U.S. patent application number 12/967816 was filed with the patent office on 2011-04-07 for anti-chemokine and associated receptors antibodies for inhibition of growth of neoplasms.
This patent application is currently assigned to MOREHOUSE SCHOOL OF MEDICINE. Invention is credited to James W. LILLARD, Shailesh Singh, Jonathan Stiles.
Application Number | 20110081407 12/967816 |
Document ID | / |
Family ID | 32326333 |
Filed Date | 2011-04-07 |
United States Patent
Application |
20110081407 |
Kind Code |
A1 |
LILLARD; James W. ; et
al. |
April 7, 2011 |
ANTI-CHEMOKINE AND ASSOCIATED RECEPTORS ANTIBODIES FOR INHIBITION
OF GROWTH OF NEOPLASMS
Abstract
The present invention provides a means of inhibiting the growth
and metastasis of cancer cells by administering anti-chemokine
antibodies. It is possible to identify the particular chemokines
which are over-expressed in the tumor using methods of the
invention and administer antibodies against that over-expressed
chemokine.
Inventors: |
LILLARD; James W.; (Smyrna,
GA) ; Singh; Shailesh; (Atlanta, GA) ; Stiles;
Jonathan; (Atlanta, GA) |
Assignee: |
MOREHOUSE SCHOOL OF
MEDICINE
Atlanta
GA
|
Family ID: |
32326333 |
Appl. No.: |
12/967816 |
Filed: |
December 14, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10712398 |
Nov 14, 2003 |
|
|
|
12967816 |
|
|
|
|
60426347 |
Nov 15, 2002 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/133.1; 424/158.1; 424/172.1; 424/489 |
Current CPC
Class: |
C07K 16/24 20130101;
A61P 35/04 20180101; A61K 39/3955 20130101; C07K 16/2866 20130101;
C07K 2317/73 20130101; A61P 35/02 20180101; A61P 35/00
20180101 |
Class at
Publication: |
424/450 ;
424/158.1; 424/172.1; 424/133.1; 424/489 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/395 20060101 A61K039/395; A61K 9/14 20060101
A61K009/14; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
[0002] This work was partially supported by the United States
government, which has certain rights in the invention.
Claims
1-14. (canceled)
15. A method of inhibiting carcinoma cell migration, comprising:
administering into said host a migration-inhibiting effective
amount of a composition comprising: (1) an anti-CCL25 antibody, a
fragment of an anti-CCL25 antibody, an anti-CCR9 antibody, or a
fragment of an anti-CCR9 antibody; and (2) a pharmaceutically
acceptable carrier, wherein said composition is administered
directly to tumor tissue.
16. The method of claim 15, wherein said anti-CCL25 antibody or
said anti-CCR9 antibody is a human antibody.
17. The method of claim 15, wherein said anti-CCL25 antibody or
said anti-CCR9 antibody is a humanized antibody.
18. The method of claim 15, wherein said anti-CCL25 antibody or
said anti-CCR9 antibody is a chimeric antibody.
19. The method of claim 15, wherein said composition comprises both
said anti-CCL25 antibody and said anti-CCR9 antibody.
20. The method of claim 15, wherein said pharmaceutically
acceptable carrier comprises a solid support.
22. The method of claim 21, wherein said solid support is a sponge
or gauze.
23. The method of claim 15, wherein said pharmaceutically
acceptable carrier comprises liposomes.
24. The method of claim 15, wherein said pharmaceutically
acceptable carrier comprises microspheres.
25. The method of claim 15, wherein said antibody is administered
directly to tumor bed during an invasive procedure.
26. The method of claim 15, wherein said anti-CCL25 antibody, a
fragment of an anti-CCL25 antibody, an anti-CCR9 antibody, or a
fragment of an anti-CCR9 antibody is administered at a dose range
of 0.01-1000 mg/kg/day.
27. The method of claim 15, wherein said anti-CCL25 antibody or
said anti-CCR9 antibody is administered at a dose range of 0.1-100
mg/kg/day.
28. The method of claim 15, wherein said pharmaceutically
acceptable carrier is saline.
29. The method of claim 28, wherein said saline is buffered
saline.
