U.S. patent application number 10/335394 was filed with the patent office on 2003-07-24 for chemokine-tumor antigen fusion proteins as cancer vaccines.
Invention is credited to Biragyn, Arya, Kwak, Larry W..
Application Number | 20030138452 10/335394 |
Document ID | / |
Family ID | 22139824 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138452 |
Kind Code |
A1 |
Kwak, Larry W. ; et
al. |
July 24, 2003 |
Chemokine-tumor antigen fusion proteins as cancer vaccines
Abstract
The present invention provides a fusion polypeptide comprising a
chemokine and either a tumor or viral antigen which is administered
as either a protein or nucleic acid vaccine to elicit an immune
response effective in treating cancer or effective in treating or
preventing HIV infection.
Inventors: |
Kwak, Larry W.; (Frederick,
MD) ; Biragyn, Arya; (Frederick, MD) |
Correspondence
Address: |
NEEDLE & ROSENBERG P C
127 PEACHTREE STREET N E
ATLANTA
GA
30303-1811
US
|
Family ID: |
22139824 |
Appl. No.: |
10/335394 |
Filed: |
December 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10335394 |
Dec 31, 2002 |
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09646028 |
Sep 12, 2000 |
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09646028 |
Sep 12, 2000 |
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PCT/US99/05345 |
Mar 12, 1999 |
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60077745 |
Mar 12, 1998 |
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Current U.S.
Class: |
424/192.1 ;
424/85.1; 435/320.1; 435/326; 435/69.5; 530/351; 530/391.1;
536/23.53 |
Current CPC
Class: |
C07K 14/4727 20130101;
A61K 39/00 20130101; Y10S 514/885 20130101; Y10S 530/806 20130101;
C07K 14/521 20130101; C07K 2319/02 20130101 |
Class at
Publication: |
424/192.1 ;
424/85.1; 435/69.5; 435/320.1; 435/326; 530/351; 530/391.1;
536/23.53 |
International
Class: |
A61K 039/00; C07H
021/04; C12P 021/02; C12N 005/06; C07K 016/46 |
Claims
What is claimed is:
1. A fusion polypeptide comprising human monocyte chemotactic
protein-3 and human Muc-1.
2. A fusion polypeptide comprising human interferon-induced protein
10 and human Muc-1.
3. A fusion polypeptide comprising human macrophage-derived
chemokine and human Muc-1.
4. A fusion polypeptide comprising human SDF-1 and human Muc-1.
5. The fusion polypeptide of any one of claims 1 to 4, further
comprising a spacer sequence having the amino acid sequence of SEQ
ID NO:11.
6. An isolated nucleic acid encoding the amino acid sequence of any
one of claims 1 to 4.
7. A vector comprising the nucleic acid of claim 6.
8. A cell comprising the vector of claim 7.
9. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:2.
10. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:1.
11. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:49.
12. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:54.
13. An isolated nucleic acid encoding the fusion polypeptide of any
one of claims 9 to 12.
14. A vector comprising the nucleic acid of claim 13.
15. A cell comprising the vector of claim 14.
16. A composition comprising the fusion polypeptide of any one of
claims 1 to 4 in a pharmaceutically acceptable carrier.
17. A composition comprising the nucleic acid of claim 6 in a
pharmaceutically acceptable carrier.
18. The composition of any one of claims 16 or 17, further
comprising an adjuvant.
19. The composition of claim 18, wherein the adjuvant is an
immunostimulatory cytokine.
20. A composition comprising the fusion polypeptide of any one of
claims 9 to 12 in a pharmaceutically acceptable carrier.
21. A composition comprising the nucleic acid of claim 13 in a
pharmaceutically acceptable carrier.
22. The composition of any one of claims 20 or 21, further
comprising an adjuvant.
23. The composition of claim 22, wherein the adjuvant is an
immunostimulatory cytokine.
24. A fusion polypeptide comprising a human chemokine and a human
immunodeficiency virus (HIV) antigen.
25. The fusion polypeptide of claim 24, wherein the chemokine is
selected from the group consisting of IP-10, MCP-1, MCP-2, MCP-3,
MCP-4, MIP 1, RANTES, SDF-1, MIG and MDC.
26. The fusion polypeptide of claim 24, wherein the HIV antigen is
selected from the group consisting of gp120, gp160, gp41, an active
fragment of gp120, an active fragment of gp160 and an active
fragment of gp41.
27. The fusion polypeptide of claim 24, further comprising a spacer
sequence having the amino acid sequence of SEQ ID NO:11.
28. A nucleic acid encoding the fusion polypeptide of claim 24.
29. A vector comprising the nucleic acid of claim 28.
30. A cell comprising the vector of claim 29.
31. A composition comprising the fusion polypeptide of claim 24 and
a pharmaceutically acceptable carrier.
32. A composition comprising the nucleic acid of claim 28 and a
pharmaceutically acceptable carrier.
33. The composition of any one of claims 31 or 32, further
comprising an adjuvant.
34. The composition of claim 33, wherein the adjuvant is an
immunostimulatory cytokine.
35. A fusion polypeptide comprising human IP-10 and HIV gp120.
36. A fusion polypeptide comprising human MCP-3 and HIV gp120.
37. A fusion polypeptide comprising human MDC and HIV gp120.
38. A fusion polypeptide comprising human SDF-1 and HIV gp120.
39. The fusion polypeptide of any one of claims 35 to 38, further
comprising a spacer sequence having the amino acid sequence of SEQ
ID NO:11.
40. An isolated nucleic acid encoding the fusion polypeptide of any
one of claims 35 to 38.
41. A vector comprising the nucleic acid of claim 40.
42. A cell comprising the vector of claim 41.
43. A composition comprising the fusion polypeptide of any one of
claims 35 to 38 and a pharmaceutically acceptable carrier.
44. A composition comprising the nucleic acid of claim 40 and a
pharmaceutically acceptable carrier.
45. The composition of any one of claims 43 or 44, further
comprising an adjuvant.
46. The composition of claim 45, wherein the adjuvant is an
immunostimulatory cytokine.
47. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:6.
48. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:7.
49. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:5.
50. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:50.
51. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:52.
52. A fusion polypeptide comprising the amino acid sequence of SEQ
ID NO:56.
53. An isolated nucleic acid encoding the fusion polypeptide of any
one of claims 47 to 52.
54. A vector comprising the nucleic acid of claim 53.
55. A cell comprising the vector of claim 54.
56. A composition comprising the fusion polypeptide of any one of
claims 47 to 52 and a pharmaceutically acceptable carrier.
57. A composition comprising the nucleic acid of claim 53 and a
pharmaceutically acceptable carrier.
58. The composition of any one of claims 56 or 57, further
comprising an adjuvant.
59. The composition claim 58, wherein the adjuvant is an
immunostimulatory cytokine.
60. A method of producing an immune response in a subject,
comprising administering to the subject the composition of any one
of claims 16, 18, 19, 20, 22 or 23.
61. A method of producing an immune response in a subject,
comprising administering to the subject the composition of any one
of claims 17, 18, 19, 21, 22 or 23 under conditions whereby the
nucleic acid of the composition can be expressed.
62. A method of producing an immune response in a subject,
comprising administering to the subject the composition of any one
of claims 31, 33, 34, 43, 45, 46, 56, 58 or 59.
63. A method of producing an immune response in a subject,
comprising adminstering to the subject the composition of any one
of claims 32, 33, 34, 44, 45, 46, 57, 58 or 59, under conditions
whereby the nucleic acid can be expressed.
64. The method of any one of claims 60 to 63, wherein the immune
response is an effector T cell (cellular) immune response.
65. A method of treating a cancer in a subject comprising
adminstering to the subject the composition of any one of claims
16, 18, 19, 20, 22 or 23.
66. A method of treating a cancer in a subject, comprising
administering to the subject the composition of any one of claims
17, 18, 19, 21, 22 or 23 under conditions whereby the nucleic acid
of the composition can be expressed.
67. A method of treating or preventing HIV infection in a subject,
comprising administering to the subject the composition of any one
of claims 31, 33, 34, 43, 45, 46, 56, 58 or 59.
68. A method of treating or preventing HIV infection in a subject,
comprising administering to the subject the composition of any one
of claims 32, 33, 34, 44, 45, 46, 57, 58 or 59, under conditions
whereby the nucleic acid can be expressed.
69. A method of treating a B cell tumor in a subject, comprising
administering to the subject a fusion polypeptide comprising a
human chemokine and a B cell tumor antigen.
70. The method of claim 69, wherein the B cell tumor antigen is
selected from the group consisting of an antibody, a single chain
antibody and an epitope of an idiotype of an antibody.
71. The method of claim 69, wherein the human chemokine is selected
from the group consisting of MCP-3, MDC and SDF-1.
72. The method of claim 69, wherein the fusion polypeptide is
selected from the group consisting of a fusion polypeptide
comprising human MCP-3 and human a single chain antibody, a fusion
polypeptide comprising human MDC and a human single chain antibody
and a fusion polypeptide comprising human SDF-1 and a human single
chain antibody.
73. The method of claim 69, wherein the fusion polypeptide is
selected from the group consisting of a polypeptide having the
amino acid sequence of SEQ ID NO:51, a polypeptide having the amino
acid sequence of SEQ ID NO:53 and a polypeptide having the amino
acid sequence of SEQ ID NO:55.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a vaccine that treats
cancer as well as a vaccine that treats or prevents human
immunodeficiency virus (HIV) infection. In particular, the present
invention provides a fusion polypeptide comprising a chemokine and
either a tumor or viral antigen which is administered as either a
protein or nucleic acid vaccine to elicit an immune response
effective in treating cancer or effective in treating or preventing
HIV infection.
[0003] 2. Background Art
[0004] Tumor cells are known to express tumor-specific antigens on
the cell surface. These antigens are believed to be poorly
immunogenic, largely because they represent gene products of
oncogenes or other cellular genes which are normally present in the
host and are therefore not clearly recognized as nonself. Although
numerous investigators have tried to target immune responses
against epitopes from various tumor specific antigens, none have
been successful in eliciting adequate tumor immunity in vivo
(71).
[0005] Humans are particularly vulnerable to cancer as a result of
an ineffective immunogenic response (72). In fact, the poor
immunogenicity of relevant cancer antigens has proven to be the
single greatest obstacle to successful immunotherapy with tumor
vaccines (73). Over the past 30 years, literally thousands of
patients have been administered tumor cell antigens as vaccine
preparations, but the results of these trials have demonstrated
that tumor cell immunization has failed to provide a rational basis
for the design or construction of effective vaccines. Even where
patients express tumor-specific antibodies or cytotoxic T-cells,
this immune response does not correlate with a suppression of the
associated disease. This failure of the immune system to protect
the host may be due to expression of tumor antigens that are poorly
immunogenic or to heterologous expression of specific antigens by
various tumor cells. The appropriate presentation of tumor antigens
in order to elicit an immune response effective in inhibiting tumor
growth remains a central issue in the development of an effective
cancer vaccine.
[0006] Chemokines are a group of usually small secreted proteins
(7-15 kDa) induced by inflammatory stimuli and are involved in
orchestrating the selective migration, diapedesis and activation of
blood-born leukocytes that mediate the inflammatory response
(23,26). Chemokines mediate their function through interaction with
specific cell surface receptor proteins (23). At least four
chemokine subfamilies have been identified as defined by a cysteine
signature motif, termed CC, CXC, C and CX.sub.3C, where C is a
cysteine and X is any amino acid residue. Structural studies have
revealed that at least both CXC and CC chemokines share very
similar tertiary structure (monomer), but different quaternary
structure (dimer) (120-124). For the most part, conformational
differences are localized to sections of loop or the N-terminus.
Monocyte chemotactic protein-3 (MCP-3) is a potent chemoattractant
of monocytes and dendritic cells, T lymphocytes, basophils and
eosinophils (10, 23, 26, 37).
[0007] There remains a great need for a method of presenting tumor
antigens, which are known to be poorly immunogenic, "self" antigens
to a subject's immune system in a manner that elicits an immune
response powerful enough to inhibit the growth of tumor cells in
the subject. This invention overcomes the previous limitations and
shortcomings in the art by providing a fusion protein comprising a
chemokine and a tumor antigen which can produce an in vivo immune
response, resulting in the inhibition of tumor cells. This
invention also overcomes previous shortcomings in the field of HIV
vaccine development by providing a fusion protein comprising a
chemokine and an HIV antigen which is effective as a vaccine for
treating or preventing HIV infection.
SUMMARY OF THE INVENTION
[0008] The present invention provides a fusion polypeptide
comprising human monocyte chemotactic protein-3 and human Muc-1, a
fusion polypeptide comprising human interferon-induced protein 10
and human Muc-1, a fusion polypeptide comprising human
macrophage-derived chemokine and human Muc-1 and a fusion
polypeptide comprising human SDF-1 and human Muc-1.
[0009] The present invention also provides a fusion polypeptide
comprising a human chemokine and a human immunodeficiency virus
(HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2,
MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the
HIV antigen can be gp120, gp160, gp41, an active fragment of gp120,
an active fragment of gp160 and/or an active fragment of gp41.
[0010] In addition, the present invention provides a method of
producing an immune response in a subject, comprising administering
to the subject any of the fusion polypeptides of this invention,
comprising a chemokine and a human immunodeficiency virus (HIV)
antigen, or a chemokine and a tumor antigen, either as a protein or
a nucleic acid encoding the fusion polypeptide.
[0011] Also provided is a method of treating a cancer in a subject
comprising adminstering to the subject any of the fusion
polypeptides of this invention, comprising a chemokine and a tumor
antigen, either as a protein or a nucleic acid encoding the fusion
polypeptide.
[0012] Further provided is a method of treating or preventing HIV
infection in a subject, comprising administering to the subject any
of the fusion polypeptides of this invention, comprising a
chemokine and a human immunodeficiency virus (HIV) antigen, either
as a protein or a nucleic acid encoding the fusion polypeptide.
[0013] A method of treating a B cell tumor in a subject is also
provided, comprising administering to the subject a fusion
polypeptide comprising a human chemokine and a B cell tumor
antigen.
[0014] Various other objectives and advantages of the present
invention will become apparent from the following detailed
description.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] As used in the claims, "a" can include multiples. For
example, "a cell" can mean a single cell or more than one cell.
[0016] The present invention is based on the unexpected discovery
that the administration of a fusion protein comprising a chemokine
and a tumor antigen or administration of a nucleic acid encoding a
fusion protein comprising a chemokine and a tumor antigen yields an
effective and specific anti-tumor immune response by converting a
"self" tumor antigen into a potent immunogen by binding to a
chemokine moiety. A further unexpected discovery of the present
invention is that the chemokine-scFv fusion polypeptide of this
invention is superior to the prototype Id-KLH vaccine in tumor
protection studies as described herein.
[0017] Thus, the present invention provides a fusion polypeptide
comprising a chemokine and a tumor antigen. The fusion polypeptide
can be present in a purified form and can induce an immune response
against the tumor antigen and inhibit the growth of tumor cells
expressing the tumor antigen. "Purified" as used herein means the
polypeptide is sufficiently free of contaminants or cell components
with which proteins normally occur to allow the peptide to be used
therapeutically. It is not contemplated that "purified"
necessitates having a preparation that is technically totally pure
(homogeneous), but purified as used herein means the fusion
polypeptide is sufficiently pure to provide the polypeptide in a
state where it can be used therapeutically. As used herein, "fusion
polypeptide" means a polypeptide made up of two or more amino acid
sequences representing peptides or polypeptides from different
sources. Also as used herein, "epitope" refers to a specific amino
acid sequence of limited length which, when present in the proper
conformation, provides a reactive site for an antibody or T cell
receptor. The identification of epitopes on antigens can be carried
out by immunology protocols that are standard in the art (74). As
further used herein, "tumor antigen" describes a polypeptide
expressed on the cell surface of specific tumor cells and which can
serve to identify the type of tumor. An epitope of the tumor
antigen can be any site on the antigen that is reactive with an
antibody or T cell receptor.
[0018] As used herein, "chemokine" means a small secreted protein,
induced by inflammatory stimuli (e.g., fibroblasts, endothelial
cells, epithelial cells, monocytes, macrophages, T cells, B cells,
PMNs, etc. stimulated by proinflammatory cytokines such as
interferon-gamma, interleukin 4, products of Th1 and Th2
lymphocytes, interleukin-1, tumor necrosis factor-alpha and
bacterial products such as lipopolysaccharide, as well as viral
infection (75,76), which orchestrates a chemotactic response
typically after binding to specific G-protein-coupled cell surface
receptors on target cells (e.g., antigen presenting cells (APC),
such as dendritic cells, monocytes, macrophages, keratinocytes and
B cells), comprising the selective migration, diapedesis and
activation of leukocytes which mediate the inflammatory response.
Four human CXC chemokine receptors (CXCR1-CXCR4), eight human CC
chemokine receptors (CCR1 -CCR8) and one CXXXC chemokine receptor
(CX.sub.3CR1) have been identified. As one example, the chemokine,
interferon-induced protein 10 (IP-10) binds to the CXCR3 receptor,
thus inducing chemotaxis of activated T cells, NK cells, etc.,
which express this receptor. As another example, the chemokine
monocyte chemotactic protein-3 (MCP-3) acts via binding to the
CCR1, CCR2 and CCR3 chemokine receptors on antigen presenting cells
(APC) such as dendritic cells, eosinophils, basophils, monocytes
and activated T cells. Thus, MCP-3 selectively targets and induces
chemotaxis of these cell types.
[0019] The chemokine of this invention can include, but is not
limited to, interferon-induced protein 10, monocyte chemotactic
protein-3, monocyte chemotactic protein-2, monocyte chemotactic
protein-1, monocyte chemotactic protein-4, macrophage inflammatory
protein 1, RANTES, SDF-1, MIG and macrophage-derived chemokine, as
well as any other chemokine now known or later identified.
[0020] It will be appreciated by one of skill in the art that
chemokines can include active fragments of chemokines which retain
the chemotactic activity of the intact molecule. For example, for
both CC and CXC chemokines, the N terminal region is the critical
region of the molecule for biological activity and leukocyte
selectivity. In particular, the N-terminal ELR motif-containing CXC
chemokines are chemotactic for neutrophils, whereas those not
containing the motif act on lymphocytes. IP-10 and MIG, for
example, do not contain the ELR motif and are known to attract
activated T cells (77). Addition of a single amino acid residue to
the amino terminus of MCP-1 decreases its biological activity up to
1000 fold and deletion of a single amino acid for that region
converts the chemokine from an activator of basophils to an
eosinophil chemoattractant (78).
[0021] A chemokine consists of two structural portions: the amino
terminal portion and the carboxy terminal portion. The amino
terminal portion is responsible for chemokine receptor binding and
the carboxy terminal end binds to heparin and heparan sulfate, for
example, in the extracellular matrix and on the surface of
endothelial cells. The chemokine gene can be fragmented as desired
and the fragments can be fused to a specific marker gene encoding
an antigen (e.g., Muc-1 VNT, lymphoma scFv, etc.). The fusion
polypeptide comprising the chemokine fragment and the tumor or
viral antigen can be produced and purified as described herein and
tested for immunogenicity according to the methods provided herein.
By producing several fusion polypeptides having chemokine fragments
of varying size, the minimal size chemokine fragment which impart
an immunological effect can be identified.
[0022] The tumor antigen moiety of the fusion polypeptide of this
invention can be any tumor antigen now known or later identified as
a tumor antigen. The appropriate tumor antigen used in the fusion
polypeptide naturally depends on the tumor type being treated. For
example, the tumor antigen can be, but is not limited to human
epithelial cell mucin (Muc-1; a 20 amino acid core repeat for Muc-1
glycoprotein, present on breast cancer cells and pancreatic cancer
cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen
(CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1,
MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP
1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA),
alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras
oncogene product, HPV E7 and melanoma gangliosides, as well as any
other tumor antigens now known or identified in the future. Tumor
antigens can be obtained following known procedures or are
commercially available (79). The effectiveness of the fusion
protein in eliciting an immune response against a particular tumor
antigen can be determined according to methods standard in the art
for determining the efficacy of vaccines and according to the
methods set forth in the Examples.
[0023] Additionally, the tumor antigen of the present invention can
be an antibody which can be produced by a B cell tumor (e.g., B
cell lymphoma; B cell leukemia; myeloma) or the tumor antigen can
be a fragment of such an antibody, which contains an epitope of the
idiotype of the antibody. The epitope fragment can comprise as few
as nine amino acids. For example, the tumor antigen of this
invention can be a malignant B cell antigen receptor, a malignant B
cell immunoglobulin idiotype, a variable region of an
immunoglobulin, a hypervariable region or complementarity
determining region (CDR) of a variable region of an immunoglobulin,
a malignant T cell receptor (TCR), a variable region of a TCR
and/or a hypervariable region of a TCR.
[0024] In a preferred embodiment, the tumor antigen of this
invention can be a single chain antibody (scFv), comprising linked
V.sub.H and V.sub.L domains and which retains the conformation and
specific binding activity of the native idiotype of the antibody
(27). Such single chain antibodies are well known in the art and
can be produced by standard methods and as described in the
Examples herein.
[0025] In addition, the tumor antigen of the present invention can
be an epitope of the idiotype of a T cell receptor, which can be
produced by a T cell tumor (e.g., T cell lymphoma; T cell leukemia;
myeloma). The epitope can comprise as few as nine amino acids.
