surface. It has been previously demonstrated, using the murine
H-2K.sup.k/influenza hemagglutinin peptide complex and a similar
antigen-presenting system that to achieve efficient killing with a
TCR-like immunotoxin molecule, a density of several thousand
particular MHC-peptide complexes are required for the selective
elimination of APCs (39). The results of described herein support
these findings, achieving a similar cytotoxic potential of a
T-cell-like immunotoxin. To improve the targeting capabilities of
these TCR-like antibody molecules, two antibody engineering
approaches can be employed: (i) to increase the affinity of the
parental antibody by affinity maturation strategies without
altering its TCR-like fine specificity (40); and (ii) to increase
the avidity of these recombinant monovalent molecules by making
them bi-valent (38). Combining these strategies will result in
second generation, improved molecules that will be valuable tools
for immunotherapeutic approaches as well as serve as innovative
research tools for studying the interaction of tumor cells and the
human immune system.
[0183] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0184] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents, patent applications and sequences identified
by a Genebank accession number mentioned in this specification are
herein incorporated in their entirety by reference into the
specification, to the same extent as if each individual
publication, patent, patent application or sequence was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
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Sequence CWU 1
1
11 1 9 PRT Artificial sequence HLA-A2-restricted peptide, gp100
(154) 1 Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 2 9 PRT Artificial
sequence HLA-A2-restricted peptide, gp100 (209) 2 Ile Met Asp Gln
Val Pro Phe Ser Val 1 5 3 9 PRT Artificial sequence
HLA-A2-restricted peptide, gp100 (280) 3 Tyr Leu Glu Pro Gly Pro
Val Thr Val 1 5 4 9 PRT Artificial sequence HLA-A2-restricted
peptide, MUC1 4 Leu Leu Leu Thr Val Leu Thr Val Leu 1 5 5 9 PRT
Artificial sequence HLA-A2-restricted peptide, HTLV-1 (TAX) 5 Leu
Leu Phe Gly Tyr Pro Val Tyr Val 1 5 6 9 PRT Artificial sequence
HLA-A2-restricted peptide, hTEroom temperature (540) 6 Ile Leu Ala
Lys Phe Leu His Trp Leu 1 5 7 9 PRT Artificial sequence
HLA-A2-restricted peptide, hTEroom temperature (865) 7 Arg Leu Val
Asp Asp Phe Leu Leu Val 1 5 8 711 DNA Artificial sequence G1 Single
chain Fv-recombinant antibody DNA sequence 8 caggtgaaac tgcaggagtc
tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag
cctctggatt cactttcagt agctatggca tgtcttgggt tcgccagact 120
ccagacaaga ggctggagtg ggtcgcaacc attagtagtg gtggtagtta cacctactat
180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa
caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt
attactgtgc aagaggtaac 300 tgggaaggat ggtacttcga tgtctggggc
caagggacca cggtcaccgt ctcctcaggt 360 ggaggcggtt caggcggagg
tggctctggc ggtggcggat cgaacatcga gctcactcag 420 tctccagcaa
tcatgtctgc atctccaggg gagagggtca ccatgacctg cagtgccagc 480
tcaagtatac gttacatata ttggtaccaa cagaagcctg gatcctcccc cagactcctg
540 atttatgaca catccaacgt ggctcctgga gtcccttttc gcttcagtgg
cagtgggtct 600 gggacctctt attctctcac aatcaaccga atggaggctg
aggatgctgc cacttattac 660 tgccaggagt ggagtggtta tccgtacacg
ttcggagggg ggacaaagtt g 711 9 237 PRT Artificial sequence G1 single
chain Fv- recombinant antibody protein sequence 9 Gln Val Lys Leu
Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35
40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Trp Glu Gly Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly
Ser Asn Ile Glu Leu Thr Gln Ser Pro Ala Ile 130 135 140 Met Ser Ala
Ser Pro Gly Glu Arg Val Thr Met Thr Cys Ser Ala Ser 145 150 155 160
Ser Ser Ile Arg Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser 165
170 175 Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Val Ala Pro Gly Val
Pro 180 185 190 Phe Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile 195 200 205 Asn Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr
Tyr Cys Gln Glu Trp 210 215 220 Ser Gly Tyr Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu 225 230 235 10 9 PRT Artificial sequence Influenza
derived HLA-restricted peptide 10 Gly Ile Leu Gly Phe Val Phe Thr
Leu 1 5 11 9 PRT Artificial sequence Hepatitis B derived
HLA-restricted peptide 11 Ser Thr Asn Arg Gln Ser Gly Arg Gln 1
5
* * * * *
References