30. The method of claim 15, wherein said pharmaceutically
acceptable carrier is glucose in saline.
31. The method of claim 15, wherein said composition further
comprises an antibody, or a fragment of an antibody, that binds to
a chemokine or chemokine receptor selected from the group
consisting of CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12,
CXCL13, CXCL16, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1.
32. A method of inhibiting carcinoma cell growth in a host,
comprising: administering into said host a growth-inhibiting
effective amount of a composition comprising: (1) an anti-CCL25
antibody, a fragment of an anti-CCL13 antibody, an anti-CCR9
antibody, or a fragment of an anti-CCR9 antibody; and (2) a
pharmaceutically acceptable carrier.
33. The method of claim 32, wherein said composition comprises (1)
an anti-CCL25 antibody or a fragment of an anti-CCL25 antibody; (2)
an anti-CCR9 antibody or a fragment of an anti-CCR9 antibody; and
(3) a pharmaceutically acceptable carrier.
34. The method of claim 32, wherein said composition further
comprises an antibody, or a fragment of an antibody, that binds to
a chemokine or chemokine receptor selected from the group
consisting of CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12,
CXCL13, CXCL16, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 10/712,398, filed on Nov. 14, 2003,
which claims priority from U.S. Provisional Patent Application
60/426,347, filed on Nov. 15, 2002. The entirety of all of the
aforementioned applications is incorporated herein by
reference.
FIELD
[0003] This invention relates to antibodies or the use of
antibodies directed against certain chemokines. The antibodies
block high affinity interactions leading to the growth or migration
of cancer cells.
BACKGROUND
[0004] Despite recent advances in cancer research, the development
of cell-specific therapies for treatment of malignancies remain
elusive. The many and complex factors which enable malignant cells
to undergo mutations, evade immune protection and promote
angiogenesis to deliver nutrients to the rapidly growing cells
complicate the development of targeted treatment modalities.
Current therapies have multiple untoward side effects. For example,
chemotherapy results in multiple painful and sometimes lethal side
effects. Advances in biotechnology have promoted the development of
targeted biologicals with fewer side effects.
[0005] Host cells have surface receptors that associate with
ligands to signal and cause host cell activities. The epidermal
growth factor receptor helps control cell growth and metastasis.
Many tumor cells express higher numbers of epidermal growth factor
receptors than normal cells. A new treatment designated IMC-225 was
specifically designed to target and block epidermal growth factor
receptors preventing cell division and repair. Recently,
trastuzumab, which is a HER-2-specific monoclonal antibody, has
proven effective at treating metastatic breast cancers. This
antibody blocks interactions on cancer cells that inhibit cell
growth. Unfortunately, HER-2 is only found on about 25 to 30
percent of breast cancer cells.
[0006] A variety of pathogens or toxins activate macrophages,
neutrophils, T cells, B cells, monocytes, NK cells, Paneth and
crypt cells, as well as epithelial cells shortly after entry into
the mucosa. Chemokines are a superfamily of small, cytokine-like
proteins that are resistant to hydrolysis, promote
neovascularization or endothelial cell growth inhibition, induce
cytoskeletal rearrangement, activate or inactivate lymphocytes, and
mediate chemotaxis through interactions with G-protein-coupled
receptors. Chemokines can mediate the growth and migration of host
cells that express their receptors. The cellular mechanisms
responsible for the function of the chemokines are often, but not
always, Ca.sup.2+ flux dependent and pertussis toxin-sensitive.