[0026] As will be appreciated by those skilled in the art, the
invention also includes peptides and polypeptides having slight
variations in amino acid sequences or other properties. Such
variations may arise naturally as allelic variations (e.g., due to
genetic polymorphism) or may be produced by human intervention
(e.g., by mutagenesis of cloned DNA sequences), such as induced
point, deletion, insertion and substitution mutants. Minor changes
in amino acid sequence are generally preferred, such as
conservative amino acid replacements, small internal deletions or
insertions, and additions or deletions at the ends of the
molecules. Substitutions may be designed based on, for example, the
model of Dayhoff et al. (80). These modifications can result in
changes in the amino acid sequence, provide silent mutations,
modify a restriction site, or provide other specific mutations. The
fusion polypeptides can comprise one or more selected epitopes on
the same tumor antigen, one or more selected epitopes on different
tumor antigens, as well as repeats of the same epitope, either in
tandem or interspersed along the amino acid sequence of the fusion
polypeptide. The tumor antigen can be positioned in the fusion
polypeptide at the carboxy terminus of the chemokine, the amino
terminus of chemokine and/or at one or more internal sites within
the chemokine amino acid sequence.
[0027] The present invention further provides a polypeptide having
the amino acid sequence selected from the group consisting of SEQ
ID NO:13 (human IP-10 fused to murine scFv38), SEQ ID NO:16 (human
MCP-3 fused to murine scFv38), SEQ ID NO:12 (human IP-10 fused to
murine scFv20A), SEQ ID NO:14 (human MCP-3 fused to murine scFv20A)
SEQ ID NO:1 (human IP-10 fused to human Muc-1 core epitope (VNT)),
SEQ ID NO:2 (human MCP-3 fused to human Muc-1 core epitope (VNT)),
SEQ ID NO:3 (murine IP-10 fused to human Muc-1 core epitope (VNT)),
SEQ ID NO:4 (murine MCP-3 fused to Muc-1 core epitope (VNT)), SEQ
ID NO:5 (human SDF-1.beta. fused to the hypervariable region of the
envelope glycoprotein, gp120, of HIV-1 (the disulfate loop V3)),
SEQ ID NO:6 (human IP-10 fused to the hypervariable region of the
envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3), SEQ
ID NO:7 (human MCP-3 fused to the hypervariable region of the
envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3), SEQ
ID NO:8 (murine IP-10 fused to the hypervariable region of the
envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3), SEQ
ID NO:52 (human IP-10 fused with HIV gp120), SEQ ID NO:56 (human
MCP-3 fused with HIV gp120), and SEQ ID NO:9 (murine MCP-3 fused to
the hypervariable region of the envelope glycoprotein gp120 of
HIV-1 (the disulfate loop V3). It would be routine for an artisan
to produce a fusion protein comprising any human chemokine region
and any human tumor antigen (e.g., human single chain antibody)
region according to the methods described herein, on the basis of
the availability in the art of the nucleic acid and/or amino acid
sequence of the human chemokine of interest and the human tumor
antigen of interest.
[0028] The present invention further provides a fusion polypeptide
comprising a first region comprising a chemokine selected from the
group consisting of interferon-induced protein 10, monocyte
chemotactic protein-2, monocyte chemotactic protein-1, macrophage
inflammatory protein 1, RANTES, SDF-1 and macrophage-derived
chemokine and a second region comprising a tumor antigen selected
from the group consisting of human epithelial cell mucin (Muc-1),
the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA),
the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3,
tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 &
2, HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein
(AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7,
melanoma gangliosides, an antibody produced by a B cell tumor
(e.g., B cell lymphoma; B cell leukemia; myeloma), a fragment of
such an antibody, which contains an epitope of the idiotype of the
antibody, a malignant B cell antigen receptor, a malignant B cell
immunoglobulin idiotype, a variable region of an immunoglobulin, a
hypervariable region or CDR of a variable region of an
immunoglobulin, a malignant T cell receptor (TCR), a variable
region of a TCR and/or a hypervariable region of a TCR.
[0029] For example, the present invention provides a fusion
polypeptide comprising an scFv cloned from a human subject's biopsy
tumor material or from a hybridoma cell line producing a lymphoma
antibody and a human chemokine moiety (e.g., MCP-3, IP-10, SDF-1,
etc.). In addition, the present invention provides a human
chemokine fused with the Muc-1 core epitope of human breast cancer
or human pancreatic cancer. Muc-1 is a glycoprotein (Mr>200,000)
abundantly expressed on breast cancer cells and pancreatic tumor
cells. A variable number of tandem (VNT) repeats of a 20 amino acid
peptide (PDTRPAPGSTAPPAHGVTSA; SEQ ID NO:40) include B and T cell
epitopes. Thus, the present invention provides a fusion protein
comprising IP-10 and Muc-1 VNT and MCP-3 and Muc-1 VNT. The
expression vector is designed so that a VNT can be changed by
routine cloning methods to produce a fusion polypeptide comprising
IP-10 or MCP-3 fused with a Muc-1 VNT dimer, trimer, tetramer,
pentamer, hexamer, etc.
[0030] In specific emobodiments, the present invention also
provides a fusion polypeptide comprising human monocyte chemotactic
protein-3 and human Muc-1, a fusion polypeptide comprising human
interferon-induced protein 10 and human Muc-1, a fusion polypeptide
comprising human macrophage-derived chemokine and human Muc-1, a
fusion polypeptide comprising human SDF-1 and human Muc 1, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:2, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:1, a fusion polypeptide comprising the amino acid sequence of
SEQ ID NO:49 (human MDC fused to human Muc-1) and a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:54
(human SDF1 fused to human Muc-1).
[0031] The present invention further provides a fusion polypeptide
comprising a human chemokine (e.g., IP-10, MCP-3, SDF-1, etc.) and
a scFv which recognizes tumor antigens, such as idiotype-specific
scFv, Muc-1, etc. Such a fusion polypeptide would allow migration,
recruitment and activation of specialized cells of the immune
system, such as natural killer (NK) cells, macrophages, dendritic
cells (DC), polymorphonuclear (PMN) leukocytes, cytotoxic
lymphocytes (CTL), etc., which would destroy the target cell.
[0032] The fusion polypeptide of this invention can further
comprise a spacer sequence between the chemokine and the tumor
antigen or viral antigen, which can have the amino acid sequence
EFNDAQAPKSLE (SEQ ID NO:11), which allows for retention of the
correct folding of the tumor antigen region of the polypeptide.
[0033] In addition, the present invention provides a composition
comprising the fusion polypeptide of this invention and a suitable
adjuvant. Such a composition can be in a pharmaceutically
acceptable carrier, as described herein. As used herein, "suitable
adjuvant" describes a substance capable of being combined with the
fusion polypeptide to enhance an immune response in a subject
without deleterious effect on the subject. A suitable adjuvant can
be, but is not limited to, for example, an immunostimulatory
cytokine, SYNTEX adjuvant formulation 1 (SAF-1) composed of 5
percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent
Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2
percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
Other suitable adjuvants are well known in the art and include
QS-21, Freund's adjuvant (complete and incomplete), alum, aluminum
phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-iso-
glutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine
(CGP 11637, referred to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl--
L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylami-
ne (CGP 19835A, referred to as MTP-PE) and RIBI, which contains
three components extracted from bacteria, monophosphoryl lipid A,
trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2%
squalene/Tween 80 emulsion. The adjuvant, such as an
immunostimulatory cytokine can be administered before the
administration of the fusion protein or nucleic acid encoding the
fusion protein, concurrent with the administration of the fusion
protein or nucleic acid or up to five days after the administration
of the fusion polypeptide or nucleic acid to a subject. QS-21,
similarly to alum, complete Freund's adjuvant, SAF, etc., can be
administered within hours of administration of the fusion
protein.
[0034] Furthermore, combinations of adjuvants, such as
immunostimulatory cytokines can be co-administered to the subject
before, after or concurrent with the administration of the fusion
polypeptide or nucleic acid. For example, combinations of
adjuvants, such as immunostimulatory cytokines, can consist of two
or more of immunostimulatory cytokines of this invention, such as
GM/CSF, interleukin-2, interleukin-12, interferon-gamma,
interleukin-4, tumor necrosis factor-alpha, interleukin-1,
hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules
and B7.2 co-stimulatory molecules. The effectiveness of an adjuvant
or combination of adjuvants may be determined by measuring the
immune response directed against the fusion polypeptide with and
without the adjuvant or combination of adjuvants, using standard
procedures, as described herein.
[0035] Furthermore, the present invention provides a composition
comprising the fusion polypeptide of this invention or a nucleic
acid encoding the fusion polypeptide of this invention and an
adjuvant, such as an immunostimulatory cytokine or a nucleic acid
encoding an adjuvant, such as an immunostimulatory cytokine. Such a
composition can be in a pharmaceutically acceptable carrier, as
described herein. The immunostimulatory cytokine used in this
invention can be, but is not limited to, GM/CSF, interleukin-2,
interleukin-12, interferon-gamma, interleukin-4, tumor necrosis
factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L,
B7.1 con-stimulatory molecules and B7.2 co-stimulatory
molecules.
[0036] The present invention further contemplates a fusion
polypeptide comprising a chemokine, or active fragment thereof, as
described herein and an antigen of human immunodeficiency virus
(HIV). For example, the HIV antigen of this invention can be, but
is not limited to, the envelope glycoprotein gp120, the third
hypervariable region of the envelope glycoprotein, gp 120 of HIV-1
(the disulfate loop V3), having the amino acid sequence:
NCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNIS (SEQ ID NO:10), any other
antigenic fragment of gp120, the envelope glycoprotein gp160, an
antigenic fragment of gp160, the envelope glycoprotein gp41 and an
antigenic fragment of gp41. For example, the nucleic acid encoding
the V3 loop can be fused to the 3' end of the nucleic acid encoding
a chemokine (e.g., IP-10, MCP-3, SDF-1, MDC) directly or separated
by a spacer sequence. The chemokine-V3 loop fusion polypeptide can
be produced in an expression system as described herein and
purified as also described herein.
[0037] In specific embodiments, the present invention provides a
fusion polypeptide comprising a human chemokine and a human
immunodeficiency virus (HIV) antigen, wherein the chemokine can be
IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or
MDC and wherein the HIV antigen can be gp120, gp160, gp41, an
active (i.e., antigenic) fragment of gp120, an active (i.e.,
antigenic) fragment of gp160 and an active (i.e., antigenic)
fragment of gp41.
[0038] Further provided in this invention is fusion polypeptide
comprising human IP-10 and HIV gp120, a fusion polypeptide
comprising human MCP-3 and HIV gp120, a fusion polypeptide
comprising human MDC and HIV gp120, a fusion polypeptide comprising
human SDF-1 and HIV gp120, a fusion polypeptide comprising the
amino acid sequence of SEQ ID NO:6 (human IP-10/gp120), a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:7
(human MCP-3/gp120), a fusion polypeptide comprising the amino acid
sequence of SEQ ID NO:5 (human SDF1/gp120), a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:52, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:56 and
a fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:50 (human MDC/gp120).
[0039] An isolated nucleic acid encoding the fusion polypeptides of
this invention as described above is also provided. By "isolated
nucleic acid" is meant a nucleic acid molecule that is
substantially free of the other nucleic acids and other components
commonly found in association with nucleic acid in a cellular
environment. Separation techniques for isolating nucleic acids from
cells are well known in the art and include phenol extraction
followed by ethanol precipitation and rapid solubilization of cells
by organic solvent or detergents (81).
[0040] The nucleic acid encoding the fusion polypeptide can be any
nucleic acid that functionally encodes the fusion polypeptide. To
functionally encode the polypeptide (i.e., allow the nucleic acid
to be expressed), the nucleic acid can include, for example,
expression control sequences, such as an origin of replication, a
promoter, an enhancer and necessary information processing sites,
such as ribosome binding sites, RNA splice sites, polyadenylation
sites and transcriptional terminator sequences. Preferred
expression control sequences are promoters derived from
metallothionine genes, actin genes, immunoglobulin genes, CMV,
SV40, adenovirus, bovine papilloma virus, etc. A nucleic acid
encoding a selected fusion polypeptide can readily be determined
based upon the genetic code for the amino acid sequence of the
selected fusion polypeptide and many nucleic acids will encode any
selected fusion polypeptide. Modifications in the nucleic acid
sequence encoding the fusion polypeptide are also contemplated.
Modifications that can be useful are modifications to the sequences
controlling expression of the fusion polypeptide to make production
of the fusion polypeptide inducible or repressible as controlled by
the appropriate inducer or repressor. Such means are standard in
the art (81). The nucleic acids can be generated by means standard
in the art, such as by recombinant nucleic acid techniques, as
exemplified in the examples herein and by synthetic nucleic acid
synthesis or in vitro enzymatic synthesis.
[0041] A vector comprising any of the nucleic acids of the present
invention and a cell comprising any of the vectors of the present
invention are also provided. The vectors of the invention can be in
a host (e.g., cell line or transgenic animal) that can express the
fusion polypeptide contemplated by the present invention.
[0042] There are numerous E. coli (Escherichia coli) expression
vectors known to one of ordinary skill in the art useful for the
expression of nucleic acid encoding proteins such as fusion
proteins. Other microbial hosts suitable for use include bacilli,
such as Bacillus subtilis, and other enterobacteria, such as
Salmonella, Serratia, as well as various Pseudomonas species. These
prokaryotic hosts can support expression vectors which will
typically contain expression control sequences compatible with the
host cell (e.g., an origin of replication). In addition, any number
of a variety of well-known promoters will be present, such as the
lactose promoter system, a tryptophan (Trp) promoter system, a
beta-lactamase promoter system, or a promoter system from phage
lambda. The promoters will typically control expression, optionally
with an operator sequence and have ribosome binding site sequences
for example, for initiating and completing transcription and
translation. If necessary, an amino terminal methionine can be
provided by insertion of a Met codon 5' and in-frame with the
protein. Also, the carboxy-terminal extension of the protein can be
removed using standard oligonucleotide mutagenesis procedures.
[0043] Additionally, yeast expression can be used. There are
several advantages to yeast expression systems. First, evidence
exists that proteins produced in a yeast secretion system exhibit
correct disulfide pairing. Second, post-translational glycosylation
is efficiently carried out by yeast secretory systems. The
Saccharomyces cerevisiae pre-pro-alpha-factor leader region
(encoded by the MF.alpha.-1 gene) is routinely used to direct
protein secretion from yeast (82). The leader region of
pre-pro-alpha-factor contains a signal peptide and a pro-segment
which includes a recognition sequence for a yeast protease encoded
by the KEX2 gene. This enzyme cleaves the precursor protein on the
carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The
polypeptide coding sequence can be fused in-frame to the
pre-pro-alpha-factor leader region. This construct is then put
under the control of a strong transcription promoter, such as the
alcohol dehydrogenase I promoter or a glycolytic promoter. The
protein coding sequence is followed by a translation termination
codon which is followed by transcription termination signals.
Alternatively, the polypeptide coding sequence of interest can be
fused to a second protein coding sequence, such as Sj26 or
.beta.-galactosidase, used to facilitate purification of the fusion
protein by affinity chromatography. The insertion of protease
cleavage sites to separate the components of the fusion protein is
applicable to constructs used for expression in yeast.
[0044] Efficient post-translational glycosylation and expression of
recombinant proteins can also be achieved in Baculovirus systems in
insect cells.
[0045] Mammalian cells permit the expression of proteins in an
environment that favors important post-translational modifications
such as folding and cysteine pairing, addition of complex
carbohydrate structures and secretion of active protein. Vectors
useful for the expression of proteins in mammalian cells are
characterized by insertion of the protein coding sequence between a
strong viral promoter and a polyadenylation signal. The vectors can
contain genes conferring either gentamicin or methotrexate
resistance for use as selectable markers. The antigen and
immunoreactive fragment coding sequence can be introduced into a
Chinese hamster ovary (CHO) cell line using a methotrexate
resistance-encoding vector. Presence of the vector RNA in
transformed cells can be confirmed by Northern blot analysis and
production of a cDNA or opposite strand RNA corresponding to the
protein coding sequence can be confirmed by Southern and Northern
blot analysis, respectively. A number of other suitable host cell
lines capable of secreting intact proteins have been developed in
the art and include the CHO cell lines, HeLa cells, myeloma cell
lines, Jurkat cells and the like. Expression vectors for these
cells can include expression control sequences, as described
above.
[0046] The vectors containing the nucleic acid sequences of
interest can be transferred into the host cell by well-known
methods, which vary depending on the type of cell host. For
example, calcium chloride transfection is commonly utilized for
prokaryotic cells, whereas calcium phosphate treatment, lipofection
or electroporation may be used for other cell hosts.
[0047] Alternative vectors for the expression of protein in
mammalian cells, similar to those developed for the expression of
human gamma-interferon, tissue plasminogen activator, clotting
Factor VIII, hepatitis B virus surface antigen, protease Nexinl,
and eosinophil major basic protein, can be employed. Further, the
vector can include CMV promoter sequences and a polyadenylation
signal available for expression of inserted nucleic acid in
mammalian cells (such as COS7).
[0048] The nucleic acid sequences can be expressed in hosts after
the sequences have been positioned to ensure the functioning of an
expression control sequence. These expression vectors are typically
replicable in the host organisms either as episomes or as an
integral part of the host chromosomal DNA. Commonly, expression
vectors can contain selection markers, e.g., tetracycline
resistance or hygromycin resistance, to permit detection and/or
selection of those cells transformed with the desired nucleic acid
sequences (83).
[0049] Additionally, the fusion polypeptides and/or nucleic acids
of the present invention can be used in in vitro diagnostic assays,
as well as in screening assays for identifying unknown tumor
antigen epitopes and fine mapping of tumor antigen epitopes.
[0050] Also provided is a method for producing a fusion polypeptide
comprising a chemokine, or an active fragment thereof and a tumor
antigen or HIV antigen, comprising cloning into an expression
vector a first DNA fragment encoding a chemokine or active fragment
thereof and a second DNA fragment encoding a tumor antigen or HIV
antigen; and expressing the DNA of the expression vector in an
expression system under conditions whereby the fusion polypeptide
is produced. The expression vector and expression system can be of
any of the types as described herein. The cloning of the first and
second DNA segments into the expression vector and expression of
the DNA under conditions which allow for the production of the
fusion protein of this invention can be carried out as described in
the Examples section included herein. The method of this invention
can further comprise the step of isolating and purifying the fusion
polypeptide, according to methods well known in the art and as
described herein.
[0051] Any of the fusion polypeptides, the nucleic acids and the
vectors of the present invention can be in a pharmaceutically
acceptable carrier and in addition, can include other medicinal
agents, pharmaceutical agents, carriers, diluents, adjuvants (e.g.,
immunostimulatory cytokines), etc. By "pharmaceutically acceptable"
is meant a material that is not biologically or otherwise
undesirable, i.e., the material may be administered to an
individual along with the selected antigen without causing
substantial deleterious biological effects or interacting in a
deleterious manner with any of the other components of the
composition in which it is contained. Actual methods of preparing
such dosage forms are known, or will be apparent, to those skilled
in this art (84).
[0052] Thus, the present invention provides a method for inducing
an immune response in a subject capable of induction of an immune
response and preferably human, comprising administering to the
subject an immune response-inducing amount of the fusion
polypeptide of this invention. As used herein, "an immune
response-inducing amount" is that amount of fusion polypeptide
which is capable of producing in a subject a humoral and/or
cellular immune response capable of being detected by standard
methods of measurement, such as, for example, as described herein.
For example, the antigenic polypeptide region can induce an
antibody response. The antibodies can treat or prevent a
pathological or harmful condition in the subject in which the
antibodies are produced or the antibodies can be removed from the
subject and administered to another subject to treat or prevent a
pathological or harmful condition. The fusion polypeptide can also
induce an effector T cell (cellular) immune response which is
effective in treating or preventing a pathological or harmful
conditions in the subject.
[0053] In an embodiment wherein the antigen moiety of the fusion
polypeptide comprises an immunoglobulin light or heavy chain or a
single chain antibody, the immune response can be the production in
the subject of anti-idiotype antibodies, which represent the image
of the original antigen and can function in a vaccine preparation
to induce an immune response to a pathogenic antigen, thereby
avoiding immunization with the antigen itself (85). The
anti-idiotype antibodies can treat or prevent a pathological or
harmful condition in the subject in which the anti-idiotype
antibodies are produced or the anti-idiotype antibodies can be
removed from the subject and administered to another subject to
treat or prevent a pathological or harmful condition.
[0054] Further provided is a method for inhibiting the growth of
tumor cells in a subject, comprising administering to the subject a
tumor cell growth-inhibiting amount of the fusion polypeptide of
this invention. The subject of this method can be any subject in
which a humoral and/or cellular immune response to a tumor can be
induced, which is preferably an animal and most preferably a human.
As used herein, "inhibiting the growth of tumor cells" means that
following administration of the fusion polypeptide, a measurable
humoral and/or cellular immune response against the tumor cell
epitope is elicited in the subject, resulting in the inhibition of
growth of tumor cells present in the subject. The humoral immune
response can be measured by detection, in the serum of the subject,
of antibodies reactive with the epitope of the tumor antigen
present on the fusion polypeptide, according to protocols standard
in the art, such as enzyme linked immunosorbent immunoassay (ELISA)
and Western blotting protocols. The cellular immune response can be
measured by, for example, footpad swelling in laboratory animals,
peripheral blood lymphocyte (PBL) proliferation assays and PBL
cytotoxicity assays, as would be known to one of ordinary skill in
the art of immunology and particularly as set forth in the
available handbooks and texts of immunology protocols (86).
[0055] The present invention also provides a method of treating
cancer in a subject diagnosed with cancer, comprising administering
to the subject an effective amount of the fusion polypeptide of the
present invention. The cancer can be, but is not limited to B cell
lymphoma, T cell lymphoma, myeloma, leukemia, breast cancer,
pancreatic cancer, colon cancer, lung cancer, renal cancer, liver
cancer, prostate cancer, melanoma and cervical cancer.