However, the precise mechanisms for chemokine-mediated events are
not known
SUMMARY
[0007] The present invention provides a means of inhibiting the
growth and metastasis of cancer cells by administering
anti-chemokine antibodies. Exemplified are anti-CXCR1, -CXCR2,
-CXCL1, -CXCL2, -CXCL3, -CXCL5, -CXCL6 -CXCL7, -CXCL8, -CXCL12,
-CXCR5a, -CXCR5b, -CXCL13, -CXCR6, -CXCL16, -CCL16, -CCL25,
-CCL25-1, -CCL25-2, -CX3CR1, and -CX3CL1 antibodies. The invention
relates to an antibody or functional fragment thereof that bind to
CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 CXCL7, CXCL8,
CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCL25,
CCL25-1, CCL25-2, CX3CR1, and CX3CL1. This invention utilizes
antibodies or antigen-binding fragment(s) that bind to epitope(s)
or peptide(s) that consists of 10 to 15 amino acids from Sequences
1 through 22. The antibodies or antigen-binding fragement(s) can be
isolated from the serum of immunized hosts, an immortalized cell
lines or tissues such as hybridomas, lymphoblastoid or cells
generated by methods of recombinant molecule biology. For increased
effectiveness, antibodies and fragments may be conjugated or linked
to other peptides, proteins, nucleic acid sequence, vitamins,
complex or simple carbohydrates or other suitable carrier
molecules.
[0008] Antibodies or antigen-binding fragments with specificity for
functional mutant or variant mammalian chemokines are appropriate.
These mutations or polymorphisms occur in nature or can be induced
by recombinant molecular biological methods to generate single,
multiple, or continuous amino acid residues, described in Sequences
1 through 22, that are deleted, added, and/or substituted for other
or no amino acids.
[0009] The antibodies for use in accord with the teachings of this
specification may be administered systemically or mucosally.
Mucosal means include oral, intranasal, ocular, intravaginal,
rectal and/or intraurethral administration in liquid or particulate
form or on solid supports. Systemic means include parenteral means
such as intravenous, subcutaneous or intramuscular
administration.
DETAILED DESCRIPTION
[0010] The invention describes a method of identifying and
isolating cancer cells from adenoma, carcinoma, leukemia, lymphoma,
melanoma, or myeloma using the antibodies in accord with the
teachings of this specification. Also, as demonstrated herein,
anti-CXCR1, -CXCR2, -CXCL1, -CXCL2, -CXCL3, -CXCL5, -CXCL6 -CXCL7,
-CXCL8,-CXCL12, -CXCR5a, -CXCR5b, -CXCL13, -CXCR6, -CXCL16, -CCL16,
-CCL25, -CCL25-1, -CCL25-2, -CX3CR1, and -CX3CL1 antibodies can to
administered to inhibit the rate of growth, metastasis and/or
angiogenesis mediated by adenomas, carcinomas, leukemias,
lymphomas, melanomas, and/or myelomas.
Materials and Methods
Primer Design
[0011] Messenger RNA sequences for CXCR1, CXCR2, CXCL1, CXCL2,
CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCL12, CXCR5a, CXCR5b, CXCL13,
CXCR6, CXCL16, CCL16,CCL25, CCL25-1, CCL25-2, CX3CR1, and CX3CL1
were obtained from the NIH-NCBI gene bank database (Sequences 23
through 44). Primers were designed using the BeaconJ 2.0 computer
program. Thermodynamic analysis of the primers was conducted using
computer programs: Primer PremierJ and MIT Primer 3. The resulting
primer sets were compared against the entire human genome to
confirm specificity.
Real Time PCR Analysis
[0012] Cancer cell lines (ATCC, Rockville, Md.) were cultured in
RMPI-1640 containing 10% fetal calf serum supplemented with
non-essential amino acids, L-glutamate, and sodium pyruvate
(complete media). Primary tumor and normal-paired matched tissues
were obtained from clinical isolates (Clinomics Biosciences,
Frederick, Md. and UAB Tissue Procurement, Birmingham, Ala.).
Messenger RNA (mRNA) was isolated from 10.sup.6 cells using
TriReagent (Molecular Research Center, Cincinnati, Ohio) according
to manufacturer's protocols. Potential genomic DNA contamination
was removed from these samples by treatment with 10 U/Fl of RNase
free DNase (Invitrogen, San Diego, Calif.) for 15 minutes at
37.degree. C. RNA was then precipitated and resuspended in RNA
Secure (Ambion, Austin, Tex.). The cDNA was generated by reverse
transcribing approximately 2 .mu.g of total RNA using Taqman7
reverse transcription reagents (Applied-Biosystems, Foster City,
Calif.) according to manufacturer's protocols. Subsequently, cDNA's
were amplified with specific human cDNA primers, to CXCR1, CXCR2,
CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCL12, CXCR5a,
CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2,
CX3CR1, and CX3CL1, using SYBR7 Green PCR master mix reagents
(Applied Biosystems) according to manufacturer's protocol. The
level of copies of mRNA of these targets were evaluated by
real-time PCR analysis using the BioRad Icycler and software
(Hercules, Calif.).