[0056] Further provided is a method of treating a B cell tumor in a
subject diagnosed with a B cell tumor, comprising administering an
effective amount of the fusion polypeptide of this invention, which
comprises an antibody or a fragment thereof, as described herein,
in a pharmaceutically acceptable carrier, to the subject.
[0057] In specific embodiments, the present invention also provides
a method of producing an immune response in a subject, comprising
administering to the subject a composition comprising a fusion
polypeptide of this invention and a pharmaceutically acceptable
carrier and wherein the fusion polypeptide can be a fusion
polypeptide comprising human monocyte chemotactic protein-3 and
human Muc-1, a fusion polypeptide comprising human
interferon-induced protein 10 and human Muc-1, a fusion polypeptide
comprising human macrophage-derived chemokine and human Muc-1, a
fusion polypeptide comprising human SDF-1 and human Muc-1, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:2, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:1, a fusion polypeptide comprising the amino acid sequence of
SEQ ID NO:49 and a fusion polypeptide comprising the amino acid
sequence of SEQ ID NO:54, thereby producing an immune response in
the subject.
[0058] Also provided is a method of producing an immune response in
a subject, comprising administering to the subject a composition
comprising a nucleic acid encoding a fusion polypeptide of this
invention and a pharmaceutically acceptable carrier and wherein the
fusion polypeptide is a fusion polypeptide comprising comprising
human monocyte chemotactic protein-3 and human Muc-1, a fusion
polypeptide comprising human interferon-induced protein 10 and
human Muc-1, a fusion polypeptide comprising human
macrophage-derived chemokine and human Muc-1, a fusion polypeptide
comprising human SDF-1 and human Muc-1, a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:2, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:1, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:49 and a fusion polypeptide comprising the amino acid sequence
of SEQ ID NO:54, under conditions whereby the nucleic acid of the
composition can be expressed, thereby producing an immune response
in the subject.
[0059] In further embodiments, the present invention also provides
a method of producing an immune response in a subject, comprising
administering to the subject a composition comprising a fusion
polypeptide of this invention and a pharmaceutically acceptable
carrier and wherein the fusion polypeptide can be a fusion
polypeptide comprising human IP-10 and HIV gp120, a fusion
polypeptide comprising human MCP-3 and HIV gp120, a fusion
polypeptide comprising human MDC and HIV gp120, a fusion
polypeptide comprising human SDF-1 and HIV gp120, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:6, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:7, a fusion polypeptide comprising the amino acid sequence of
SEQ ID NO:52, a fusion polypeptide comprising the amino acid
sequence of SEQ ID NO:56, a fusion polypeptide comprising the amino
acid sequence of SEQ ID NO:5, and/or a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:50, thereby
producing an immune response in the subject.
[0060] Also provided is a method of producing an immune response in
a subject, comprising administering to the subject a composition
comprising a nucleic acid encoding a fusion polypeptide of this
invention and a pharmaceutically acceptable carrier and wherein the
fusion polypeptide is a fusion polypeptide comprising human IP-10
and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV
gp120, a fusion polypeptide comprising human MDC and HIV gp120, a
fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:6, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:7, a fusion polypeptide comprising the amino acid sequence of
SEQ ID NO:5, a fusion polypeptide comprising the amino acid
sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino
acid sequence of SEQ ID NO:56, and/or a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:50, under
conditions whereby the nucleic acid of the composition can be
expressed, thereby producing an immune response in the subject.
[0061] Also provided is a method of producing an immune response in
a subject, comprising administering to the subject a composition
comprising a fusion polypeptide and a pharmaceutically acceptable
carrier and wherein the fusion polypeptide is a fusion polypeptide
comprising a human chemokine and a human immunodeficiency virus
(HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2,
MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the
HIV antigen can be gp120, gp160, gp41, an active (i.e., antigenic)
fragment of gp120, an active (i.e., antigenic) fragment of gp160
and/or an active (i.e., antigenic) fragment of gp41, thereby
producing an immune response in the subject.
[0062] The present invention also provides a method of producing an
immune response in a subject, comprising administering to the
subject a composition comprising a nucleic acid encoding a fusion
polypeptide comprising a human chemokine and a human
immunodeficiency virus (HIV) antigen, wherein the chemokine can be
IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or
MDC and wherein the IIIV antigen can be gp120, gp160, gp41, an
active (i.e., antigenic) fragment of gp120, an active (i.e.,
antigenic) fragment of gp160 and/or an active (i.e., antigenic)
fragment of gp41, and a pharmaceutically acceptable carrier, under
conditions whereby the nucleic acid can be expressed, thereby
producing an immune response in the subject.
[0063] In any of the methods provided herein which recite the
production of an immune response, the immune response can be
humoral and/or an effector T cell (cellular) immune response, as
determined according to methods standard in the art.
[0064] In another embodiment, the present invention provides a
method of treating a cancer in a subject comprising adminstering to
the subject a composition comprising a fusion polypeptide of this
invention and a pharmaceutically acceptable carrier and wherein the
fusion polypeptide is a fusion polypeptide comprising human
monocyte chemotactic protein-3 and human Muc-1, a fusion
polypeptide comprising human interferon-induced protein 10 and
human Muc-1, a fusion polypeptide comprising human
macrophage-derived chemokine and human Muc-1, a fusion polypeptide
comprising human SDF-1 and human Muc-1, a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:2, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:1, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:49 and a fusion polypeptide comprising the amino acid sequence
of SEQ ID NO:54, thereby treating a cancer in the subject.
[0065] Additionally provided is a method of treating a cancer in a
subject, comprising administering to the subject a composition
comprising a nucleic acid encoding a fusion polypeptide of this
invention and a pharmaceutically acceptable carrier and wherein the
fusion polypeptide is a fusion polypeptide comprising human
monocyte chemotactic protein-3 and human Muc-1, a fusion
polypeptide comprising human interferon-induced protein 10 and
human Muc-1, a fusion polypeptide comprising human
macrophage-derived chemokine and human Muc-1, a fusion polypeptide
comprising human SDF-1 and human Muc-1, a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:2, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:1, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:49 and a fusion polypeptide comprising the amino acid sequence
of SEQ ID NO:54, under conditions whereby the nucleic acid of the
composition can be expressed, thereby treating a cancer in the
subject.
[0066] Further provided is a method of treating or preventing HIV
infection in a subject, comprising administering to the subject a
composition comprising a human chemokine and a human
immunodeficiency virus (HIV) antigen, wherein the chemokine can be
IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or
MDC and wherein the HIV antigen can be gp120, gp160, gp41, an
active (i.e., antigenic) fragment of gp120, an active (i.e.,
antigenic) fragment of gp160 and/or an active (i.e., antigenic)
fragment of gp41, and a pharmaceutically acceptable carrier,
thereby treating or preventing HIV infection in the subject.
[0067] In addition, a method of treating or preventing HIV
infection in a subject is provided herein, comprising administering
to the subject a composition comprising a nucleic acid encoding a
fusion polypeptide comprising a human chemokine and a human
immunodeficiency virus (HIV) antigen, wherein the chemokine can be
IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or
MDC and wherein the HIV antigen can be gp120, gp160, gp41, an
active (i.e., antigenic) fragment of gp120, an active (i.e.,
antigenic) fragment of gp160 and/or an active (i.e., antigenic)
fragment of gp41, and a pharmaceutically acceptable carrier, under
conditions whereby the nucleic acid can be expressed, thereby
treating or preventing HIV infection in the subject.
[0068] Further provided is a method of treating or preventing HIV
infection in a subject, comprising administering to the subject a
composition comprising a fusion polypeptide comprising human IP-10
and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV
gp120, a fusion polypeptide comprising human MDC and HIV gp120, a
fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:6, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:7, a fusion polypeptide comprising the amino acid sequence of
SEQ ID NO:5, a fusion polypeptide comprising the amino acid
sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino
acid sequence of SEQ ID NO:56 and/or a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:50, and a
pharmaceutically acceptable carrier, thereby treating or preventing
HIV infection in the subject.
[0069] In addition, a method of treating or preventing HIV
infection in a subject is provided herein, comprising administering
to the subject a composition comprising a nucleic acid encoding a
fusion polypeptide comprising human IP-10 and HIV gp120, a fusion
polypeptide comprising human MCP-3 and HIV gp120, a fusion
polypeptide comprising human MDC and HIV gp120, a fusion
polypeptide comprising human SDF-1 and HIV gp120, a fusion
polypeptide comprising the amino acid sequence of SEQ ID NO:6, a
fusion polypeptide comprising the amino acid sequence of SEQ ID
NO:7, a fusion polypeptide comprising the amino acid sequence of
SEQ ID NO:5, a fusion polypeptide comprising the amino acid
sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino
acid sequence of SEQ ID NO:56, and/or a fusion polypeptide
comprising the amino acid sequence of SEQ ID NO:50, and a
pharmaceutically acceptable carrier, under conditions whereby the
nucleic acid can be expressed, thereby treating or preventing HIV
infection in the subject.
[0070] In a further embodiment, the present invention provides a
method of treating a B cell tumor in a subject, comprising
administering to the subject a fusion polypeptide comprising a
human chemokine and a B cell tumor antigen, wherein the B cell
tumor antigen can be an antibody, a single chain antibody or an
epitope of an idiotype of an antibody, wherein the human chemokine
can be MCP-3, MDC or SDF-1, wherein the fusion polypeptide can be a
fusion polypeptide comprising human MCP-3 and human a single chain
antibody, a fusion polypeptide comprising human MDC and a human
single chain antibody or a fusion polypeptide comprising human
SDF-1 and a human single chain antibody and wherein the fusion
polypeptide can be a polypeptide having the amino acid sequence of
SEQ ID NO:51 (human MCP-3/human scFV fusion), a polypeptide having
the amino acid sequence of SEQ ID NO:53 (human MDC/human scFv
fusion) and/or a polypeptide having the amino acid sequence of SEQ
ID NO:55 (human SDF-1 human scFv fusion), thereby treating a B cell
tumor in the subject.
[0071] Also provided is a fusion polypeptide comprising the human
chemokine, SDF-1.beta., and the V3 loop of HIV-1 envelope
glycoprotein, gp120, as well as a fusion protein comprising
SDF-1.beta. and gp160 of HIV-1, a fusion protein comprising
SDF-1.beta. and gp41 of HIV-1, a fusion protein comprising
SDF-1.beta. and an active fragment of gp120, a fusion protein
comprising SDF-1.beta. and an active fragment of gp160 and a fusion
polypeptide comprising SDF-1.beta. and an active fragment of
gp41.
[0072] The methods of this invention comprising administering the
fusion protein of this invention to a subject can further comprise
the step of administering one or more adjuvants, such as an
immunostimulatory cytokine to the subject. The adjuvant or
adjuvants can be administered to the subject prior to, concurrent
with and/or after the administration of the fusion protein as
described herein.
[0073] The subject of the present invention can be any animal in
which cancer can be treated by eliciting an immune response to a
tumor antigen. In a preferred embodiment, the animal is a mammal
and most preferably is a human.
[0074] To determine the effect of the administration of the fusion
polypeptide on inhibition of tumor cell growth in laboratory
animals, the animals can either be pre-treated with the fusion
polypeptide and then challenged with a lethal dose of tumor cells,
or the lethal dose of tumor cells can be administered to the animal
prior to receipt of the fusion polypeptide and survival times
documented. To determine the effect of administration of the fusion
polypeptide on inhibition of tumor cell growth in humans, standard
clinical response parameters can be analyzed.
[0075] To determine the amount of fusion polypeptide which would be
an effective tumor cell growth-inhibiting amount, animals can be
treated with tumor cells as described herein and varying amounts of
the fusion polypeptide can be administered to the animals. Standard
clinical parameters, as described herein, can be measured and that
amount of fusion polypeptide effective in inhibiting tumor cell
growth can be determined. These parameters, as would be known to
one of ordinary skill in the art of oncology and tumor biology, can
include, but are not limited to, physical examination of the
subject, measurements of tumor size, X-ray studies and
biopsies.
[0076] The present invention further provides a method for treating
or preventing HIV infection in a human subject, comprising
administering to the subject an HIV replication-inhibiting amount
of the chemokine/HIV antigen fusion polypeptide of this invention.
As used herein, "a replication-inhibiting amount" is that amount of
fusion polypeptide which produces a measurable humoral and/or
effector T cell (cellular) immune response in the subject against
the viral antigen, as determined by standard immunological
protocols, resulting in the inhibition of HIV replication in cells
of the subject, as determined by methods well known in the art for
measuring HIV replication, such as viral load measurement, which
can be determined by quantitative PCR (QPCR) and branched DNA
(bDNA) analysis; reverse transcriptase activity measurement, in
situ hybridization, Western immunoblot, ELISA and p24 gag
measurement (87,88,89,90,91). The fusion polypeptide can be
administered to the subject in varying amounts and the amount of
the fusion polypeptide optimally effective in inhibiting HIV
replication in a given subject can be determined as described
herein.
[0077] The fusion polypeptide of this invention can be administered
to the subject orally or parenterally, as for example, by
intramuscular injection, by intraperitoneal injection, topically,
transdermally, injection directly into the tumor, or the like,
although subcutaneous injection is typically preferred.
Immunogenic, tumor cell growth inhibiting and HIV replication
inhibiting amounts of the fusion polypeptide can be determined
using standard procedures, as described. Briefly, various doses of
the fusion polypeptide are prepared, administered to a subject and
the immunological response to each dose is determined (92). The
exact dosage of the fusion polypeptide will vary from subject to
subject, depending on the species, age, weight and general
condition of the subject, the severity of the cancer or HIV
infection that is being treated, the particular antigen being used,
the mode of administration, and the like. Thus, it is not possible
to specify an exact amount. However, an appropriate amount may be
determined by one of ordinary skill in the art using only routine
screening given the teachings herein.
[0078] Generally, the dosage of fusion protein will approximate
that which is typical for the administration of vaccines, and
typically, the dosage will be in the range of about 1 to 500 .mu.g
of the fusion polypeptide per dose, and preferably in the range of
50 to 250 .mu.g of the fusion polypeptide per dose. This amount can
be administered to the subject once every other week for about
eight weeks or once every other month for about six months. The
effects of the administration of the fusion polypeptide can be
determined starting within the first month following the initial
administration and continued thereafter at regular intervals, as
needed, for an indefinite period of time.
[0079] For oral administration of the fusion polypeptide of this
invention, fine powders or granules may contain diluting,
dispersing, and/or surface active agents, and may be presented in
water or in a syrup, in capsules or sachets in the dry state, or in
a nonaqueous solution or suspension wherein suspending agents may
be included, in tablets wherein binders and lubricants may be
included, or in a suspension in water or a syrup. Where desirable
or necessary, flavoring, preserving, suspending, thickening, or
emulsifying agents may be included. Tablets and granules are
preferred oral administration forms, and these may be coated.
[0080] Parenteral administration, if used, is generally
characterized by injection. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. A more recently revised approach for
parenteral administration involves use of a slow release or
sustained release system, such that a constant level of dosage is
maintained. See, e.g., U.S. Pat. No. 3,710,795, which is
incorporated by reference herein.
[0081] For solid compositions, conventional nontoxic solid carriers
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talc, cellulose,
glucose, sucrose, magnesium carbonate, and the like. Liquid
pharmaceutically administrable compositions can, for example, be
prepared by dissolving, dispersing, etc. an active compound as
described herein and optional pharmaceutical adjuvants in an
excipient, such as, for example, water, saline, aqueous dextrose,
glycerol, ethanol, and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be
administered may also contain minor amounts of nontoxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like, for example, sodium acetate, sorbitan
monolaurate, triethanolamine sodium acetate, triethanolamine
oleate, etc. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this (84).
[0082] The present invention also provides a method for producing
single chain antibodies against tumor antigens comprising producing
a fusion polypeptide comprising a chemokine region and a region
comprising a tumor antigen; immunizing animals with an amount of
the fusion polypeptide sufficient to produce a humoral immune
response to the fusion polypeptide; isolating spleen cells
expressing immunoglobulin specific for the fusion polypeptide;
isolating the immunoglobulin variable genes from the spleen cells;
cloning the immunoglobulin variable genes into an expression
vector; expressing the immunoglobulin variable genes in a
bacteriophage; infecting E. coli cells with the bacteriophage;
isolating bacteriophage from the E. coli cells which express the
immunoglobulin variable genes and isolating the immunoglobulin
variable gene products for use as single chain antibodies.
[0083] The chemokine-scFv fusion proteins described herein would be
better targets than tumor cells or purified tumor antigen peptides
for antibody selection approaches such as phage displayed scFv
production. For example, there are two ways to produce specific Fv
displayed on the surface of phage: (1) Immunize mice with tumor
cells; isolate immunoglobulin variable fragment genes from spleen
cells by RT/PCR; clone the genes into bacteriophage in frame with
genes coding phage surface proteins (e.g., major coat protein
subunits gpVIII or gp III of the filamentous bacteriophage)
(93,94); and (2) Construct semisynthetic antibody libraries by PCR
as described (95). The specific phage producing scFv are selected
by several rounds of binding elution and infection in E. coli,
using biotin labeled chemokine-tumor antigen (e.g., Muccore). The
biotin enables selection of high affinity scFv-phage through
binding to streptavidin conjugated magnetic beads. This approach
provides simple, fast and efficient production of specific
anti-tumor epitope scFv.
[0084] As described herein, the present invention also provides a
nucleic acid which encodes a fusion polypeptide of this invention
and a vector comprising a nucleic acid which encodes a fusion
polypeptide of this invention, either of which can be in a
pharmaceutically acceptable carrier. Such nucleic acids and vectors
can be used in gene therapy protocols to treat cancer as well as to
treat or prevent HIV infection in a subject.
[0085] Thus, the present invention further provides a method of
treating a cancer in a subject diagnosed with a cancer comprising
administering the nucleic acid of this invention to a cell of the
subject under conditions whereby the nucleic acid is expressed in
the cell, thereby treating the cancer.
[0086] A method of treating a B cell tumor in a subject diagnosed
with a B cell tumor is also provided, comprising administering the
nucleic acid of this invention, encoding a chemokine and an
antibody or fragment thereof, in a pharmaceutically acceptable
carrier, to a cell of the subject, under conditions whereby the
nucleic acid is expressed in the cell, thereby treating the B cell
tumor.
[0087] The methods of this invention comprising administering
nucleic acid encoding the fusion protein of this invention to a
subject can further comprise the step of administering a nucleic
acid encoding an adjuvant such as an immunostimulatory cytokine to
the subject, either before, concurrent with or after the
administration of the nucleic acid encoding the fusion protein, as
described herein.
[0088] The nucleic acid can be administered to the cell in a virus,
which can be, for example, adenovirus, retrovirus and
adeno-associated virus. Alternatively, the nucleic acid of this
invention can be administered to the cell in a liposome. The cell
of the subject can be either in vivo or ex vivo. Also, the cell of
the subject can be any cell which can take up and express exogenous
nucleic acid and produce the fusion polypeptide of this invention.
Thus, the fusion polypeptide of this invention can be produced by a
cell which secretes it, whereby it binds a chemokine receptor and
is subsequently processed by an antigen presenting cell and
presented to the immune system for elicitation of an immune
response. Alternatively, the fusion polypeptide of this invention
can be produced in an antigen presenting cell where it is processed
directly and presented to the immune system.
[0089] If ex vivo methods are employed, cells or tissues can be
removed and maintained outside the body according to standard
protocols well known in the art. The nucleic acids of this
invention can be introduced into the cells via any gene transfer
mechanism, such as, for example, virus-mediated gene delivery,
calcium phosphate mediated gene delivery, electroporation,
microinjection or proteoliposomes. The transduced cells can then be
infused (e.g., in a pharmaceutically acceptable carrier) or
transplanted back into the subject per standard methods for the
cell or tissue type. Standard methods are known for transplantation
or infusion of various cells into a subject.
[0090] For in vivo methods, the nucleic acid encoding the fusion
protein can be administered to the subject in a pharmaceutically
acceptable carrier as described herein.
[0091] In the methods described herein which include the
administration and uptake of exogenous DNA into the cells of a
subject (i.e., gene transduction or transfection), the nucleic
acids of the present invention can be in the form of naked DNA or
the nucleic acids can be in a vector for delivering the nucleic
acids to the cells for expression of the nucleic acid to produce
the fusion protein of this invention. The vector can be a
commercially available preparation, such as an adenovirus vector
(Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of
the nucleic acid or vector to cells can be via a variety of
mechanisms. As one example, delivery can be via a liposome, using
commercially available liposome preparations such as LIPOFECTIN,
LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT
(Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec,
Inc., Madison, Wis.), as well as other liposomes developed
according to procedures standard in the art. In addition, the
nucleic acid or vector of this invention can be delivered in vivo
by electroporation, the technology for which is available from
Genetronics, Inc. (San Diego, Calif.) as well as by means of a
SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson,
Ariz.).
[0092] Vector delivery can also be via a viral system, such as a
retroviral vector system which can package a recombinant retroviral
genome (see e.g., 96,97). The recombinant retrovirus can then be
used to infect and thereby deliver to the infected cells nucleic
acid encoding the fusion polypeptide. The exact method of
introducing the exogenous nucleic acid into mammalian cells is, of
course, not limited to the use of retroviral vectors. Other
techniques are widely available for this procedure including the
use of adenoviral vectors (98), adeno-associated viral (AAV)
vectors (99), lentiviral vectors (100), pseudotyped retroviral
vectors (101). Physical transduction techniques can also be used,
such as liposome delivery and receptor-mediated and other
endocytosis mechanisms (see, for example, 102). This invention can
be used in conjunction with any of these or other commonly used
gene transfer methods.