Anti-Sera Preparation
[0013] The 15 amino acid peptides from CXCR1, CXCR2, CXCL1, CXCL2,
CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCL12, CXCR5a, CXCR5b, CXCL13,
CXCR6, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2, CX3CR1, and CX3CL1
(Sequences 1 through 22) were synthesized (Sigma Genosys, The
Woodlands, Tex.) and conjugated to hen egg lysozyme (Pierce,
Rockford, Ill.) to generate the "antigen" for subsequent
immunizations for anti-sera preparation or monoclonal antibody
generation. The endotoxin levels of chemokine peptide conjugates
were quantified by the chromogenic Limulus amebocyte lysate assay
(Cape Cod, Inc., Falmouth, Mass.) and shown to be <5 EU/mg. 100
.mu.g of the antigen was used as the immunogen together with
complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first
immunization in a final volume of 1.0 ml. This mixture was
administered in 100 ml aliquots on two sites of the back of the
rabbit subcutaneously and 400 ml intramuscularly in each hind leg
muscle. Three to four weeks later, rabbits received 100 .mu.g of
the antigen in addition to incomplete Freund's adjuvant for 3
subsequent immunizations. Anti-sera were collected when anti-CXCR1,
-CXCR2, -CXCL1, -CXCL2, -CXCL3, -CXCL5, -CXCL6 -CXCL7, -CXCL8,
-CXCL12, -CXCR5a, -CXCR5b, -CXCL13, -CXCR6, -CXCL16, -CCL16,
-CCL25, -CCL25-1, -CCL25-2, -CX3CR1, and -CX3CL1 antibody titers
reached 1:1,000,000. Subsequently, normal or anti-sera were
heat-inactivated and diluted 1:50 in PBS.
Monoclonal Antibody Preparation
[0014] The 15 amino acid peptides from CXCR1, CXCR2, CXCL1, CXCL2,
CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCL12, CXCR5a, CXCR5b, CXCL13,
CXCR6, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2, CX3CR1, and CX3CL1
(Sequences 1 through 22) were synthesized (Sigma Genosys) and
conjugated to hen egg lysozyme (Pierce) to generate the "antigen"
for subsequent immunizations for anti-sera preparation or
monoclonal antibody generation. The endotoxin levels of chemokine
peptide conjugates were quantified by the chromogenic Limulus
amebocyte lysate assay (Cape Cod, Inc., Falmouth, Mass.) and shown
to be <5 EU/mg. 100 .mu.g of the antigen was used as the
immunogen together with complete Freund's adjuvant Ribi Adjuvant
system (RAS) for the first immunization in a final volume of 200
.mu.l. This mixture was subcutaneously administered in 100 .mu.l
aliquots at two sites of the back of a rat, mouse, or
immunoglobulin-humanized mouse. Two weeks later, animals received
100 .mu.g of the antigen in addition to incomplete Freund's
adjuvant for 3 subsequent immunizations. Serum were collected and
when anti-CXCR1, -CXCR2, -CXCL1, -CXCL2, -CXCL3, -CXCL5, -CXCL6
-CXCL7, -CXCL8, -CXCL12, -CXCR5a, -CXCR5b, -CXCL13, -CXCR6,
-CXCL16, -CCL16, -CCL25, -CCL25-1, -CCL25-2, -CX3CR1, or -CX3CL1
antibody titers reached 1:2,000,000, hosts were sacrificed and
splenocytes were isolated for hybridoma generation. Briefly, B
cells from the spleen or lymph nodes of immunized hosts were fused
with immortal myeloma cell lines (e.g., YB2/0). Hybridomas were
next isolated after selective culturing conditions (i.e.,
HAT-supplemented media) and limiting dilution methods of hybridoma
cloning. Cells that produce antibodies with the desired specificity
were selected using ELISA. Hybridomas from normal rats or mice were
humanized with molecular biological techniques in common use. After
cloning a high affinity and prolific hybridoma, antibodies were
isolated from ascites or culture supernatants and adjusted to a
titer of 1:2,000,000 and diluted 1:50 in PBS.