[0093] Various adenoviruses may be used in the compositions and
methods described herein. For example, a nucleic acid encoding the
fusion protein can be inserted within the genome of adenovirus type
5. Similarly, other types of adenovirus may be used such as type 1,
type 2, etc. For an exemplary list of the adenoviruses known to be
able to infect human cells and which therefore can be used in the
present invention, see Fields, et al. (103). Furthermore, it is
contemplated that a recombinant nucleic acid comprising an
adenoviral nucleic acid from one type adenovirus can be packaged
using capsid proteins from a different type adenovirus.
[0094] The adenovirus of the present invention is preferably
rendered replication deficient, depending upon the specific
application of the compounds and methods described herein. Methods
of rendering an adenovirus replication deficient are well known in
the art. For example, mutations such as point mutations, deletions,
insertions and combinations thereof, can be directed toward a
specific adenoviral gene or genes, such as the E1 gene. For a
specific example of the generation of a replication deficient
adenovirus for use in gene therapy, see WO 94/28938 (Adenovirus
Vectors for Gene Therapy Sponsorship) which is incorporated herein
in its entirety.
[0095] In the present invention, the nucleic acid encoding the
fusion protein can be inserted within an adenoviral genome and the
fusion protein encoding sequence can be positioned such that an
adenovirus promoter is operatively linked to the fusion protein
nucleic acid insert such that the adenoviral promoter can then
direct transcription of the nucleic acid, or the fusion protein
insert may contain its own adenoviral promoter. Similarly, the
fusion protein insert may be positioned such that the nucleic acid
encoding the fusion protein may use other adenoviral regulatory
regions or sites such as splice junctions and polyadenylation
signals and/or sites. Alternatively, the nucleic acid encoding the
fusion protein may contain a different enhancer/promoter (e.g., CMV
or RSV-LTR enhancer/promoter sequences) or other regulatory
sequences, such as splice sites and polyadenylation sequences, such
that the nucleic acid encoding the fusion protein may contain those
sequences necessary for expression of the fusion protein and not
partially or totally require these regulatory regions and/or sites
of the adenovirus genome. These regulatory sites may also be
derived from another source, such as a virus other than adenovirus.
For example, a polyadenylation signal from SV40 or BGH may be used
rather than an adenovirus, a human, or a murine polyadenylation
signal. The fusion protein nucleic acid insert may, alternatively,
contain some sequences necessary for expression of the nucleic acid
encoding the fusion protein and derive other sequences necessary
for the expression of the fusion protein nucleic acid from the
adenovirus genome, or even from the host in which the recombinant
adenovirus is introduced.
[0096] As another example, for administration of nucleic acid
encoding the fusion protein to an individual in an AAV vector, the
AAV particle can be directly injected intravenously. The AAV has a
broad host range, so the vector can be used to transduce any of
several cell types, but preferably cells in those organs that are
well perfused with blood vessels. To more specifically administer
the vector, the AAV particle can be directly injected into a target
organ, such as muscle, liver or kidney. Furthermore, the vector can
be administered intraarterially, directly into a body cavity, such
as intraperitoneally, or directly into the central nervous system
(CNS).
[0097] An AAV vector can also be administered in gene therapy
procedures in various other formulations in which the vector
plasmid is administered after incorporation into other delivery
systems such as liposomes or systems designed to target cells by
receptor-mediated or other endocytosis procedures. The AAV vector
can also be incorporated into an adenovirus, retrovirus or other
virus which can be used as the delivery vehicle.
[0098] As described above, the nucleic acid or vector of the
present invention can be administered in vivo in a pharmaceutically
acceptable carrier. By "pharmaceutically acceptable" is meant a
material that is not biologically or otherwise undesirable, i.e.,
the material may be administered to a subject, along with the
nucleic acid or vector, without causing any undesirable biological
effects or interacting in a deleterious manner with any of the
other components of the pharmaceutical composition in which it is
contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject, as would be well known to one of skill
in the art.
[0099] The mode of administration of the nucleic acid or vector of
the present invention can vary predictably according to the disease
being treated and the tissue being targeted. For example, for
administration of the nucleic acid or vector in a liposome,
catheterization of an artery upstream from the target organ is a
preferred mode of delivery, because it avoids significant clearance
of the liposome by the lung and liver.
[0100] The nucleic acid or vector may be administered orally as
described herein for oral administration of the fusion polypeptides
of this invention, parenterally (e.g., intravenously), by
intramuscular injection, by intraperitoneal injection,
transdermally, extracorporeally, topically or the like, although
intravenous administration is typically preferred. The exact amount
of the nucleic acid or vector required will vary from subject to
subject, depending on the species, age, weight and general
condition of the subject, the severity of the disorder being
treated, the particular nucleic acid or vector used, its mode of
administration and the like. Thus, it is not possible to specify an
exact amount for every nucleic acid or vector. However, an
appropriate amount can be determined by one of ordinary skill in
the art using only routine experimentation given the teachings
herein (84).
[0101] As one example, if the nucleic acid of this invention is
delivered to the cells of a subject in an adenovirus vector, the
dosage for administration of adenovirus to humans can rahge from
about 10.sup.7 to 10.sup.9 plaque forming units (pfu) per
injection, but can be as high as 10.sup.12 pfu per injection
(104,105). Ideally, a subject will receive a single injection. If
additional injections are necessary, they can be repeated at six
month intervals for an indefinite period and/or until the efficacy
of the treatment has been established.
[0102] Parenteral administration of the nucleic acid or vector of
the present invention, if used, is generally characterized by
injection. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions. A more recently revised approach for parenteral
administration involves use of a slow release or sustained release
system such that a constant dosage is maintained. See, e.g., U.S.
Pat. No. 3,610,795, which is incorporated by reference herein in
its entirety.
[0103] The present invention is more particularly described in the
following examples which are intended as illustrative only since
numerous modifications and variations therein will be apparent to
those skilled in the art.
EXAMPLES
[0104] Mice and tumor. C3H/HeN female mice 6 to 12 weeks of age
were obtained from the Animal Production Area of the National
Cancer Institute-Frederick Cancer Research and Development Center
(NCI-FCRDC, Frederick, Md.). The cell line 38c13 is a
carcinogen-induced murine B cell tumor cell line (125). The 38c13
tumor cell secretes and expresses IgM(.kappa.) on the cell surface
and is MHC class I positive but class II negative. 38c13 cells from
a common frozen stock were passaged in vitro 3 days before use in
RPMI 1640 supplemented with 100 U/ml of penicillin and
streptomycin, 2.times.10.sup.-5 M 2-mercaptoethanol and heat
inactivated 10% fetal bovine serum (BioWhitaker).
[0105] Construction of expression vectors. Two types of expression
systems have been used to produce scFv and scFv fusions. In one
system, nucleic acid encoding the fusion protein was expressed in a
modified pet11d vector (Stratagene) and purified from inclusion
bodies of E.coli. In the second system, the nucleic acid encoding
the fusion polypeptide was cloned into a pCMVE/AB (Arya Biragyn)
vector under regulatory elements of the early promoter and enhancer
of CMV and expressed in the epidermis of mice as a naked DNA
vaccine.
[0106] Fv fragments were cloned from two different B cell
lymphomas, 38C13 and A20, respectively (106,107) by RT/PCR and
produced as recombinant fusion peptides with either IP-10,
respectively designated as IP10scFv38 and IP10scFv20A, or
MCP3scFv38 and MCP3scFv20A. Specifically, lymphoma specific Vh and
V1 fragments were cloned by RT/PCR techniques as single chain
antibody from total RNA of 38c13 and A20 tumor cells, designated
scFv38 and scFv20A respectively, using the following primers.
1 PRVh-5': PRV.sub.H38-5': CTCGAGG TGAAGCTGGTGGAGTCTGGA (SEQ ID
NO:17) PRVh-3': PRV.sub.H38-3': AGAGGAGA CTGTGAGAGTGGTGCCTT (SEQ ID
NO:18) PRVL-5': PRV.sub.L38-5': GACATCCAGATGACACAGTCTCCA (SEQ ID
NO:19) PRV1-3': PRV.sub.L38-3':
GGATCCTTTTATTTCCAGCTTGGTCCCCCCTCCGAA (SEQ ID NO:20)
PRV.sub.H20A-5': CCATGGTCCAAC TGCAGCAGTCAGGGCCTGAC (SEQ ID NO:21)
PRV.sub.H20A-3': TGAGGAGACTGTGAGTTCGGTACCT- T GGCC (SEQ ID NO:22)
PRV.sub.L20A-5': GATGTTGTGATGACGCAGACTCCACTC (SEQ ID NO:23)
PRV.sub.L20A-3': GGATCCTT TGACTTCCAGCTTTGTGCCTCCA (SEQ ID
NO:24)
[0107] The resulting scFv contained a (Gly.sub.4Ser).sub.3 linker
and was cloned into the expression vector pET11d, which was
modified to fuse in frame with c-myc and the His tag peptide
sequences, followed by an amber stop codon. The resulting scFv
contained a 17 a.a. residue linker, GGGGSGGGGSGGGGSGS
(Gly.sub.4Ser).sub.3GlySer (SEQ ID NO:57) (108).
[0108] Constructs for the nDNA vaccination were fused in frame to a
leader sequence of IP-10 in pCMVE/AB to enable secretion. The
carboxy-terminus of scFv was fused in frame with the tag sequence
encoding c-myc peptide and six His residues, respectively: GGA TCC
GCA GAA GAA CAG AAA CTG ATC TCA GAA GAG GAT CTG GCC CAC CAC CAT CAC
CAT CAC TAA CCCGGG (SEQ ID NO:25). Genes for the mature sequence of
murine chemokines, IP-10 and MCP-3, were cloned by RT/PCR technique
from RNA of the LPS-induced murine monocyte cell line ANA-1 (109)
utilizing the following primers:
2 PRmIP10-5': CCATGGCCATCCCTCTCGCAAGGACGGTCCGC (SEQ ID NO:26)
PRmIP10-3': GAATTCAGG AGCCCTTTTAGACCTTTTTTG (SEQ ID NO:27)
PRmMCP3-5': ACCATGGCCCAACCAGATGGGCC CAATGCA (SEQ ID NO:28)
PRmMCP3-3': GAATTCAGGCTTTGGAGTTGGGGTTTTCAT (SEQ ID NO:29)
[0109] The cDNA for human monocyte chemotactic protein-3 was PCR
amplified and subcloned using the specific primers:
3 PRhMCP3-5': ACCATGGCGCAACCGGTAGGTATAAACACAAGCA (SEQ ID NO:30)
PRhMCP3-3': GAATTCCAGTTTCGGCGTCTGTGTCTTTTTA (SEQ ID NO:31)
[0110] Human IP10 was PCR amplified and subcloned using specific
primers:
4 PrhIP10-1: CCCATGGTACCTCTCTCTAGAACCGTA (SEQ ID NO:32) PrhIP10-R1:
GGATCCTTAAGGAGATCTTTTAGACATTTCCTTGCTAACT (SEQ ID NO:33)
[0111] IP-10, MCP-3 or control viral epitope (PreS2 and DomA)
fusions were made by fusing them to amino-terminus of scFv through
a short spacer sequence: 5'GAA TTC AAC GAC GCT CAG GCG CCG AAG AGT
CTC GAG 3' (SEQ ID NO:34), encoding the amino acid sequence:
EFNDQAPKSLE (SEQ ID NO:11). Two unique restriction endonuclease
sites were introduced at the ends of the space to facilitate
cloning: EcoRI at the 5.dbd. end (underlined) and XhoI at the 3'
end (underlined). All constructs were verified by DNA
dideoxy-sequencing method, using T7 SEQUENASE kit (Amersham).
[0112] IP10 or MCP-3 chemokines were cloned into the scFv38
expression vector through NcoI and XhoI restriction sites. The
resulting fusion nucleic acid contained the chemokine gene ligated
to the 5'-end of the scFv38 gene and separated with a short spacer
sequence, as described above.
[0113] Bacterial expression and scFv purification. The recombinant
proteins were expressed in BL21(DE3) cells (InVitrogen) as
inclusion bodies after 8 hours of induction in Super-Broth with 0.8
mM IPTG in the presence of 150 .mu.g/ml carbenicillin and 50
.mu.g/ml ampicillin at 30.degree. C. IP10-scFv38, MCP3-scFv38 and
scFv38 were purified from the inclusion bodies with a modified
method (110). Briefly, inclusion bodies, denatured in 6M GuHCl, 100
mM NaH.sub.2PO.sub.4, 10 mM Tris-HCl, pH 8.0, were reduced in 0.3M
DTE and refolded at a concentration of 80 .mu.g/ml in the refolding
solution (Tris-HCl, pH 8.0, 0.5M arginine-HCL, 4 mM GSSG and 2 mM
EDTA) for 72 hours at 10.degree. C. The refolded solution was
dialyzed in 100 mM Urea and 20 mM tris-HCl, pH 7.4 and the
recombinant protein was purified by binding to heparin-sepharose
resins (Pharmacia, Biotech, Uppsala, Sweden). The integrity and
purity of the recombinant protein was tested by PAGE gel
electrophoresis in reducing conditions and by Western blot
hybridization with mAb 9E10. The purification yielded 2-20 mg/l of
the soluble protein with greater than 90% purity.
[0114] Purified fusion polypeptide was tested for the ability to
inhibit binding of native IgM 38c13 (Id38), as compared to positive
sera from mice immunized with Id38-KLH. ELISA plates were coated
with 10 .mu.g/ml Id38, then wells were incubated with anti-Id38
positive sera (1:500) and titrated amounts of scFv. Id38 (10 .mu.g
/ml) and IP10scFv20 (IP-10 fused to an irrelevant scFv) were used
as positive and negative control samples, respectively.
[0115] Recombinant fusion proteins purified from E coli were
characterized for proper idiotype folding by their ability to
inhibit 38c13 IgM binding to a monoclonal (SIC5 mAb) or polyclonal
anti-idiotypic sera. These results suggest that IP-10 and MCP-3
fusion did not interfere with the proper conformation of scFv38.
Next, receptor binding experiments demonstrated that both IP-10 and
MCP-3 fused scFv, but not control viral epitope DomA fused scFv38
(DomAscFv38), bound to their respective chemokine receptors on
unfractionated murine splenocytes and purified T cells. The native
ligands IP-10 or MCP-3 inhibited only binding of respective
chemokine-scFv. Moreover, no binding was detected with truncated
IP10TFBscFv38, which contained an intact heparin binding domain of
IP-10 with a deleted (amino-terminal 9 a.a. residues) chemokine
receptor binding portion. Next, chemotactic activity of the fusion
proteins was tested. All chemokine-scFv proteins, but not a viral
epitope fused scFv38 control (PreS2scFv38), induced in vitro
chemotaxis of murine lymphocytes in a dose dependent manner.
Chemotactic activity also was confirmed in vivo by histologic
evaluation of the skin at the site of injection, which showed that
the dermis and subcutaneous layers injected with IP10FBscFv38 or
MCP3FBscFv38, but not with control PreS2scFv38, contained
significant numbers of infiltrating mononuclear cells and some PMN.
Therefore, IP-10 and MCP-3 fused scFv proteins were biologically
active and retained functional properties of their corresponding
chemokines.
[0116] In vivo immunization and tumor protection. Six- to nine-week
old female C3H/HeN mice were immunized intraperitoneally (i p) with
100 to 200 .mu.g of the soluble protein in PBS and control
immunogen Id38-KLH two times at two week intervals or were shaved
and immunized by Accell gene delivery device (Agracetus, Inc.,
Middleton, WN) with 1.mu. gold particles carrying 1-3 .mu.g plasmid
DNA. Sera were collected by orbital bleeding two weeks after each
vaccination. Serum anti-idiotypic (anti-Id) antibody levels were
tested as described (111) over microtiter plates coated with 10
.mu.g/ml native IgM 38c13. Two weeks after the last immunization,
mice were inoculated with 2000 38c13 tumor cells i.p. Survival was
determined, and significance with the respect to time to death, was
assessed using BMDP IL software (BMDP statistical software, Los
Angeles). Mice were observed daily for any signs of toxicity and
date of death and animals surviving >80 days after tumor
challenge were killed and were reported as long-term survivors.
[0117] Mice were immunized either with a plasmid coding for
MCP3scFv38 fusion or a mixture of DNA constructs expressing
unlinked scFv38 and MCP3scFv20A (scFv38D+MCP3scfv20AD).
[0118] Ten mice per group were immunized with two types of scFv38
fused to IP-10, respectively IP10scFv38 or IP10scFv38(INV),
differing only in orientation of variable genes in scFv. Control
mice received IgM-KLH (Id38-KLH) and IP10 fusion to A20 lymphoma
scFv (IP10scFv20A). Ten mice per group were immunized i.d. with
plasmid coding either for chemokine fusion vaccine (MCP3scFv38D),
or free scFv (scFv38D), or viral epitope preS2 fused scFv
(PreS2scFv38D).
[0119] Effector CD8.sup.+ and CD4.sup.+ cells were depleted two
weeks after the last immunization with three i.p. injections of 400
.mu.g .alpha.-CD8 mAb 53.6.72, or .alpha.-CD4 mAb GK1.5 (both
ammonium sulfate purified ascites, Biological Resource Branch,
NCI-FCRDC) (32,34), or control rat IgG (Sigma). Control mice were
immunized with plasmid expressing MCP3 fused to A20 scFv
(MCP3scFv20AD). The .alpha.-CD8.sup.+ depletion resulted in a drop
of CD8.sup.+ cells from 9.5% to 0.7%, while CD4.sup.+ cell
proportions remained unchanged, about 16%, as monitored by
fluorescence activated cell sorting (FACS) staining. Similarly,
.alpha.-CD4.sup.+ mAb treatment decreased the proportion of
CD4.sup.+ cells from 18% to 1.8%, while it did not affect the
CD8.sup.+ cell count, which remained at 10.6%.
[0120] Ten Balb/C mice per group were immunized i.p. twice with 100
.mu.g of IP-10 or MCP-3 fused with scFv20A protein in PBS
(IP10scFv20A and MCP3scFv20A, respectively) and challenged i.p.
with 10.sup.5 A20 tumor cells. To determine the role of free versus
linked chemokine, IP10scFv20A (which failed to protect, but
expressed the correct scFv20A) was co-injected with MCP-3 fused to
an irrelevant scFv38 (IPscFv20A+MCP3scFv38). Control mice were
immunized with A20 IgM-KLH (Id20A-KLH).
[0121] Immunoassays and serum anti-idiotypic antibody. The
assessments for correct folding of purified scFv38 and fusion
scFv38 were determined by ELISA with mAbs and by inhibition assay
with Id38-KLH sera (immunized with native IgM 38c13 conjugated to
KLH). Briefly, microtiter plates (Nunc, Naperville, Ill.) were
coated overnight at 4.degree. C. with 10 .mu.g/ml anti-c-myc mAb
9E10 in carbonate buffer (50 mM NaHCO.sub.3, pH 9.0). The wells
were blocked with 5% nonfat dry milk in PBS for 30 min. Plates were
washed in 0.05% Triton X-100 in PBS, and serially diluted scFv
(starting from 10 .mu.g/ml in 50 .mu.l 2% BSA/PBS) was applied,
after which plates were incubated 40 min at room temperature. After
washing, the wells were incubated with 50 .mu.l of 1:300 diluted
biotinylated anti-Id38 mAb in 2% BSA/PBS for 30 min at room
temperature. Wells were washed and incubated with streptavidin-HRP
conjugate (1:5000) in 2% BSA/PBS for 30 min at room temperature.
Then, wells were washed and incubated with ABTS peroxidase
substrate (KPL, Gaithersburg, Md.) and the absorbance at 405 nm was
measured.
[0122] Inhibition assays were performed as described above, except
plates were coated with 10 .mu.g/ml of native IgM 38c13, then,
wells were incubated for 30 min at room temperature with a 1:2
dilution of positive Id38-KLH sera mixed with serially diluted
purified scFv proteins starting from 50 .mu.g/ml in 2% BSA/PBS. The
bound antibodies from the sera were assayed by incubating wells for
30 min at room temperature with anti-mouse IgG-HRP mAb
(Jackson).
[0123] Serum anti-idiotypic (anti-Id) antibody levels were tested
as described (37). Briefly, mouse serum was serially diluted over
microtiter plates coated with 10, .mu.g/ml native IgM 38c13.
Binding of antibodies in the serum to 38c13 IgM was detected by
goat anti-mouse IgG-HRP. Serum anti-Id antibody levels were
quantitated by comparing sera titration curves with a standard
curve obtained with a known concentration of a mixture of purified
monoclonal anti-Id antibodies. Antibody levels were expressed in
g/ml of serum for individual mice. In each ELISA, sera obtained
from mice immunized with control IgM-KLH were included as negative
controls. Such sera never showed any titration binding activity on
Id-38c13.
[0124] In vitro and in vivo chemotaxis assays. Single cell
suspensions were prepared from spleens of untreated C3H/HeJ mice.
Murine T cell enrichment columns (R&D System, Minneapolis,
Minn.) were then used to prepare a purified murine T cell
population via high-affinity negative selection according to the
manufacturer's instructions. The isolation procedure typically
yielded over 89% CD3.sup.+ T cells, as determined by FACS analysis.
T cell migration in vitro was assessed by 48-well microchemotaxis
chamber technique. Briefly, a 26 .mu.l aliquot of the recombinant
scFv fusion protein serially diluted in the chemotaxis medium (RPMI
1640, 1% BSA, 25 mM HEPES) was placed in the lower compartment and
50 .mu.l of cell suspension (5.times.10.sup.6 cells/ml) was placed
in the upper compartment of the chamber. The two compartments were
separated by a polycarbonate filter (5 .mu.m pore size; Neuroprobe,
Cabin John, Md.) coated with 10 .mu.g/ml of fibronectin (Sigma, St.
Luis, Mo.) and incubated overnight at 4.degree. C. or for 2 hours
at 37.degree. C. The chemotaxis assay was performed at 37.degree.
C. for 2 hours. Then the filter was removed, fixed and stained with
Diff-Quik (Harlew, Gibbstown, N.J.). The number of migrated cells
in three high power fields (400 x) was counted by light microscopy
after coding the samples. The results are expressed as the mean
.+-.SE value of the migration in triplicate samples.
[0125] T cell migration in vitro was assessed by the 48-well micro
chemotaxis chamber technique as described (112). Single cell
suspensions were prepared from spleens of untreated C3H/HeJ mice.
Murine T cell enrichment columns (R&D System, Minneapolis,
Minn.) were then used to prepare a purified murine T cell
population via high-affinity negative selection according to the
manufacturer's instruction. The isolation procedure typically
yielded over 89% CD3.sup.+ T cells, as determined by FACS
analysis.
[0126] In order to test in vivo effects on cell accumulation,
C3H/HeN mice were injected s.c. with a single 10 .mu.g dose of scFv
fusion proteins. Portions of the skin from the site of injection
were removed 72 hours after the injection, fixed in 10% neutral
buffered formalin, embedded in paraffin, sectioned at 5 .mu.m and
stained with hematoxylin and eosin (H&E). Slides were evaluated
microscopically without knowledge of the experimental
treatment.
[0127] In vivo cellular infiltration into murine skin. The numbers
of PMN and mononuclear (MN) cells infiltrated into murine skin were
graded as following: -, no significant lesion; 1, mild; 2,
moderate; 3 severe; F, focal; MF, multi focal. Mice were injected
with 10 .mu.g of IP10scFv38 (N8), MCP3-scFv38 (N21), preS2scFv38
(N18), or PBS, subcutaneously. After 72 h, the injection site was
excised and examined histologically on coded slides to determine
the extent of infiltration. The amount of endotoxin injected with
samples was 0.5 1 units.
[0128] Chemokine binding assay and confocal microscopy. Chemokine
binding assays were performed using laser confocal microscopy
(113). Purified T cells or spleen cells from C3H mice were used at
.about.1.times.10.sup.6 per ml and were incubated with 100 nM
chemokine-scFv (N6IP10scFv38, N21MCP3scFv38), control viral
epitope-scFv (N2 DomAscFv38), or truncated IP10scFv38
(N16IP10TscFv38) for 1 hour at 37.degree. C. For the ligand
competition assay, 100 nM chemokine-scFv was incubated with 500 nM
of the corresponding chemokine (IP-10 or MCP-3). Samples were
washed 2.times. in PBS and fixed in suspension with 2%
paraformaldehyde. The samples were incubated at RT for 15 min.
Slides containing the samples were incubated in 9E10 anti c-myc mAb
primary antibody at a 1:50 dilution in wash buffer (0.25% gelatin,
0.15% saponin, 1% goat serum in PBS). Slides were then incubated
with goat anti-mouse IgG F(ab')2-FITC (Boehringer-Mannheim) at a
1:50 dilution for 30 min at RT in a humidified chamber. Slides were
washed 3.times.5 min in 0.25% gelatin, 0.15% saponin in TBS. Slides
were then incubated for 10 min in a 1:100 dilution of DAPI, washed
2.times. briefly in TBS, then 1.times. briefly in dH.sub.2O,
air-dried and mounted using aqueous mounting medium appropriate for
immunofluorescence (Gel/Mount, Biomeda).
[0129] The traditional approach to enhance immunogenicity by cross
linking to KLH is not effective. Several different approaches were
used for the production of single chain antibody fragments from
38c13 cells (scFv38) in E.coli. Yield of scFv38 differed
significantly depending on the method used. Production of scFv38
through a secretory path using a PelB leader sequence as a native
protein was least efficient. The problem was solved when scFv38 was
produced as insoluble "inclusion" bodies, which yielded about 2-8
mg of refolded scFv per liter of the batch culture with greater
than 90% purity. Folding properties of the produced scFv38 were
monitored by either (i) inhibition assay with native Id38; or (ii)
modified ELISA assay where scFv38 was captured through an
anti-c-myc tag and detected with the biotinylated monoclonal
anti-Id38 antibody (anti-Id38 mAb does not recognize linear or
incorrectly folded epitope). These experiments demonstrated that
scFv38, but not irrelevant scFv20A, specifically binds to
anti-Id38c mAb and inhibits binding of the native Id38c to
anti-Id38c mAb, 50% binding inhibition by 10-15 fold excess of
scFv38. In addition, positive sera from Id38c-KLH immunized mice
specifically recognized purified scFv38. These data indicate that
purified scFv38 is folded correctly and imitates the idiotype of
the native antibody (Id38c) of B cell lymphoma 38c13.
[0130] Immunization experiments showed that scFv38, similarly to
the native Id38c IgM, is a poor immunogen. Attempts were made to
convert scFv38 into a potent immunogen by chemical cross linking
with KLH, in analogy to the native Id38c. However, in contrast to
Id38-KLH, i.p. immunizations of syngeneic mice with 100 .mu.g of
scFv38-KLH did not elicit any anti-Id38c specific antibody
response. This inability to induce anti-Id38 response correlates
with the loss of ability to affect binding of anti-Id38 mAb (SIC5)
to Id38c by samples containing scFv38-KLH, while a control sample
of an equimolar mixture of non-cross linked scFv38 and KLH
(scFv38+KLH) inhibited anti-Id38/Id38c binding similarly to pure
scFv38. These data indicate that a fragile Id conformation of
scFv38 was removed by KLH cross linking and that this traditional
approach is not applicable for the enhancement of immunogenicity of
scFv38.
[0131] Design and Production of Chemokine Fused scFv38. Murine IP10
was subcloned from LPS induced monocyte cell line ANA-1 by RT/PCR
using specific primers as described herein and inserted in frame in
front of the scFv38 DNA sequence. The resulting fusion gene was
designated as IP10FBscFv38MH. Similarly MCP-3 fused scFv38 was
constructed and designated as MCP3FBscFv38MH. In order to evaluate
input of the immunoglobulin V chain specific orientation, two
variants of fusion chemokine-scFv genes were designed, one
containing a V.sub.H-V.sub.L and one containing a V.sub.L-V.sub.H
sequence, respectively designated as scFv38MH and
scFv38(INV)MH.
[0132] All fusion proteins used in these experiments were purified
from inclusion bodies of E.coli, solubilized and refolded as
described herein. A spacer sequence, as described herein, was
introduced into the chemokine fusion proteins and correct folding
was tested for each recombinant protein. These tests demonstrated
that both IP-10 and MCP-3 fused scFv38 recombinant proteins folded
correctly, thereby imitating the structure of native idiotype of
38c13 lymphoma Ig.
[0133] IP10- and MCP3-scFv38 fusion proteins retain functional
properties of chemokines. The ability of the fusion proteins to
induce chemotaxis in vitro of spleen cells or purified T cells from
C3H/HeN was tested. Both chemokine fusion proteins IP10FBscFv38MH
and MCP3FBscFv38MH induced chemotaxis of murine lymphocytes in a
dose dependent manner, demonstrating a typical bell shape curve,
with the maximum activity at 100 .mu.g/ml concentration. Therefore,
these data indicate that scFv38 became chemotactic due to the
presence of the fused chemokine. A control sample, the viral
epitope, but not chemokine, fused to scFv38 (PreS2FBscFv38MH) did
not cause any in vitro chemotaxis.
[0134] The ability of IP10FBscFv38MH and MCP3FBscFv38MH proteins to
induce chemotaxis in vivo in C3H/HeN mice was also tested. Mice
were s.c. injected once with 10 .mu.g of the fusion protein and
after 72 hours, the skin around the site of injection was removed
and analyzed as described herein. The endotoxin contamination level
of samples injected was less than 0.5-1 units. Histologic
evaluation of the skin at the site of the injection showed that the
dermis and subcutaneous layer contained significant amounts of
monocytes and less PMN infiltration in mice injected with
IP10FBscFv38MH and MCP3FBscFv38MH. In contrast, the skin from mice
injected with control PreS2FBscFv38MH showed no significant
cellular infiltration. These data demonstrate that scFv38 is
converted into a chemotactic protein by fusion with IP-10 or MCP-3
chemokines and that these proteins are able to induce chemotaxis in
vivo.
[0135] Chemokine binding competition experiments were performed on
purified murine T cells and spleen cells. Confocal microscopy
experiments demonstrated that both chemokines IP-10 and MCP-3 fused
with scFv38, but not control viral epitope DomA fused with scFv38
(DomAFBscFv38), bound to purified murine T cells and spleen cells.
The binding was specifically inhibited by incubation with the
corresponding native chemokine, IP-10 or MCP-3, respectively. Thus,
chemokine fused scFv38 acts as a chemotactic protein via binding to
the corresponding chemokine receptor.
[0136] Chemokine fusion enables conversion of scFv into a good
immunogen. To test the potency of the chemokine fused to scFv38,
syngeneic C3H mice were i.p. injected with 100 .mu.g of the
purified fusion protein, without any adjuvants. No significant
anti-Id38 antibody response was induced by repeated injections of
scFv38 alone (up to 200 .mu.g). However, the anti-Id38 antibody
response was induced by injection of IP10FBscFv38 and MCP3FBscFv38
into mice. This response was specific to 38c13 lymphoma Id, because
only immune sera from groups IP10FBscFv38, MCP3FBscFv38 and
Id38-KLH reacted in ELISA with the IgM from 38c13, but not with an
isotype matched irrelevant IgM. The positive anti-Id38 response was
not detected when mice were immunized with a IP10-scFv38, which had
indistinguishable chemotactic activity and an incorrectly folded
scFv38.
[0137] The single immunization of 50-100 .mu.g of IP10FBscFv38
induced detectable levels of anti-Id38 titers. However,
significantly higher amounts of the specific antibody were detected
usually after three immunizations. Interestingly, despite
comparable levels of Id folding, as detected in vitro, IP10FBscFv38
was a much more potent anti-Id38 inducer, than MCP3FBscFv38. Three
immunizations with IP10FBscFv38 produced about 15 to 233 .mu.g/ml
anti-Id38, while MCP3FBscFv38 produced from 1 to 25 .mu.g/ml.
However, these levels of anti-Id38 production were lower than the
amount produced after a single i.p. injection of 50 .mu.g Id38-KLH
antibody, which was within a range of 400-800 .mu.g/ml.
[0138] Immunization with chemokine fused scFv38 can protect against
tumor challenge. In the 38c13 model, a tumor challenge dose as low
as 100 cells is lethal in 100% of control immunized mice (111). It
has been shown that a single i.p. immunization with 25-50 .mu.g of
tumor-derived Id conjugated to KLH in syngeneic C3H mice produced
modest resistance against a subsequent minimum lethal dose i.p.
tumor challenge 2 weeks later, with no significant differences
between different routes of immunization (111).
[0139] Ten animals per group were immunized with 100 .mu.g of
IP10FBscFv38 or IP10FBscFv38(INV) three times at biweekly
intervals. Control group animals were injected either with IP-10
fused to the irrelevant lymphoma scFv (IP10FBscFv20A), scFv38
alone, or PBS. The survival rate of the control group of animals
immunized with IP10FBscFv20A was indistinguishable from the
survival rate of the group that received scFv38 alone or PBS
injections, suggesting the lack of any antitumor effects of IP10
immunization (all animals died within 14 days). However,
immunization with either variant of scFv fused with IP-10
significantly increased survival of mice (P=0.0002, respectively,
groups 1 and 2 vs. group 3; n=10 mice per group). Survival data
were not different between groups 1 and 2, indicating that linkage
in either orientation of variable chains in scFv38 resulted in an
equivalent Id38 folding and consequently induced a similar
anti-tumor response. A positive control group of animals immunized
with native antibody Id38 cross-linked to KLH demonstrated the
highest survival with the median at 28 days.
[0140] In contrast to IP-10 fused to scFv38, mice immunized with
MCP3FBscFv38 were not protected reproducibly, although in some
experiments, an increase in survival was observed, which correlates
with the ability of this protein to induce an anti-Id38 antibody
response. The animals immunized with the incorrectly folded scFv38
fused to IP-10, IP10scFv38, could not elicit an anti-Id38 response
and demonstrated no protection against tumor challenge. These data
indicate that scFv38 is converted into a potent immunogen through
fusion with chemokine IP-10 and that it can induce significant
tumor protection. The protection depended mostly from the ability
to elicit higher titers of anti-Id38 antibody.
[0141] Naked DNA vaccination with IP-10 fused scFv38. In an attempt
to improve the potency of the anti-tumor response, expression
plasmid vectors were constructed, consisting of the
promoter-enhancer sequence from the CMV early gene linked to either
a scFv gene alone or fused with MCP-3 or IP-10 for
particle-mediated DNA vaccine delivery. Mice received three
biweekly i.d. immunizations, consisting of four shots of 0.5 mg
gold particles carrying 1-3 .mu.g of plasmid DNA each. Mice
receiving control plasmids scFv38D, PreS2scFv38D (50 a.a. preS2
region of the middle surface antigen of HBV, a non-chemokine
carrier), or scFv20AD (MCP-3 fusion to the A20 scFv) generated no
anti-idiotypic antibody. In contrast, immunization with MCP3 or
IP-10 fusions elicited high levels of anti-Id38 antibody (mean
909.+-.625 and 752.+-.660 .mu.g/ml, respectively). Furthermore,
these levels of antibody were comparable to those elicited by
IgM-KLH (mean 576.+-.104). Groups of ten immunized mice were
challenged with tumor two weeks after the final immunization.
Significant protective immunity was demonstrated in mice immunized
with MCP3scFv38D (40% survival, log rank P=0.005 vs. PreS2scFv38D
control). The survival of mice receiving either of control plasmids
PreS2scFv38D, free scFv38D or MCP-3 fusion to the irrelevant
scFv20D was not significantly different from those receiving PBS.
Furthermore, the magnitude of protection with MCP3scFv38D exceeded
that demonstrated with IgM-KLH (P<0.03, chi-square analysis of
pooled data). In addition, to determine if the chemokine moiety
must be linked to scFv to render its immunogenicity, mice were
immunized with a mixture of DNA constructs expressing unlinked
scFv38 and MCP3scFv20A in a separate experiment. Neither antibody
response, nor survival was observed. Thus, this effect required
that the chemokine be physically linked with scFv.
[0142] In vivo depletion of T-cell subsets. The MCPscFv38D induced
protective immunity mediated by effector CD8.sup.+ or CD4.sup.+ T
cells was also investigated. Two weeks after immunization with
MCP3scFv38nDNA, groups of ten mice were randomly assigned to
treatment with specific mAb depleting CD8.sup.+ or CD4.sup.+ T
cells, or with normal rat IgG as a control every other day for
three doses before challenge. Comparison of treated and untreated
mice immunized with MCP3scFv38D revealed a loss of protection for
the groups receiving either anti-CD8 mAb (0% vs 40% survival,
respectively, log rank P=0.08) or anti-CD4 mAb (0% vs 30% survival,
respectively). These data indicate that immunization with a naked
DNA construct expressing MCP-3 fused to scFv38 elicited efficient
anti-Id38 antibody production sufficient to delay tumor growth but
complete protection was dependent upon T cells at the effector
phase of the response. Thus, these data indicate that, in addition
to inducing a vigorous humoral response, MCP3scFv38D induces
effector CD8.sup.+ and CD4.sup.+ T cells, which are required for
protective anti-tumor immunity elicited by pMCP3scFv38 (P<0.004
by Fisher's exact test, logrank P=0.02, T-cell depleted vs. rat IgG
treated).
[0143] MCP-3 fusion also elicits protective anti-tumor immunity in
a second lymphoma model. Comparable results were observed with the
MCP3scFv20A fusion protein and plasmid MCP3scFv20AD, which also
elicited protective immunity against corresponding A20 lymphoma
cells. Of particular importance, the potency of MCP3scFv20A fusions
was also superior to that of IgM-KLH in this second lymphoma model
(40% survival vs 0%, log rank P=0.05). One difference observed
between the two models is that IP-10 fusion scFv20a did not produce
protective immunity. Thus, the observation that mixing MCP3scFv38
with IP10scFv20A did not produce protection (0% vs 40% survival for
MCP3scFv20A, log rank P=0.03), provides further evidence that the
chemokine must be physically linked to scFv.
[0144] In summary, these data demonstrate that fusion of chemokines
MCP-3 and IP-10 to a self tumor antigen can convert non-immunogenic
scFv into a potent immunogen. Chemokine fusion did not interfere
with correct folding of the native idiotype, the resulting
chemokine-scFv was fully biologically active (induced in vitro and
in vivo chemotaxis and bound specifically to the corresponding
receptors). These data also indicate that the immune response to
the chemokine-scFv is mediated through an interaction with the
chemokine receptor, as this effect is only seen when chemokine and
scFv were physically linked. Immunizations with the truncated
IP10TscFv38, which lost its ability to bind to the chemokine
receptor and induce T cell chemotaxis in vitro, could not elicit
efficient anti-Id production (mean 4.+-.2 .mu.g/ml). These data
also demonstrated significantly better tumor protection when
immunization was performed by the gene gun delivery of naked DNA
expressing MCP-3-scFv38.
[0145] The induction of protective anti-tumor immunity required
both CD8.sup.+ and CD4.sup.+ effector T cells. However, a minimum
threshold level of anti-idiotypic antibody was probably also
important since the improved survival depended on an efficient
anti-Id antibody response (114,115). No anti-Id38 antibody response
or tumor protection is detected in mice immunized with incorrectly
folded idiotype fusion protein or nDNA fusion protein constructs
expressing incorrectly folded idiotype. Furthermore, the protective
anti-tumor immunity elicited by MCP3scFv fusions as either protein
or DNA vaccines was superior to that of Id-KLH protein, the
formulation currently in clinical testing (116,117), in both tumor
models (P<0.03 and P<0.05 by chi-square test for pooled data
and MCP3-scFv vs. Id-KLH for 38c13 and A20, respectively).
Moreover, this effect does not require use of adjuvants. Finally,
both the superior potency of MCP3-scFv fusions relative to Id-KLH
protein and its ability to induce a critical effector CD8.sup.+ and
CD4.sup.+ T cells responses distinguish these fusion proteins from
other idiotype proteins and DNA vaccines (118,119).
[0146] Production of fusion polypeptides comprising a human
chemokine and a human tumor antigen or HIV antigen. To produce the
fusion polypeptides of the present invention which comprise a human
chemokine region and a human tumor antigen region or HIV antigen
region, the following procedures are carried out: Tumor or viral
antigen is cloned by PCR or RT/PCR from DNA or RNA of biopsy cells
of a patient, using specific primer. The primers are made using
standard methods for selecting and synthesizing primer sequences
from analysis of known sequences of the genes of interest (e.g.,
from GenBank, Kabat Ig sequence database and other available
genetic databases, as are known in the art). For example, lymphoma
or myeloma-specific scFv is cloned by RT/PCR from the nucleic acid
from a patient's lymphoma or myeloma biopsy cells or from nucleic
acid from hybridoma cells expressing the patient's immunoglobulin.
Several sets of primers are used to clone human variable (V) genes
based on GenBank and Kabat IG sequence data. As in cloning murine
scFv, human tumor V fragments are cloned and sequenced using a
family-specific primer or primer mixture for leader and constant
region sequences. Next, scFv is constructed using primers based on
the sequence of each V gene cloned. These primers can have specific
restriction endonuclease sites to facilitate routine cloning, or
scFv is made by overlapping PCR, according to methods well known in
the art. The vector expressing the fusion polypeptide can contain
several unique restriction endonuclease sites (e.g., XhoI, BamHI)
between the 3' end of the spacer sequence and the 5' end of the
c-myc and six His tag sequences, or the 5' end of the polyA
transcription terminator region (if a SmaI site is used), thus
enabling routine cloning of any scFv, tumor antigen or viral
antigen.
[0147] As described herein, nucleic acid encoding the human
chemokine-tumor antigen fusion polypeptides of this invention is
expressed in yeast (e.g., Saccharomyces cerevisiae; Pichia
pastoris, etc.) or in mammalian cell culture according to methods
standard in the art. The proteins produced in these systems are
affinity purified with anti-c-myc antibodies (e.g., 9E10; M5546,
Sigma) or anti-poly-His antibodies (e.g., H1029, Sigma).
Alternatively, immobilized metal chelate affinity chromatography
(Ni-NTA resin, Qiagen) is used for purification of soluble or
refolded fusion polypeptides.
[0148] Administration of fusion polypeptides to human subjects.
Immunity and suppression of tumor growth in a human subject. To
elicit a tumor cell growth-inhibiting response in a human subject,
a fusion polypeptide comprising a human chemokine and a tumor
antigen which is present in the human subject is administered to
the subject subcutaneously in a dose ranging from 1 to 500 .mu.g of
the fusion polypeptide once weekly for about eight weeks or once
monthly for about six months. Within the first month following the
initial immunization, blood samples can be taken from the subject
and analyzed to determine the effects of administration of the
fusion polypeptide. Particularly, the presence in the subject's
serum, of antibodies reactive with the tumor antigen in the fusion
protein can be determined by ELISA, Western blotting or
radioimmunoprecipitation, or other methods for detecting the
formation of antigen/antibody complexes as would be standard
practice for one of ordinary skill in the art of immunology. Also,
a cellular immune response to the tumor antigen in the fusion
polypeptide can be detected by peripheral blood lymphocyte (PBL)
proliferation assays, PBL cytotoxicity assays, cytokine
measurements, or other methods for detecting delayed type
hypersensitivity and cellular immune response, as would be standard
practice for one of ordinary skill in the art of immunology.