Anti-Sera or Monoclonal Antibody Treatment
[0015] The NIH-III mice (8 to 12 weeks old, Charles River
Laboratory, Wilmington, Mass.), which lack T, B, and NK cells,
received 1.times.10.sup.6 cancer cells, subcutaneously, for the
establishment of a tumor. Correspondingly, freshly isolated or
liquid nitrogen frozen 1 g of tumor tissue were surgically
implanted in the intestinal adipose tissue for the generation of
tumor. Once the xenografted tumor growth reached 5 mm in size the
NIH-III mice received 200.mu.l intraperitoneal injections of either
antisera or monoclonal antibodies every three days and the tumor
was monitored for progression or regression of growth.
Data Analysis
[0016] SigmaStat 2000 (Chicago, Ill.) software was used to analyze
and confirm the statistical significance of data. The data were
subsequently analyzed by the Student's t-test, using a two-factor,
unpaired test. In this analysis, treated samples were compared to
untreated controls. The significance level was set at
p<0.05.
Results
[0017] Semiquantitative RT-PCR Identification of Molecular
Targets
[0018] The RT-PCR products obtained using CXCR1-, CXCR2-, CXCL1-,
CXCL2-, CXCL3-, CXCL5-, CXCL6-, CXCL7-, CXCL8-, CXCL12-, CXCR5a-,
CXCR5b-, CXCL13-, CXCR6-, CXCL16-, CCL16-, CCL25-, CCL25-1-,
CCL25-2-, CX3CR1-, or CX3CL1-specific primer sets did not cross
react with other gene targets due to exclusion of primers that
annealed to host sequences (NIH-NCBI Genebank). The primers used
produced different size amplicon products relative the
polymorphisms that resulted in CXCR5a versus CXCR5b and CCL25,
CCL25-1, versus CCL25-2. To this end, RT-PCR analysis of adenoma,
carcinoma, leukemia, lymphoma, melanoma, and/or myeloma cell lines
and tumor tissue revealed that CXCR1, CXCR2, CXCL1, CXCL2, CXCL3,
CXCL5, CXCL6 CXCL7, CXCL8, CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13,
CXCR6, CXCL16, CCL16, CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1, and
CX3CL1 were differentially expressed by cancer cells.
In vitro Growth Studies
[0019] The adenoma, carcinoma, leukemia, lymphoma, melanoma, and/or
myeloma cell lines were grown in complete media in the presence or
absence of antibodies specific for CXCR1, CXCR2, CXCL1, CXCL2,
CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCR4, CXCL12, CXCR5a, CXCR5b,
CXCL13, CXCR6, CXCL16, CCL16, CCR9, CCL25, CCL25-1, CCL25-2,
CX3CR1, or CX3CL1. The growth of cancer cell lines expressing CXCR1
and/or CXCR2 were inhibited by antibodies to CXCR1, CXCR2, CXCL1,
CXCL2, CXCL3, CXCL5, CXCL6 CXCL7, or CXCL8. Similarly, the growth
of cancer cell lines expressing CXCR4 were inhibited by antibodies
to CXCR4 or CXCL12. The growth of cancer cell lines expressing
CXCR5a or CXCR5a were inhibited by antibodies to CXCR5a, CXCR5b, or
CXCL13. The proliferation of cancer cell lines expressing CXCR6 or
were inhibited by antibodies to CXCR6 or CXCL16. The growth of
cancer cell lines expressing CCR9 were inhibited by antibodies to
CCR9 or CCL25, CCL25-1, and CCL25-2. The propagation of cancer cell
lines expressing CX3CR1 were inhibited by antibodies to CX3CR1 or
CXC3L1. Of interest, antibodies against the soluble ligands, CXCL1,
CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CXCL13, CXCL16,
CCL16, CCL25, CCL25-1, CCL25-2, or CX3CL1, were more effective at
growth inhibition that those directed against the membrane
receptors.