Additionally, the kinetics of tumor growth and inhibition of tumor
cell growth can be determined by monitoring the subject's clinical
response, through physical examination, tumor measurement, x-ray
analysis and biopsy. The exact dosage can be determined for a given
subject by following the teachings as set forth herein, as would be
standard practice for one of ordinary skill in the art of vaccine
development.
[0149] As an example of how the vaccine of this invention can be
administered to a patient to treat cancer or to treat or prevent
HIV infection (with the additional administration of adjuvants,
such as immunostimulatory cytokines, if desired), the following is
a complete protocol for a clinical trial describing the
administration of Id-KLH and GM-CSF to patients to treat follicular
lymphoma. The same study design can be employed for the
administration of the chemokine-tumor antigen fusion polypeptide or
the chemokine-viral antigen fusion polypeptide of the present
invention or nucleic acids encoding the fusion polypeptides of this
invention, with appropriate modifications, as would be apparent to
one of skill in the art. In particular, studies to test the
efficacy of HIV vaccines are well known in the art and the clinical
protocol described herein can be readily modified by one of skill
in the art as appropriate to test the efficacy of the HIV fusion
polypeptide or HIV fusion polypeptide-encoding nucleic acid of this
invention according to well known protocols for testing HIV
vaccines (126,127).
[0150] 1.1 Background and Rationale
[0151] The development of a vaccine against human malignancies has
been a long-sought goal which has yet to be achieved. Many of the
efforts toward this end have been frustrated by the lack of
identification of a tumor-specific antigen which would allow tumor
cells to be distinguished from normal cells. Conceptually, such an
antigen could be used as a vaccine to induce the hosts immune
system to reject cells bearing that antigen.
[0152] Immunoglobulin (Ig) molecules are composed of heavy and
light chains, which possess highly specific variable regions at
their amino termini. The variable regions of heavy and light chains
combine to form the unique antigen recognition site of the Ig
protein. These variable regions contain determinants that can
themselves be recognized as antigens, or idiotopes. B-cell
malignancies are composed of clonal proliferations of cells
synthesizing a single antibody molecule with unique variable
regions in the heavy and light chains. B-cell lymphomas are
neoplasms of mature resting and reactive lymphocytes which
generally express synthesized Ig at the cell surface. The idiotypic
determinants of the surface Ig of a B-cell lymphoma can thus serve
as a tumor-specific marker for the malignant clone.
[0153] Studies in experimental animals, as well as in man, have
demonstrated the utility of the Ig idiotype as a tumor-specific
antigen for the study of the biology of B-cell lymphoma in vitro
and as a target for passive immunotherapy in vivo (1,2,3).
Furthermore, active immunization against idiotypic determinants on
malignant B cells has been demonstrated to produce resistance to
tumor growth in a number of syngeneic experimental tumor models, as
well as specific anti-tumor therapy against established tumors
(4-13). These results, taken together, provided the rationale for
testing autologous tumor-derived idiotypic surface Ig (Id) as a
therapeutic "vaccine" against human B-cell lymphoma. Furthermore,
preclinical studies in subhuman primates demonstrated that optimal
immunization with human lymphoma-derived Id required conjugation of
the protein to an immunogenic protein carrier (keyhole limpet
hemocyanin; KLH) and emulsification in an adjuvant (14).
[0154] Guided by these observations, nine patients with B-cell
lymphoma were immunized with autologous Id protein (15). These
patients received no anti-tumor therapy during the time of the
study. They were either in complete remission or in a state of
minimal residual disease following conventional chemotherapy. In
addition, three patients with rapidly progressive recurrent
lymphoma were enrolled in a separate safety study; all three
required reinstitution of chemotherapy shortly after enrollment,
did not complete the immunization series, and were not studied
further. They received intramuscular injections of 0.5 mg of Id
conjugated to KLH at 0, 2, 6, 10 and 14 weeks, followed by two
booster injections at 24 and 28 weeks. Patients in the first trial
(five patients) received Id-KLH alone for the first three
immunizations, then Id-KLH emulsified in a Pluronic polymer-based
adjuvant vehicle formulation for all subsequent immunizations.
Because no idiotype-specific immune responses were observed prior
to the addition of the adjuvant to the program in this first group
of patients, patients in the second trial (four patients) received
the entire series of immunizations with this adjuvant. All patients
were analyzed for idiotype-specific antibody production and
peripheral blood mononuclear cell (PBMC) proliferative responses in
vitro immediately before each immunization and at one to two month
intervals following the last immunization. The KLH carrier provided
a convenient internal control for immunocompetence of the patients
and all patients demonstrated both humoral and PBMC proliferative
responses to the KLH protein, with the exception of one patient,
who demonstrated only the latter. Seven of the nine patients
demonstrated either a humoral (n=2) or a cell-mediated (n=4)
anti-idiotypic immunological response, or both (n=1).
[0155] Anti-idiotypic antibody responses were detected by analysis
of pro- and hyper-immune sera in either direct, or competition,
ELISA. The immunization with autologous Id protein induced
significant titers of anti-idiotypic antibody that either directly
bound or inhibited the binding of a murine anti-idiotype monoclonal
antibody (anti Id mAb) to Id on the plate. The specificity of the
humoral response for the Ig idiotype was demonstrated by the lack
of significant binding of hyperimmune serum to a panel of
isotype-matched human Igs of unrelated idiotype, or by the lack of
significant inhibition of a panel of heterologous Id-anti-Id
systems, respectively. Peak humoral responses were obtained after
the fifth immunization and persisted for at least nine months. The
anti-idiotypic antibody produced by patient 1 was affinity-purified
and shown to contain heterogeneous light chains as well as
immunoglobulin G heavy chains. This patient's antibody titer was
successfully boosted with a single administration of Id-KLH in
adjuvant after a decline of the humoral response after 15
months.
[0156] Cellular immune responses were measured by the proliferation
of PBMC to KLH and to autologous Id separately at concentrations
ranging from 1-100 .mu.g per milliliter of soluble protein in five
day in vitro cultures. None of the pre-immune PBMC demonstrated any
preexisting proliferation to autologous Id above that to culture
medium alone. Hyperimmune PBMC from all patients demonstrated
strong proliferative responses to the KLH carrier. Of primary
interest, significant hyperimmune proliferative responses to Id
were detected in five patients. Although their responses were of
lower magnitude than parallel responses to KLH, patients 3, 4, 6, 8
and 9 were classified as responders on the basis of reproducible
increases in counts-per minute (cpm) .sup.3H-thymidine
incorporation in wells containing Id, compared with medium alone,
that were sustained over multiple time points. Patients
demonstrating occasional increases in cpm in wells containing Id
compared with medium alone were classified as non-responders
(patients 1 and 5).
[0157] Flow cytometry analysis of cultures demonstrating
proliferation to Id revealed a predominance of cells staining
positively for CD4 (>95%), suggesting the phenotype of the
responding cell subpopulation. These cultures could be successfully
expanded for approximately four weeks by stimulation alternatively
with interleukin-2 (IL-2) and Id-pulsed autologous irradiated PBMC
as antigen-presenting cells. Specificity of the responses for Ig
idiotype was confirmed by the lack of significant proliferation to
an isotype-matched human Ig of unrelated idiotype compared with
medium alone. Such idiotype-specific PBMC proliferative responses
were observed only after the addition of the adjuvant to the
program and also persisted for at least 9-14 months.
[0158] The ability of the idiotype-specific humoral response to
bind autologous tumor cells was also tested. This was shown by the
inhibition of binding of a labeled murine anti-idiotype mAb to
tumor cells from a pre-treatment lymph-node specimen from patient 8
by hyperimmune, but not by pre-immune, serum from this patient. In
addition, affinity purified anti-idiotypic antibodies from the
hyperimmune sera of the two other patients who demonstrated
idiotype specific humoral responses were demonstrated by flow
cytometry to bind autologous tumor.
[0159] All patients were also closely monitored for disease
activity with physical examinations and routine laboratory and
radiographic studies. Of the two patients with measurable tumor at
the initiation of Id immunization, one (patient 1) experienced
complete regression of a single 2.5 cm left submandibular lymph
node, and the other (patient 4) experienced complete regression of
a 4.5 cm cutaneous lymphomatous mass on the right arm. This
clinical response in patient 4 correlated with an Id-specific, PBMC
proliferative response in vivo. Correlating with the duration of
their immunological responses, the clinical responses in both
patients have continued at 24 and 10 months, respectively, after
completion of the immunization series. Moreover, with a median
follow up time of 10 months, the only case of tumor recurrence
among those patients who were in remission and completed the
immunization series occurred in patient 5, who was one of the two
patients who failed to demonstrate an idiotype-specific
immunological response.
[0160] Toxicity was minimal in all twelve patients. All patients
experienced transient local reactions characterized by mild
erythema, induration, and discomfort, without skin breakdown, at
the injection sites. Splitting the components of the vaccine
(Id-KLH and adjuvant) in one patient who had experienced a moderate
local reaction and in another patient who had experienced a
moderate systemic reaction, characterized by fever, rigors and
diffuse arthralgias, established the adjuvant as the component
associated with these reactions. Both of these moderate reactions
resolved completely after 24-48 hours. The only laboratory
abnormality associated with Id immunization was a mild elevation
(less than twice the normal value) of serum creatine phosphokinase
24 hours after immunization in an occasional case.
[0161] These results demonstrate that patients with B-cell lymphoma
can be induced to make sustained idiotype-specific immune responses
by active immunization with purified autologous tumor-derived
surface Ig. They show that autologous Id, made immunogenic by
conjugation to KLH, can serve as an immunogen (antigen) to elicit
host immunological responses. The induction of low levels of
idiotype-specific immunity was demonstrated in the setting of
minimal tumor burden following conventional chemotherapy. These
results, taken together with the induction of relatively stronger
immune responses to the KLH carrier, and exogenous antigen, suggest
that chemotherapy-induced immunosuppression is not an obstacle to
active immunotherapy administered adjunctively to cytoreductive
drug therapy in this manner.
[0162] This initial study also established the requirement for an
immunological adjuvant, as no Id-specific responses were observed
prior to the addition of an adjuvant to the program. The objective
of further clinical trials using tumor derived Id as a therapeutic
vaccine is to further optimize the immunogenicity of this vaccine.
To this end, this study will focus on the use of novel
immunological adjuvants which are 1) more potent and 2) more
effective in the induction of cell-mediated immune responses,
compared with the pluronic polymer-based adjuvant used in the
study.
[0163] The 38C13 B cell tumor is used as a model system to screen
promising immunological adjuvants. A number of these have included
cytokines and among these, GM-CSF has emerged as a promising
adjuvant for idiotypic Ig antigen. In these experiments (10 mice
per group), syngeneic mice were immunized with 50 .mu.g Id-KLH
derived from the tumor, either alone or in combination with GM-CSF
mixed together with the antigen and administered subcutaneously.
Three additional daily doses of GM-CSF were administered s.c. as
close to the original site of immunization as possible. Mice
immunized with an irrelevant Id-KLH (4C5 IgM) served as negative
controls for the vaccine. Two weeks after this single immunization,
all mice were challenged with a single preparation of 38C13 tumor
cells (5.times.10.sup.3 cells i.p.) and followed for survival. The
results demonstrated that the augmented survival benefit afforded
by immunization with relevant Id-KLH alone can be significantly
enhanced by the addition of GM-CSF at either the 100 or 10,000 unit
dose. The loss of this protective effect at a higher dose of GM-CSF
of 50,000 units was also observed. These data suggest that GM-CSF
may have a potent adjuvant effect in vivo for Id-KLH antigen,
especially at relatively low doses.
[0164] Current Treatment of Follicular Lymphomas
[0165] The follicular lymphomas are follicular small cleaved cell
(FSC) and follicular mixed lymphoma (FM). Stage I and II patients
comprise only 10% to 15% of all cases of follicular lymphomas and
are best managed with radiation therapy. Eight-five percent of
patients with follicular lymphomas present with stage III or IV
disease. The optimal management of these patients remains
controversial and has generally followed two divergent approaches
(16, 17). One is an aggressive approach, which has included
radiation therapy, combination chemotherapy, or combined modality
therapy and the other is a conservative approach that involves no
initial treatment followed by a single-agent chemotherapy or
involved-field radiotherapy when required (18; 19). Most forms of
systemic therapy have the capacity to produce high complete
response rates. However, they have failed to produce long-term
disease-free survival or to prolong overall survival; thus, it has
become clear that the vast majority of patients with this disease
will relapse and die of their lymphoma, despite its usually
indolent course.
[0166] The NCI study (MB-110, BRMP 8903) begun in 1978, is a
prospective randomized study comparing these two distinct
approaches to the management of stage III or IV indolent histology
lymphoma. Most patients were randomized between no initial therapy
or aggressive combined modality therapy with ProMACE/MOPP
flexitherapy followed by low dose (2400cGy) total nodal
irradiation. Among the 149 patients treated thus far, 125 (84%)
were randomized; 62 to watch and wait (W & W) and 63 to
aggressive treatment. Among the 62 patients on the watch and wait
arm, 29 continue to be observed for periods up to 10+ years. The
median time to cross over to aggressive therapy is 23 months.
[0167] It is apparent that patients in whom therapy is initiated
after the development of symptoms have a significantly lower
complete response rate to therapy than patients randomized to
receive the same therapy at diagnosis (74% vs 40%, P.sub.2=0.0039).
The complete responder (CR) rate of patients randomized to initial
aggressive treatment is comparable to those obtained in patients
with advanced-stage intermediate grade lymphoma receiving the same
treatment. The CR rate in indolent lymphoma does not appear to be
significantly higher than what can be achieved with other
combination regimens. For patients randomized to watch and wait,
median follow-up of CRs is shorter because of the delay in
initiating treatment. However, the median duration of remission has
not been reached at five years and 57% of patients are projected to
be disease-free>8 years and 44% are projected to be in a CR at
12 years. The disease-free survival curves are not significantly
different between the two arms. Thus, allowing the patient to reach
a greater tumor burden before instituting systemic therapy reduces
the likelihood of obtaining a CR, but once achieved, CRs are
comparably durable to those obtained from primary aggressive
therapy. The lengthening of the remission duration, however, has
not resulted in a survival advantage for patients randomized to
receive primary aggressive chemotherapy. Furthermore, even though a
minority of complete responders have relapsed, the probability of
relapse appears to be continuous over time, and the vast majority
of patients are expected to eventually succumb to their
disease.
[0168] Thus, even immediate aggressive therapy has not resulted in
improved survival. Therefore, although patients diagnosed with
follicular lymphoma enjoy relatively longer survival times compared
with patients with solid tumors, follicular lymphoma remains an
incurable disease. Novel experimental therapies designed to improve
the durability of the remissions already effectively induced by
chemotherapy are justified.
[0169] 1.3 Summary of Treatment Plan
[0170] The goal is to treat patients with follicular lymphomas to
complete remission or maximal response with ProMACE chemotherapy.
After the completion of chemotherapy, in an effort to reduce the
relapse rate (by eradicating microscopic disease resistant to
chemotherapy), patients will receive an autologous Id vaccine
administered in combination with GM-CSF.
[0171] The goal of this study is to evaluate the ability of the Id
vaccine to clear the bone marrow of malignant cells detectable by
pathologic (morphologic) examination or molecular examination
(polymerase chain reaction, PCR) in patients with PCR amplifiable
translocations. All patients have serial bone marrow and peripheral
blood samples collected to search for clonal abnormalities by PCR.
Patients are followed after vaccine therapy and their remission
status correlated with clinical vs. molecular determinations of
response. There should be three categories of complete responders:
those who had a clinical complete response before the vaccine but
had an abnormal clone by PCR that cleared after the vaccine; those
with a clinical CR before the vaccine who were also PCR negative
before the vaccine; and those who achieved a clinical complete
response but had PCR positive marrows before and after the vaccine.
It is a goal of this study to assess whether "molecular complete
responses" can be achieved using the vaccine in patients following
chemotherapy.
[0172] 2.0 Objectives
[0173] The objectives of this trial are to:
[0174] 2.1 To induce cellular and humoral immunity against the
unique idiotype expressed on the surface of patients' B-cell
lymphomas.
[0175] 2.2 To determine the ability of Id immunization to eradicate
bcl-2 positive tumor cells from the bone marrow as detected by
PCR.
[0176] 2.3 As a secondary objective, to determine the more
biologically active of the two GM-CSF doses as an adjuvant, as
measured by the endpoints in 2.1 and 2.2.
[0177] 2.4 To determine the impact of Id immunization on disease
free survival of patients achieving a CR with chemotherapy.
[0178] 3.0 Patient Selection
[0179] 3.1 Patient Sample
[0180] A. Sample size, approximately 42 patients
[0181] B. Sex distribution: male and female
[0182] C. Age: patients must be .gtoreq.18 years old
[0183] 3.2 Eligibility Criteria
[0184] Patient must meet all of the following eligibility
criteria:
[0185] A. Tissue diagnosis of: follicular small cleaved cell, or
follicular mixed lymphoma with surface IgM, IgG or IgA phenotype
with a monoclonal heavy and light chain. Pathology slides must be
submitted to the NIH Pathology Department for review.
[0186] B. Stage III or IV lymphoma.
[0187] C. Only previously untreated patients are eligible.
[0188] D. Previous treatment with radiation alone (less than TBI)
is permissible.
[0189] E. A single peripheral lymph node of at least 2 cm size
accessible for biopsy/harvest.
[0190] F. Karnfsky status .gtoreq.70%.
[0191] G. Life expectancy of .gtoreq.one year.
[0192] H. Serum creatinine .ltoreq.1.5 mg/dl unless felt to be
secondary to lymphoma.
[0193] I. Bilirubin .ltoreq.1.5 mg/dl unless felt to be secondary
to lymphoma or Gilbert's disease. SGOT/SGPT <3.5.times. upper
limit of normal.
[0194] J. Ability to give informed consent. Ability to return to
clinic for adequate follow-up for the period that the protocol
requires.
[0195] 3.3 Patient Exclusion Criteria
[0196] The presence of any exclusion criteria (listed below) will
prohibit entry into study:
[0197] A. Prior total body irradiation.
[0198] B. Presence of antibodies to HIV, hepatitis B surface
antigen or other active infectious process.
[0199] C. Pregnancy or lactation. Fertile men and women must plan
to use effective contraception. A beta-HCG level will be obtained
in women of childbearing potential.
[0200] D. Patients with previous or concomitant malignancy,
regardless of site, except curatively treated squamous or basal
cell carcinoma of the skin, or effectively treated carcinoma in
situ of the cervix.
[0201] E. Patient unwilling to give informed consent.
[0202] F. Failure to meet any of the eligibility criteria in
Section 3.2.
[0203] G. Any medical or psychiatric condition that in the opinion
of the protocol chairman would compromise the patient's ability to
tolerate this treatment.
[0204] H. Patient with CNS lymphoma (current or previously treated)
will not be eligible.
[0205] 4.0 Clinical Evaluation
[0206] 4.1 Complete history and physical examination.
[0207] 4.2 CBC, diff., platelet count.
[0208] 4.3 Serum chemistry, .beta..sub.2-microglobulin.
[0209] 4.4 PT/PTT
[0210] 4.5 Quantitative immunoglobulins, serum protein
electrophoresis, immunoelectrophoresis.
[0211] 4.6 HIV antibody, HBsAg.
[0212] 4.7 Urinalysis.
[0213] 4.8 Serum .beta.-HCG in women of child-bearing
potential.
[0214] 4.9 EKG and MUGA.
[0215] 4.10 5 TT for serum storage.
[0216] 4.11 Leukapheresis to obtain 3.times.10.sup.9 lymphocytes.
These samples will be used for baseline studies of T-call
activation and response to Id.
[0217] 4.12 Tumor Biopsy--prior to therapy, all patients must
undergo biopsy/harvest of a clinically involved peripheral lymph
node to obtain tissue for morphological classification,
immunophenotypic characterization, determination of immunoglobulin
gene rearrangements, bcl-2 translocation, cytogenetics, and to
provide starting material for an Id vaccine. The sample should be
at least 2 cm in size. Only patients with tumors that are surface
immunoglobulin positive with a monoclonal heavy and light chain
will be accepted as study candidates. Use standard lymphoma vaccine
biopsy orders. See section 11.1 of protocol. Leftover tumor biopsy
samples may be used for basic studies of lymphoma biology in vitro.
Such future studies may be done without re-consenting the subjects
only if the studies involve risks already outlined in the original
consent form.
[0218] 4.13 CXR--PA and LAT.
[0219] 4.14 CT scan of abdomen and pelvis.
[0220] 4.15 Lymphangiogram, unless contraindicated by massive pedal
edema, severe chronic lung disease, ethiodal sensitivity (Note:
sensitivity to other iodine compounds, e.g., renograffin, are
relative, but not absolute contraindications).
[0221] 4.16 Other tests (CT chest, ultrasound, liver scan, bone
scan, upper and lower GI series, IVP, MRI) should be performed as
needed to evaluate all disease sites adequately.
[0222] 4.17 Examination of pleural fluid or ascites when
present.
[0223] 4.18 Bilateral bone marrow aspirates and biopsies--In
addition to the normal aspirate and biopsy, 5 cc of marrow will be
aspirated from each side into 0.5 ml of PFH for PCR analysis. The
procedure should be performed in the usual manner with a biopsy
performed first. Then a small volume (0.5-1 cc) can be aspirated
for the smear and clot tube. A separate Rosenthal needle with bevel
should be used for the aspirate. The 5 cc sample for PCR can be
obtained from the same site as the initial aspirate.
[0224] 4.19 CT scan of the head and lumbar puncture with CSF
analysis if clinically indicated.
[0225] 5.0 Patient Registration
[0226] 5.1 Patients will be registered prior to the initiation of
therapy at which time eligibility criteria will be reviewed.
Stratification and randomization are described in detail in Section
15.0 Statistical considerations.
[0227] 6.0 Study Design
[0228] (See Schema)
[0229] 6.1
5 ProMACE Day 0 Day 7 Day 28 Cyclophosphamide Cyclophosphamide Next
cycle begins 650 mg/m.sup.2 IV 650 mg/m.sup.2 IV Doxorubicin
Doxorubicin 25 mg/m.sup.2 IV 25 mg/m.sup.2 IV Etoposide VP-1 6
Etoposide BP-1 6 120 mg/m.sup.2 IV 120 mg/m.sup.2 IV Prednisone 60
mg/m.sup.2 po qd .times. 14 (days 0 to 13) Bactrim one double
strength tablet po BID throughout therapy
[0230] 6.1.1 All patients will be treated until a complete
remission is obtained and two additional cycles of chemotherapy
have been given, or until disease has been stable for two cycles of
chemotherapy, or progressive disease develops. A minimum of six
cycles will be given to each complete responder before therapy is
discontinued. Patients with more than 90% PR or a full CR will be
continued on the vaccination part of the protocol. Patients with
less than 90% PR or progressive disease will be taken off of the
study.
[0231] 6.2 Postinduction Therapy--Three to six months (or whenever
a customized GMP vaccine is available, up to a maximum period of 12
months) after the completion of chemotherapy, all patients in whom
either a complete clinical remission or minimal disease status
(.gtoreq.90% partial response) has been achieved will receive a
series of five injections of a vaccine consisting of 0.5 mg
autologous tumor derived immunoglobulin (Id) conjugated to KLH. The
vaccine will be administered together with GM-CSF as an
immunological adjuvant. Both the vaccine and GM-CSF will be
administered subcutaneously according to the following
schedule:
6 Schedule: At 0, 1, 2, 3 and 5 months Id-KLH (0.5 mg s.c.) day 0
adjuvant (s.c.) days 0-3 Cohort 1: GM-CSF 500 mcg/m.sup.2/d s.c.
for 4 days Cohort 2: GM-CSF 100 mcg/m.sup.2/d s.c. for 4 days
[0232] The sites of injection will be rotated between the upper and
lower extremities. Each dose of vaccine or GM-CSF will be split
equally between the two upper or lower extremities. All GM-CSF
injections will be given in close proximity to the vaccination
site, as close to the exact site of injection as possible. If local
reactions to GM-CSF are severe, GM-CSF injections may be given
elsewhere. Patients will be observed in the clinic for two hours
following Id-KLH and/or GM-CSF administration. During the
observation period, vital signs will be taken every minutes during
the first hour and every 30 minutes during the second hour.
[0233] 7.0 Supportive Care
[0234] 7.1 G-CSF 5 mcg/kg/d SC may be used in all patients who are
hospitalized for the treatment of febrile neutropenia, regardless
of how long the neutropenia persists.
[0235] 8.0 Grading and Management of Toxicity
[0236] 8.1 Chemotherapy: Dose modification of chemotherapy will be
based on the granulocyte count done at the time of drug
administration (day 0 or 7 of each cycle). The percentage of drugs
administered may be further modified based on toxicity in prior
cycles (see below). If the granulocyte count is <1200, and the
patient is due for day 0 drugs, delay day 0 for one week until
appropriate parameters are met. In general, delays of up to one
week are preferable to starting G-CSF. If after a one week delay,
appropriate parameters are still not met, then G-CSF may be started
as above. Also, in general, delays of up to one week are preferable
to dose reductions. Full doses of all drugs should be given on time
if blood count suppression is due to bone marrow involvement with
disease.
[0237] 8.1.1 Dose Modification for Hematologic Toxicity
7 THEN DOSE AS IF GRANULOCYTE COUNT IS: FOLLOWS: On Day 0
.gtoreq.1200 100% all drugs .ltoreq.1200 Day 0 Delay
[0238] For neutrophil nadir <500 or platelet count <25,000 on
previous cycle, 75% of cyclophosphamide, doxorubicin, and etoposide
should be considered. For neutrophil nadir (day 21 counts) >750
on a previous cycle, dose escalation of cyclophosphamide,
doxorubicin, and etoposide by 10-20% should be prescribed.
8 IF PLATELET COUNT IS: THEN DOSE AS FOLLOWS >100,000 100% of
all drugs 50-99,999 100% Prednisone 75% Etoposide 50%
Cyclophosphamide, Doxorubicin <50,000 Delay
[0239] 8.1.2 Dose Modification for Non-hematologic Toxicity
[0240] 8.1.2.1 Assessment of non-hematologic toxicity will be
graded according to the CRB/DCS/NCI Common Toxicity Criteria.
Chemotherapy will be withheld in patients experiencing grade 2 or
greater non-hematologic toxicity until the patient has completely
recovered from the toxicity. For nausea/vomiting 2: grade 2, drug
therapy should be continued with non-steroid antiemetics.
[0241] 8.1.2.2 Doxorubicin dosage should be adjusted as follows in
the presence of the following LFT abnormalities:
9 % Dose Bilirubin SGOT 100 <1.5 mg/dl <75 U 50 1.5-2.9 mg/dl
75-150 U 25 3.0-5.9 mg/dl 151-300 U 0 .gtoreq.6.0 mg/dl >300
U
[0242] 8.2 Immunotherapy
[0243] 8.2.1 Id-KLH Vaccine
[0244] Based on previous experience with autologous Id-KLH
vaccines, little or no toxicity is expected from the Id-KLH
component of the vaccine (15). Nevertheless, any local skin
reactions will be carefully noted and scored for erythema,
induration, pain and disruption of the barrier surface. If any
patient has a reaction suggestive of sensitization, the vaccine may
be split into its component parts; specifically, the patient will
be tested with Id-KLH alone and then GM-CSF alone. Toxicities will
be graded according to the CRBINCI/DCS common toxicity
criteria.
[0245] 8.2.2 GM-CSF
[0246] Anticipated toxicities from GM-CSF administration in this
dose range are expected to be mild based on previous experience.
Potential toxicities include fever, chills, myalgias, arthralgias,
nausea, vomiting, diarrhea, dyspnea, tachycardia, arrhythmias,
elevation of liver function tests, elevation of BUN and creatinine.
However, local skin reactions, such as erythema and induration, may
be observed and will be carefully noted. Attempts will be made to
maintain these patients as outpatients. For grade IV fever (not
responsive to Indocin or Tylenol), or grade III vomiting
(unresponsive to therapy), GM-CSF will be held until toxicity is
less than grade II and will be restarted at 50% of the original
dose level for the rest of that weekly injection cycle and for
subsequent cycles. For neurologic toxicity that affects daily
function (unable to carry on simple routine duties, or grade II in
the toxicity grading scale), hold treatment until symptoms resolve,
then reduce GM-CSF by 50%. If symptoms persist, the adjuvant should
be removed for subsequent immunizations. Patients with grade III
neurotoxicity will be removed from the study.
[0247] For well-documented evidence of cardiac toxicity (i.e.,
grade III, including evidence of ischemia or ventricular
arrhythmia, but not supraventricular tachycardia or atrial
fibrillation controlled by digoxin or calcium channel blocking
agents), the adjuvant will be removed for subsequent
immunizations.
[0248] Asymptomatic elevations in serum bilirubin and creatinine
(not resulting in hyperkalemia) will be tolerated. For SGOT or SGPT
>10.times. normal, GM-CSF will be held until values return to
<5.times. normal, then resumed at 50% of the GM-CSF dose for all
remaining doses.
[0249] 8.2.3 Fever and chills associated with vaccine
administration and/or GM-CSF will be treated with TYLENOL and/or
DEMEROL. The use of non-steroidal antiinflammatory drugs and/or
steroids should be avoided. Should non-steroidals or steroids be
required for unrelated medical conditions for a course exceeding 2
weeks, the patient will be taken off of the study.
9.0 Adverse Drug Reactions
[0250] 9.1 All toxicities and adverse events will be recorded on
the study flow sheet and appropriately graded as to severity and
cause. Toxicities that are related to the underlying disease should
be clearly differentiated from drug toxicities.
[0251] 9.2 Adverse drug reactions related to chemotherapy will be
submitted based on guidelines for commercial drugs.
[0252] 9.3 Reports of adverse reactions to Id-KLH and GM-CSF will
be made using the Division of Cancer Treatment Common Toxicity
Criteria for reference according to the guidelines published by the
DCT, NCI. These guidelines can be summarized as follows:
[0253] A. Report by telephone to IDB within 24 hours (301)
230-2330
[0254] 1. All life-threatening events (grade 4, except for grade 4
myelosuppression) which may be due to administration of the
investigational drug(s),
[0255] 2. All fatal events (grade 5),
[0256] 3. All first occurrences of any previously unknown toxicity
(regardless of grade).
[0257] B. A written report should follow within 10 working
days.
[0258] C. All adverse drug reactions will also be reported in
writing to the NCI Institutional Review Board within 10 working
days.
[0259] D. All adverse drug reactions will also be reported to the
FDA in accordance with Federal regulations.
[0260] E. Data will be submitted at least every two weeks.
[0261] 10. Study Parameters
[0262] 10.1 During Chemotherapy
[0263] 10.1.1 Weekly: CBC, diff. platelets; except day 14, i.e. CBC
on day 0, 7, 21, and 28.
[0264] 10.1.2 Beginning of each cycle: Chem 20, CXR, LAG follow-up
(KUB), CT scans (only after 4 cycles, then every 2 cycles).
[0265] 10.1.3 Bilateral bone marrow aspirate and biopsy after four
cycles and every additional two cycles thereafter. Include 5 cc of
aspirate in PFH from each side for PCR analysis.
[0266] 10.2 At Maximal Response to Chemotherapy
[0267] 10.2.1 If residual disease is obvious, record measurements
and perform bone marrows as above.
[0268] 10.2.2 For complete responders, complete restaging should be
performed. This should include all studies that were positive at
initial staging evaluation with the exception of repeat thoracotomy
or laparotomy. Bilateral bone marrows should be performed as
above.
[0269] 10.3 During Vaccine Therapy
[0270] 10.3.1 If residual disease is obvious, record measurements
and perform bone marrows as above.
[0271] 10.3.2 PT-PTT day 0
[0272] 10.3.3 UA, .beta..sub.2 microglobulin day 0 of each
immunization.
[0273] 10.3.4 Leukapheresis is performed on the day of initiation
of vaccine therapy (prior to the first cycle only) to obtain
pre-vaccine lymphocytes for storage. Five tiger top tubes are drawn
at this time to obtain serum for storage.
[0274] 10.3.5 Two tiger top tubes and peripheral blood (60 cc in
PFH) are collected on day 0 of each monthly cycle, for preparation
of serum and lymphocytes, respectively.
[0275] 10.3.6 Skin Biopsy is obtained near a planned immunization
site on day 0 prior to the first cycle (baseline sample) and again
on day 1, 2, or 3 of cycle 3 at an active site of erythema and/or
induration as close to the original biopsy site as possible.
[0276] 10.3.7 DTH--Delayed type hypersensitivity test (DTH) to
autologous idiotype protein is performed during cycle 4 and again
following completion of the immunization regimen, i.e., during or
after cycle 5. The DTH-test is performed by intradermal injection
of 0.5 mg of idiotype protein in 0.1-0.2 ml of NS. To ascertain the
specificity of a positive reaction, 0.5 mg of a heterologous
isotype matched Id-protein (from another patient on the same study)
in the same volume will be used as a negative control.
[0277] The control idiotypes used on these two occasions will be
from two different patients, also in the study, in order to
minimize the possibility of eliciting an immunologic response
against a particular irrelevant idiotype.
[0278] A skin biopsy will also be obtained at the site of the
intradermal injection of idiotype protein and at the control site,
one to three days, after the intradermal injections.
[0279] 10.3.8 Fine needle aspiration or core biopsy (with or
without CT guidance) of any enlarged lymph node draining the
vaccination sites is performed to obtain lymphocytes for in vitro
assays.
[0280] 10.4 At Discontinuation of Vaccine
[0281] 10.4.1 Restaging as described for Chemotherapy in Section
10.2.
[0282] 10.4.2 Bilateral bone marrow aspirates and biopsies at
completion of therapy and every six months for two years after
completing therapy and yearly thereafter.
[0283] 10.4.3 10 cc of serum for storage and 60 cc of peripheral
blood in PFH is collected at completion of therapy and every three
months for a year.
[0284] 11.0 Specimen Processing and Immunological Assays
[0285] 11.1 Lymph Node Harvest/Biopsy
[0286] Each lymph node biopsy will be divided as follows: (a)
one-third of the specimen will be sent in saline to the
Hematopathology Section, Laboratory of Pathology, NIH. Biopsies are
processed for routine histopathy and for immunophenotypic
characterization, particularly with respect to monotypic heavy and
light chain expression; and (b) two-thirds of the specimen is sent
in sterile saline in a sterile container to Clinical Immunology
Services, NCI FCRDC, where it is processed into a single-cell
suspension and cryopreserved.
[0287] 11.2 Blood and Bone Marrow Samples
[0288] All peripheral blood and bone marrow aspirate samples are
sent in an expedited manner to Clinical Immunology Services,
NCI-FCRDC. Tiger top tubes are spun down and serum divided into 1
ml aliquots for frozen storage. Peripheral blood mononuclear cells
(PBMC) are isolated prior to freezing by Ficoll-hypaque
centrifugation using standard protocols.
[0289] 11.3 Assay for Serum Antibody
[0290] In a direct enzyme-linked immunosorbent assay (ELISA),
preimmune and hyperimmune serum samples from each patient are
diluted over wells of a microtiter plate that are coated with
either autologous immunoglobulin idiotype or a panel of
isotype-matched human tumor immunoglobulins of unrelated idiotype.
Bound antibody is detected with horseradish peroxidase-goat
antihuman light-chain antibodies directed against the light chain
not present in the immunoglobulin idiotype (Caltag Laboratories,
South San Francisco).
[0291] 11.4 Assay for Idiotype-Specific Proliferative Response
[0292] Whenever feasible, fresh PBMC, isolated above, are used on
the same day they are obtained. Stored frozen PBMC are available as
a back-up. PBMC are washed and plated at a concentration of
4.times.10.sup.5 cells per well in Iscove's modified Dulbecco's
medium (IMDM) with 1 percent human AB7 serum (IMDM-1 percent AB).
KLH, autologous immunoglobulin idiotype, or a panel of isotype
matched immunoglobulins of irrelevant idiotypes at concentrations
of 0 to 100 .mu.g per milliliter in IMDM-1 percent AB preparation
are added in triplicate. After the cells are incubated for three
days at 37.degree. C. in an atmosphere containing 5 percent carbon
dioxide, they are transferred to a preparation of IMDM and 5
percent fetal-.calf serum containing recombinant interleukin-2 (30
U per milliliter). The plates are incubated for two days and pulsed
for 16 to 20 hours with .sup.3H-labeled thymidine (1 .mu.Ci per
well). Data are expressed as mean (.+-.SEM) counts per minute of
[.sup.3H]thymidine incorporation. Initial five-day cultures of
PBMCs established as described above are expanded in IMDM-5 percent
fetal-calf serum containing interleukin-2 (30 U per milliliter).
Harvested cells are replaced in IMDM-1 percent AB containing
autologous immunoglobulin idiotype and fresh irradiated (5000 R)
autologous PBMCs (4.times.10.sup.5 cells per well) as
antigen-presenting cells for five days, before pulsing with
.sup.3[H]thymidine.
[0293] 11.5 Cytotoxicity Assays
[0294] The potential cytotoxicity of PBMC cultured with Id as
above, or with irradiated fresh cryopreserved tumor cells, is
assayed against either autologous lymphoblastoid cell lines (LBL)
pulsed with Id or fresh cryopreserved tumor targets. Autologous LBL
pulsed with soluble antigen have been used successfully as targets
to detect gp 160-specific cytotoxic T-lymphocytes (20).
Historically, the inability to establish long-term cultures of
follicular lymphoma has hindered their availability as targets.
However, two recent reports have described the use of fresh
cryopreserved lymphoma cell targets, with levels of spontaneous
incorporated radioisotope release in the acceptable range of
<35% (21-22). Standard four hour .sup.51Cr release, as well as
18-24 hour .sup.111In release assays are used.
[0295] Autologous LBL are prepared from pre-immune PBMC by the AIDS
Monitoring Laboratory, NCI-FDRDC, using published methods.
[0296] 11.6 Monitoring of T-cell Receptor (TCR) Status
[0297] Pre-chemotherapy and pre- and postimmunization serum samples
are assayed for TCR status by Western blot assay. Approximately
7.times.10.sup.6 purified T-cells from PBMC are lysed for 5 minutes
at 4.degree. C. in lysis buffer (25 mM Tris, pH 7.4 [Sigma Chemical
Co., St Louis, Mo.], 300 mM NaCl, 0.05 Triton X-100, 1 mM Na
orthovanadate, 10 .mu.g/ml aprotinin, 10 .mu.g/ml leupeptin, 10 mM
nitrophenol-guanidine benzoate [NPGB] and 5 mM EDTA). The lysates
are centrifuged at 12,000 rpm at 4.degree. C. for 5 minutes and
supernatant is removed with a micropipettor, making sure the
nuclear pellet is not disturbed. A sample of the supernatant is
then used to quantitate protein using the BCA protein assay
(Pierce, Rockford, Ill.). The rest of the lysate is boiled with
3.times.reducing sample buffer for 5 minutes and placed on ice
before its use in Western blot.
[0298] Varying concentrations of cellular lysate ranging between 1
and 30 .mu.g are electrophoresed in 14% Tris-glycine gels (Novex
Experimental Technology, CA) under reducing conditions and then
transferred to Imobilon-p PVDF transfer membranes (Millipore Co.,
Bedford, Mass.). The membranes are incubated with a 5% solution of
non-fat dried milk for one hour and then blotted for one hour at
room temperature with anti-TCR.zeta. anti-serum (Onco-Zeta 1,
OncoTherapeuties, Cranbury, N.J.) at a 1:2000 dilution. The
membranes are washed with TBS-T buffer [1 M Tris base, 5M NaCl,
0.1% Tween 20 (pH 7.5)] and incubated with anti-rabbit or
anti-mouse Ig horseradish peroxidase (Amersham, Buckinghamshire,
UK). After washing with TBS-T, the membranes are developed with the
chemiluminescence kit ECL (Amersham, UK) for 1-5 minutes. X-OMAT AR
film (Kodak Co., Rochester, N.Y.) is used to detect the
chemiluminescence.
[0299] 11.7 PCR Amplification of Rearranged bcl-2
[0300] Nested oligonucleotide amplification is performed at the MBR
or mcr of the bcl-2/IgH hybrid gene using previously published
methods (23). Briefly, samples containing 1 .mu.g of genomic DNA
are initially amplified for 25 cycles in a final volume of 50 .mu.g
containing 50 mmol/L KCl, 10 mmol/L Tris HCL, 2.25 mmol/L
MgCl.sub.2, 200 mmol/L oligonucleotide primers, 200 mmol/L each of
dGTP, dCTP, dTTP and dATP, and 1.5 U Taq polymerase(Cetus,
Emeryville, Calif.). Reamplification of an aliquot of product is
performed for 30 cycles in a final volume of 50 .mu.l using
identical conditions to the original amplification, with
oligonucleotide primers internal to the original primers. Aliquots
of the final product are analyzed by gel electrophoresis in 4%
agarose gels containing ethidium bromide and visualized under UV
light. DNA is
[0301] Southern blotted onto Zeta-probe blotting membrane (BioRad.
Richmond, Calif.) and bcl-2-specific DNA is detected by
hybridization with oligonucleotide probes radiolabeled with
.sup.32P(ATP) using T4 polynucleotide kinase.
[0302] 12.0 Removal of Patients From Protocol Therapy
[0303] Patients will be removed from protocol for any of the
following reasons:
[0304] 12.1 Unacceptable toxicity (as defined in Section 8.0).
[0305] 12.2 The patient declines further therapy.
[0306] 12.3 The patient experiences progressive lymphoma.
[0307] 12.4 It is deemed in the best interest of the patient. In
this instance,
[0308] 12.4.1 The Principal Investigator should be notified.
[0309] 12.4.2 The reasons for withdrawal should be noted in the
flow sheet.
[0310] 13.0 Response Criteria
[0311] Patients will be reevaluated for tumor response after every
two cycles of chemotherapy using the following criteria:
[0312] 13.1 Complete Response--disappearance of all clinical and
laboratory (excluding PCR) signs and symptoms of active disease for
a minimum of one month.
[0313] 13.2 Partial Response--a 50% or greater reduction in the
size of the lesions as defined by the sum of the products of the
longest perpendicular diameters of all measured lesions lasting for
a minimum of one month. No lesions may increase in size and no new
lesions may appear.
[0314] 13.3 Minimal Residual Response--a .gtoreq.90% partial
response. For most patients in this category, this will mean
.ltoreq.10% residual bone marrow involvement by lymphoma.