In vitro Angiogenesis Studies
[0020] The microvascular endothelial cells (Cell Systems, Wirkland,
Wash.) were grown according to manufacturer's protocols and allowed
to form microvascular venules in an in vitro assay for angiogenesis
(BD-Biocoat, Hercules, in the presence or absence of antibodies
specific for CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 CXCL7,
CXCL8, CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16,
CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1, or CX3CL1. The angiogenesis
was inhibited by antibodies against CXCR1, CXCR2, CXCL1, CXCL2,
CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCR4, CXCL12, CXCR6 or
CXCL16.
In vivo Growth Studies
[0021] Cancer cell lines or primary tumor tissue were adoptively
transferred into NIH-III mice and allowed to form the xenograft
tumor of interest. Antibodies directed against CXCR1, CXCR2, CXCL1,
CXCL2, CXCL3, CXCL5, CXCL6 CXCL7, CXCL8, CXCR4, CXCL12, CXCR5a,
CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCR9, CCL25, CCL25-1,
CCL25-2; CX3CR1, or CX3CL1 differentially affected the progression
and regression of tumor size. In certain cases, antibodies directed
towards CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 CXCL7,
CXCL8, CXCR4, CXCL12, CXCR6 or CXCL16 effectively lead to the both
regression and impeding progression of tumor growth. Antibodies
directed against CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CCL16,
CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1, or CX3CL1 were effective at
inhibiting the progression of tumor size.
[0022] The protein sequences of the chemokines used herein are
recorded in NIH-NCBI Genebank as: (1) CXCR1 (ACCESSION# NM 000634),
(2) CXCR2(ACCESSION# NM 001557), (3) CXCL1 (ACCESSION#
NM.sub.--001511), (4) CXCL2 (ACCESSION# NM.sub.--001557), (5) CXCL3
(ACCESSION# NM.sub.--002090), (6) CXCL5 (ACCESSION#
NM.sub.--002994), (7) CXCL6 (ACCESSION# NM.sub.--002993), (8) CXCL7
(ACCESSION# NM.sub.--002704), (9) CXCL8 (ACCESSION#
XM.sub.--170504), (10) CXCR4 (ACCESSION# NM.sub.--003467), (11)
CXCL12 (ACCESSION# NM.sub.--000609), (12) CXCR5A (ACCESSION#
NM.sub.--032966), (13) CXCR5B (ACCESSION# NM.sub.--001716), (14)
CXCL13 (ACCESSION# NM.sub.--006419), (15) CXCR6 (ACCESSION#
NM.sub.--006564), (16) CXCL16 (ACCESSION# NM022059), (17) CCL16
(ACCESSION# NM.sub.--004590), (18) CCL25 (ACCESSION#015444), (19)
CCL25-1 (ACCESSION# NM.sub.--005624), (20) CCL25-2 (ACCESSION#
NM.sub.--148888), (21) CX3CR1 (ACCESSION# NM.sub.--001337), and
(22) CX3CL1 (ACCESSION# NM.sub.--002996).
[0023] The cDNA sequences are known and are available in NIH-NCBI
Genebank under the following accession numbers: (23) CXCR1
(ACCESSION# NM 000634), (24) CXCR2 (ACCESSION# NM 001557), (25)
CXCL1 (ACCESSION# NM.sub.--01511). (26) CXCL2 (ACCESSION#
NM.sub.--001557), (27) CXCL3 (ACCESSION# NM.sub.--002090), (28)
CXCL5 (ACCESSION# NM.sub.--002994), (29) CXCL6 (ACCESSION#
NM.sub.--002993), (30) CXCL7 (ACCESSION# NM.sub.--002704), (31)
CXCL8 (ACCESSION# XM.sub.--170504), (32) CXCR4 (ACCESSION#
NM.sub.--003467), (33) CXCL12 (ACCESSION# NM.sub.--000609), (34)
CXCR5A (ACCESSION# NM.sub.--032966), (35) CXCRSB (ACCESSION#
NM.sub.--001716) (36) CXCL13 (ACCESSION# NM.sub.--006419), (37)
CXCR6 (ACCESSION# NM.sub.--006564), (38) CXCL16 (ACCESSION#
NM.sub.--022059), (39) CCL16 (ACCESSION# NM.sub.--004590), (40)
CCL25 (ACCESSION#015444), (41) CCL25-1 (ACCESSION#
NM.sub.--005624), (42) CCL25-2 (ACCESSION# NM.sub.--148888), (43)
CX3CR1 (ACCESSION# NM.sub.--001337), and (44) CX3CL1 (ACCESSION#
NM.sub.--002996).