[0315] 13.4 Progressive Disease--an increase of 25% or more in the
sum of the products of the longest perpendicular diameters of all
measured indicator lesions compared to the smallest previous
measurement or the appearance of a new lesion.
[0316] 14.0 Drug Formulation and toxicity Data
[0317] 14.1 Cyclophosphamide (CTX. Cytoxan)-NSC #26271
[0318] 14.1.1 Source and Pharmacology--CTX is an alkylating agent,
related to nitrogen mustard, which is biochemically inert until it
is metabolized to its active components by the liver
phosphoramidases. It is non-phase-specific. The drug is excreted
exclusively by the kidney after parenteral administration.
[0319] 14.1.2 Formulation and Stability--CTX is supplied as a 100,
200, 500, 1000 mg and a 2 gram lyophilized powder with 75 mg
mannitol per 100 mg (anhydrous) cyclophosphamide. The vials are
stored at room temperature (59-86.degree. F.) and reconstituted
with sterile water for injection to yield a final concentration of
20 mg/ml as described in the package insert. Reconstituted
cyclophosphamide is stable for at least 6 days under refrigeration
and for 24 hours at room temperature. Reconstituted drug and
diluted solutions should be stored under refrigeration.
[0320] 14.1.3 Supplier--Commercially available.
[0321] 14.1.4 Route of Administration--The cyclophosphamide used in
this regimen is given IV over 30 minutes and is diluted in 100 cc
of either D.sub.5W or NSS.
[0322] 14.1.5 Toxicity--Toxicities described with cyclophosphamide
include nausea, vomiting, myelosuppression, gonadal failure in both
males and females, alopecia, interstitial pneumonitis, pulmonary
fibrosis. hemorrhagic cystitis, cardiac events (cardiomyopathy),
syndrome of inappropriate antidiuretic hormone secretion (SIADH)
and rarely, anaphylaxis.
[0323] 14.2 Prednisone (Deltasone. Meticorten, Liquid Pred)
NSC#10023
[0324] 14.2.1 Source and Pharmacology--Prednisone is the synthetic
congener of hydrocortisone, the natural adrenal hormone. It binds
with steroid receptors on the nuclear membrane, blocks mitosis, and
inhibits protein synthesis. It kills primarily during the S-phase
of the cell cycle. It is catabolized in the liver and excreted in
the urine. Peak blood levels occur within two hours after oral
intake. Plasma half-life is 3-6 hours. (Biologic half-life is 12-30
hours.)
10 Cortisone 25 Equivalent Hydrocortisone 20 strength in mg
Prednisone 5 Decadron 0.75
[0325] 14.2.2 Formulation and Stability - Available in 1, 2.5,
5,10, 20 and 50 mg tablets; 5 mg/5 ml liquid.
[0326] 14.2.3 Supplier--Prednisone is commercially available.
[0327] 14.2.4 Route of Administration--PO; NOTE: May cause GI
upset; take with meals or snacks. Take in the morning prior to 9
a.m.
[0328] 14.2.5 Toxicity--Toxicities described with prednisone
include fluid and electrolyte changes, edema, hypertension,
hyperglycemia, gastritis, osteoporosis, myopathy, behavioral and
mood changes, poor wound healing, and Cushing's syndrome (moon
face, buffalo hump, central obesity, acne, hirsutism and
striae).
[0329] 14.3 VP-16 (Etoposide.VePesid) NSC#141540
[0330] 14.3.1 Source and Pharmacology--VP-16 is a semisynthetic
derivative of podophyllotoxin which inhibits toposomerase II and
functions as mitotic inhibitor, but does not bind microtubules. Its
main effect appears to be in the S and G.sub.2-phase of the cell
cycle. The mean terminal half-life is 11.5 hours, with a range of 3
to 15 hours. It is primarily excreted in the urine.
[0331] 14.3.2 Formulation and Stability--VP-16 is supplied in vials
containing either 100 or 500 mg of etoposide (20 mg/ml) in a
polyethylene vehicle. VP-16 is diluted in either 500 cc of 5%
dextrose or 0.9% Sodium Chloride Injection. Diluted solutions
(concentrations of 0.2, 0.4 mg/ml and 1 mg/ml) are stable for 96,
48 hours and 2 hours, respectively at room temperature under normal
room fluorescent light in both glass and plastic containers. Do not
refrigerate etoposide-containing solutions.
[0332] 14.3.3 Supplier--VP-16 is commercially available.
[0333] 14.3.4 Route of Administration--Etoposide is administered as
an IV infusion over 60 minutes.
[0334] 14.3.5 Toxicity--Toxicities described with etoposide
administration include rnyelosuppression (neutropenia), nausea,
vomiting, mucositis, allergic reactions characterized by
anaphylactic symptoms and hypotension and alopecia.
[0335] 14.4 Doxorubricin (Adriamycin) NSC #123127
[0336] 14.4.1 Source and Pharmacology--Doxorubicin is an
anthracycline antibiotic isolated from cultures of Streptomyces
peucetius. It binds to DNA and inhibits nucleic acid synthesis,
with its major lethal effect occurring during the S-phase of the
cell cycle. Since it is primarily excreted by the liver, any liver
impairment may enhance toxicity. Some of the drug has a very short
.alpha. T 1/2 of <20 minutes and a .beta.1/2 of 17 hours. Animal
studies indicate cytotoxic levels persist in tissue for as long as
24 hours. Biliary excretion also is a source of elimination for
Doxorubicin; therefore, patients with
hyperbilirubinemia/cholestasis caused by something other than
lymphoma should have dosage modification.
[0337] 14.4.2 Formulation and stability--Doxorubicin is available
as a freeze-dried powder in 10, 50 and 150 mg vials. The drug is
stored at room temperature, protected from light, and is
reconstituted with sodium chloride 0.9% (NSS) to yield a final
concentration of 5 mg/ml. The reconstituted solution is stable for
7 days at room temperature (15-30.degree. C.) or if stored under
refrigeration (2-8.degree. C.).
[0338] 14.4.3 Supplier--Doxorubicin is commercially available.
[0339] 14.4.4 Route of Administration--Doxorubicin is given as a
slow IV injection over 5-7 minutes through an established line with
a free flowing IV. Special precautions: Avoid extravasation and
local contact with skin or conjunctiva.
[0340] 14.4.5 Toxicity--Toxicities described with doxorubicin
administration includemyelosuppression, nausea, vomiting,
mucositis, stomatitis, alopecia. diarrhea, facial flushing,
dose-related congestive cardiomopathy, arrhythmias, vein streaking
(hypersensitivity reaction), radiation-recall dermatitis, local
cellulitis, vesication and tissue necrosis upon extravasation (SQ
and dermal necrosis).
[0341] 14.5 ID-KLH Vaccine
[0342] 14.5.1 Source--Idiotype protein from the individual B cell
lymphomas is obtained from tissue culture, purified, and covalently
coupled to keyhole limpet hemocyanin (KLH) as previously described.
Each batch is produced according to Good Manufacturing Practices
standards and tested for sterility, endotoxin contamination, and
general safety prior to its use in any patient. The preparation and
quality control/quality assurance testing of the Id-KLH conjugate
is performed by TSI Washington under CRB contract. The IND for the
Id-KLH vaccine will be held by the Drug Regulatory Affairs Section,
CTEP.
[0343] 14.5.2 How supplied--Formulated product for subcutaneous
administration contains 0.5 mg of Id and KLH each per ml of normal
saline. Id-KLH is supplied as a 1 ml vial.
[0344] 14.5.3 Storage--Prior to administration, Id-KLH is stored at
-20.degree. C.
[0345] 14.5.4 Administration--After thawing and gentle agitation,
the vial contents are drawn up using an 18-gauge needle on a
syringe. After the entire contents have been drawn up, the 18-gauge
needle is replaced by a 25-gauge needle for injection. This
procedure is important to ensure that all particulates (normal
components of this vaccine) are obtained from the vial.
[0346] 14.5.5 Toxicity--Toxicities described with Id-KLH vaccine
administration include local site reactions (erythema, induration,
swelling and tenderness), fever,chills, rash, myalgias and
arthralgias. Mild elevations in creatinine phosphokinase (CPK) have
been observed.
[0347] 14.6 GM-CSF (Sargramostim: NSC #613795;.BB-IND 2632
[0348] 14.6.1 Source and Pharmacology--The GM-CSF used in this
study is glycosylated, recombinant human GM-CSF. This GM-CSFis an
altered form of the native molecule; the position 23 arginine has
been replaced with a leucine to facilitate expression of the
protein in yeast (Saccharomyces cerevisiae).
[0349] 14.6.2 Formulation and Stability--The GM-CSF is formulated
as a white lyophilized cake and is provided in vials containing 500
.mu.g of the GM-CSF protein as well as 10.0 mg of sucrose, 40.0 mg
of mannitol, and 1.2 mg of Tris (Trimethamine).
[0350] To prepare a vial of GM-CSF for direct subcutaneous use,
aseptically inject 1.0 ml of Sterile Water for Injection, USP, into
the vial to dissolve the lyophilized cake. The diluent should be
directed against the side of the vial to avoid excess foaming.
Avoid vigorous agitation of the vial; do not shake. This yields a
solution containing 500 .mu.g/ml. The unreconstituted material
should be kept refrigerated at 2-8.degree. C. and is stable for at
least eighteen months. Once reconstituted, the solution is stable
for at least 24 hours at 2-8.degree. C. or at 18-25.degree. C.
Because the product does not contain a preservative, vials should
be treated as unit-dose containers; reconstituted solution should
be held at 2-8.degree. C. and discarded after no more than six
hours. Do not freeze GM-CSF.
[0351] 14.6.3 Supplier. Manufactured by Immunex.
[0352] 14.6.4 Route of Administration--The appropriate total dose
is withdrawn into and administered from a plastic tuberculin
syringe. The GM-CSF is injected subcutaneously as close as possible
to the Id-KLH injection site. All GM-CSF doses for each patient are
administered by the nursing staff in the outpatient unit.
[0353] 14.6.5 Toxicity--Toxicities described in patients receiving
GM-CSF include: fever, chills, diaphoresis, myalgias, fatigue,
malaise, headache, dizziness, dyspnea, bronchospasm, pleural
effusion, anorexia, indigestion, nausea, vomiting, diarrhea,
injection site tenderness, urticaria, rash, pruritus,
hypersensitivity reaction, bone pain, thromboembolic events,
phlebitis, hypotension, peripheral edema, leukocytosis,
thrombocytosis or thrombocytopenia, hepatic enzyme abnormalities,
and bilirubin elevation. The first administration of GM-CSF has
provoked a syndrome of dyspnea and hypotension within two hours
after GM-CSF injection in a single patient receiving yeast-derived
GM-CSF; this type of reaction has more frequently been observed in
patients receiving GM-CSF produced in E. coli. One report of a
vascular leak-like syndrome occurring after autologous bone marrow
transplant in a patient receiving continuous IV infusion of GM-CSF
has been recorded.
[0354] 14.7 Unconjugated Lymphoma Immunoglobulin Idiotype (for
intradermal skin testing) NSC# 684151
[0355] 14.7.1 Source--The patient-specific purified idiotype
protein, previously produced according to GMP standards as
described above in 14.5, is vialed as a separate product by TSI
Washington Laboratories and will be supplied by CTEP, DCT, NCI.
This vialed product is tested separately for sterility, endotoxin,
and mycoplasma, according to IND specifications previously
discussed with the FDA. Each vial of patient-specific unconjugated
idiotype will be labeled to include the following information:
[0356] Purified sterile immunoglobulin idiotype
[0357] patient-specific lot
[0358] final volume and concentration of product
[0359] patient-specific immunoglobulin subtype
[0360] storage conditions
[0361] fill date
[0362] patient identification (first name/last initial)
[0363] 14.7.2 How Supplied--This product is available as a solution
containing 0.2-0.3 ml of unconjugated idiotype diluted in sodium
chloride 0.9%. The solution is contained inside a sterile vial. The
final solution contains 0.5 mg of patient-specific immunoglobulin
idiotype protein. Intact vials are stored at -20.degree. C.
[0364] 14.7.3 Toxicity--The toxicities associated with
administration of unconjugated Id protein are anticipated to be
identical to those described with the Id-KLH vaccine. The safety
issues regarding the injection of heterologous idiotype protein
isolated from other patients' B-cell tumors have already been fully
addressed in CRB # 9407 (NCI T94-0085; Active immunization of
Healthy Sibling Marrow Transplant Donors With Myeloma-derived
Idiotype) and are felt to be minimal, because of the highly
purified nature of the protein. Briefly, an immune response of any
consequence to the isotype matched idiotype used as a negative
control during the second skin test is not likely, based on:
[0365] 1. The isotype matched idiotype will only be administered
once and is not conjugated to a carrier protein. These minimize the
chance of eliciting a sustained immune response to the protein.
[0366] 2. Any immune response specifically directed against the
idiotype (i.e., variable region) on the control idiotype protein is
not likely to cross-react with host cells and is therefore not
likely to be of any consequence.
[0367] 3. An autoimmune response against constant region or
allotype determinants shared between the idiotype of the patient's
own tumor and that of the control idiotype tumor is theoretically
possible. However no evidence of such autoimmune responses have
been observed either in vivo or in vitro during the course of
immunization of sibling bone marrow transplant donors with purified
myeloma protein.
[0368] Furthermore, a safety precedent exists for immunizing
patients with material derived from tumor cells from other
patients. For example, in attempting to develop immune responses
against metastatic melanomas, patients were immunized with 1)
intact melanoma cells; 2) shed antigens fractionated by detergent
treatment and ultracentrifugation; 3) melanoma cells infected with
vaccinia virus and melanoma cells freeze thawed and mechanically
disrupted, all using a pool of allogeneic melanoma cell lines
(24-28).
[0369] 14.8 Bactrim will be supplied by the Clinical Center.
[0370] 14.9 Filgrastim (G-CSF)/Neupogen
[0371] 14.9.1 Source and Pharmacology--The G-CSF to be used in this
study is the recombinant methionyl human granulocyte-colony
stimulating factor (r-methi-HuG-CSF). G-CSF is a hematopoietic
growth factor with effects on both immature bone marrow progenitors
and mature myeloid cells. It acts by supporting growth of human
bone marrow derived colony forming units and enhancing neutrophil
growth and proliferation.
[0372] 14.9.2 Formulation and Stability--The G-CSF is formulated as
a clear, sterile solution and is provided in vials at a final
concentration of 300 mcg/ml. The commercial vials are available in
300 and 480 mcg sizes. The intact vials are stored under
refrigeration (2-8.degree. C.) prior to use and must not be frozen
and are stable at this temperature for at least one year.
[0373] 14.9.3 Supplier--Manufactured by Amgen; supplied by the
Clinical Center.
[0374] 14.9.4 Route of Administration--The appropriate total dose
is withdrawn into and administered from a plastic tuberculin
syringe. The G-CSF is injected as a subcutaneous injection. The
patient or other care-giver is instructed on proper injection
technique.
[0375] 14.9.5 Toxicities--Toxicities described with G-CSF include:
transient bone pain (sternal/pelvic) myalgias, fatigue, mild
elevations in uric acid, LDH and alkaline phosphate, fluid
retention, transient hypotension, local inflammation at injection
site, rarely cutaneous vasculitis, rarely pericardial effusion and
rare anaphylactic reactions with first dose.
[0376] 15.0 Statistical Considerations
[0377] Statistical issues to be addressed include identification of
significant endpoints, sample size determination, power
considerations, stratification, randomization and design.
[0378] The design of this study is viewed primarily within the
framework of a Single Arm Phase II trial. However, as the purpose
is also to investigate possible differences between GM-CSF doses as
adjuvants, it incorporates design elements characteristic of a
Multiple Arm Phase II or a randomized Phase III trial. Statistical
methods that are appropriate to both single and double arm designs
are described.
[0379] Patients receive combination chemotherapy to best response
followed by Id-KLH combined with GM-CSF. Several outcome measures
(endpoints) are evaluated in order to meet the objectives of this
study. They include:
[0380] 1) The clinical complete response rate (in contradistinction
to the molecular or PCR response rate) of all patients to
ProMACE--a percentage indicated by the disappearance of all
clinical and laboratory signs and symptoms of active disease,
excluding PCR, for a minimum of one month.
[0381] 2) The Polymerase Chain Reaction (PCR) response rate
(molecular-complete response rate)--the percentage of patients who,
having achieved a clinical complete response still remain PCR (+)
at the end of chemotherapy, and who then become PCR (-) with the
administration of immunotherapy.
[0382] 3) Disease Free Survival Rate--computed by Kaplan-Meier
curves and related survival measures.
[0383] The PCR response rate is taken as the primary outcome
variable of interest to ascertain the following: (1) to determine
the ability of Id immunization to eradicate bcl-2 positive tumor
cells from the bone marrow and; (2) to identify the more
biologically active of the two doses of GM-CSF. In this endeavor,
the plan is to accrue 42 patients. It is estimated that
approximately 38 (90%) of these patients will be bcl-2 (+) and thus
evaluable for molecular response rate. The other four patients may
still be evaluable for a molecular response rate based on Ig gene
amplification using allele-specific (CDR3) primers by PCR. From
previous experience with ProMACE-based regimens, it is estimated
that 32 (85%) of these patients will achieve either a complete
response (complete clinical response, CCR) or a partial response in
which a >90% partial remission has been obtained (high partial
response, HPR). The accuracy of these estimates are of some
interest. For the 42 (90%) patients anticipated to be bcl-2 (+),
lower and upper 95% confidence intervals are 77% and 96%. For the
38 (85%) patients anticipated to achieve either a complete clinical
response or a high partial response, the lower and upper confidence
intervals are 70% and 93%.
[0384] Patients are stratified on the basis of their ProMACE
treatment performance as either a complete clinical responder (CCR)
or as a high partial responder (HPR). It is not known exactly what
percentage of these 32 patients will be CCRs and what percentage
will be HPR'S. Hence a block size of four (4) is used in the
randomization scheme to assure a reasonably balanced allocation to
each dose group. Given the patients allocation stratum, he (she) is
randomly assigned to one of the adjuvant groups according to the
envelope method (29). Specifically, a block of four assignments is
placed in four separate envelopes. The block of four is placed in
one of the two allocation strata, say CCR. Another block of four is
placed in the other allocation strata, say CCR. Another block of
four is placed in the other allocation stratum, HPR. When a patient
is to be randomized, a call is made to the biostatistician who,
after being informed of the patients status as either a CCR or an
HPR, randomly draws an envelope from the appropriate stratum to
determine the patients dose group assignment. After the four
envelopes pertinent to a particular stratum have been exhausted,
the next batch of four envelopes is made available for use. This
procedure is continued until a total of 32 patients have been
assigned to the two dose groups.
[0385] For example, it is estimated that 50-80 percent of
pathological complete responders will fall into the CCR category.
If 75% of 32, or 24 patients were to be classified as CCRs, six
blocks of four envelopes would be required to randomly assign 12
patients to cohort 1 and 12 patients to cohort 2. A similar
procedure would occur concurrently with the 8 patients classified
as HPRs. Two blocks of four envelopes would be required to randomly
assign 4 patients to cohort 1 and 4 patients to cohort 2. At no
time could the number of patients in each dose group differ by more
than four.
[0386] At the time of data analysis, approximately 16 subjects will
comprise each dose group and a test for the difference in PCR
response rates between the two groups will be conducted. By
hypothesis, neither dose group is predicted to have a higher PCR
response rate than the other; hence, a two-tailed test is
appropriate. Power calculations show that, with the groups limited
to 16 patients, the difference in PCR response rates will have to
be large (30, 31). For example, to detect a difference at the
a=0.05 level of significance with power (1-.beta.) equal to 80%,
the response rates must differ by 55%; with power equal to 50%, the
response rates must differ by 50%. In the event that no significant
difference is detected, the subjects will be pooled and the overall
PCR response rate will be assessed. With a total of 32 CCRs and
HPRs treated with vaccine, the width of a two-tailed 95% confidence
interval for a response rate of 50% will not exceed 17 percentage
points. If the actual response rate is higher or lower than 50%,
the confidence interval will be smaller.
[0387] Disease-free survival distributions are estimated by the
Kaplan-Meier (product-limit) method and dose groups are compared
using the log rank test. If no dose group differences are detected,
the subjects from both groups are pooled and the Kaplan-Meier
estimate of the survivorship function and related functions are
evaluated. If suggested by the data analysis, parametric
distributions (e.g., Weibull, log-normal) are fit as well (32,
33).
[0388] 15.1 Research ethics: Subjects from [both genders and] all
racial/ethnic groups are eligible for this study if they meet the
eligibility criteria outlined in Section 2.0. To date, there is not
information that suggests that differences in grud metabolism or
disease response would be expected in one group compared to
another. Efforts are made to extend accrual to a representative
population, but in this preliminary study, a balance must be struck
between patient safety considerations and limitations on the number
of individuals exposed to potentially toxic and/or ineffective
treatments on the one hand and the need to explore gender and
ethnic aspects of clinical research on the other hand. If
differences in outcome that correlate to gender or to ethnic
identity are noted, accrual can be expanded or a follow-up study
can be written to investigate those differences more fully.
Alternatively, substantial scientific data exist demonstrating that
there is no significant difference in outcome between genders or
various ethnic groups.
[0389] 16.0 Records to be Kept and Quality Assurance
[0390] 16.1 Consent form: The original signed informed consent
documents will be kept with the patient's other study documentation
(e.g., the research chart). A copy of the informed consent document
will also be retained by the Data Management Section.
[0391] 16.2 The Clinical Coordinator, Data Management Section, will
ascertain the dates of the IRB approvals before registering the
first patient.
[0392] Although the present process has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims.
[0393] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
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Sequence CWU 0
0
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