[0024] As shown in the table below, the particular chemokines which
are most which any tumor expresses may vary. It is possible, using
methods of the invention, to customize treatment for the particular
patient, depending on the chemokines over-expressed by the
patient's own tumor. It is possible to identify the particular
chemokines which are over-expressed in the tumor using methods of
the invention and administer antibodies against that over-expressed
chemokine. The tailoring of treatment for the cancer patient is
novel, and is a particularly valuable aspect of the invention.
[0025] The method consists of 1) exposing samples of malignant
tissue or products therefrom to an array of antibodies to different
chemokines, allowing the antibodies to bind to the malignant
tissue, then measure the amount of the chemokines by measuring the
amount of chemokine bound to particular antibodies to identify the
level of expression of each chemokine. The patient is then given
the antibodies against the over-expressed chemokine(s). However,
the level of each chemokine may also be evaluated using PCR
technologies. Such direct evaluation is now easily done, as shown
above. See particularly the section entitled, "Semiquantitative
RT-PCR identification of molecular targets".
[0026] Antibodies of the invention can be administered in the
usually accepted pharmaceutically acceptable carriers. For example,
compositions containing antibodies can be prepared in accord with
the section entitled "Monoclonal antibody preparation". Acceptable
carriers include, but are not limited to, saline, buffered saline,
glucose in saline. Solid supports, liposomes or microspheres may
also be used as carriers for administration of the antibodies.
Antibodies of the invention may be administered directly to target
tissue. For example, compositions containing the compositions
containing antibodies as prepared under the heading "anti-sera
preparation" can be administered intravenously, rectally vaginally,
intrathecally, by inhalation, transvaginally, transurethrally or
directly to tissue during surgery. The anti-sera preparations may
also be placed on a solid support such as a sponge or gauze for
administration of antibodies against the target chemokine to the
affected tissues, including administration directly to the tumor
bed during invasive procedures. The table on the following page
indicates the differing amounts of particular chemokines
over-expressed in particular tumors that were studied.
TABLE-US-00001 TABLE I Chemokine, Chemokine Receptor and Cancer
Association (dependent of stage of disease) Cancer Chemokine
Chemokine Receptor Carci- CCL1, CCL4, CCL17, CCL19, CCL21, CCR7,
CCR8, noma CCL22, CCL25 CCR9 CXCL12, CXCL13, CXCL16 CXCR4, CXCR5,
CXCR6, CX3CR1 Leuke- CCL1, CCL4, CCL17, CCL19, CCL21, CCR7, CCR8,
mia CCL22, CCL25 CCR9 CXCL12 CXCR4 Lym- CXCL12, CXCL13 CXCR4, CXCR5
phoma Mela- CCL25, CCL27 CCR9, CCR10 noma CXCL1, CXCL2, CXCL3,
CXCL5, CXCR1, CXCR2, CXCL6, CXCL7, CXCL8, CXCL12, CXCR4, CXCR5,
CXCL13, CXCL16 CXCR6 CX3CL1 CX3CR1 Sar- CCL1, CCL3, CCL4, CCL5,
CCL7, CCR3, CCR5, coma CCL8, CCL11, CCL13, CCL17, CCR8 CCL22, CCL24
CXCL12 CXCR4 CX3CL1 CX3CR1
[0027] Dosage would more commonly be in the range of 0.01 to 1000
mg/kg/da, more often in the range of 0.1 to 100 mg/kg/da. As
expected, the dosage will be dependant on the condition, size, age
and condition of the patient.
* * * * *