WO1996016986A1 - Mel-cam ligand and its use - Google Patents

Mel-cam ligand and its use Download PDF

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Publication number
WO1996016986A1
WO1996016986A1 PCT/US1995/015812 US9515812W WO9616986A1 WO 1996016986 A1 WO1996016986 A1 WO 1996016986A1 US 9515812 W US9515812 W US 9515812W WO 9616986 A1 WO9616986 A1 WO 9616986A1
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WO
WIPO (PCT)
Prior art keywords
ligand
mel
cam
cells
endothelial cells
Prior art date
Application number
PCT/US1995/015812
Other languages
French (fr)
Inventor
Meenhard Herlyn
David Speicher
Original Assignee
The Wistar Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Wistar Institute filed Critical The Wistar Institute
Priority to AU44162/96A priority Critical patent/AU4416296A/en
Publication of WO1996016986A1 publication Critical patent/WO1996016986A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Mel-CAM also known as MUC18 and A32 antigen
  • immunoglobulin supergene family Lehmann et al., APTOC. Nail. Acad. Sciences USA 86:9891-9895 (1989); Shih et al., Cancer Research 54:2514-2520 (1994). It is an integral membrane glycoprotein expressed by most human malignant melanomas and nevi, but not on normal epidermal melanocytes. Of the normal tissues tested, only endothelial cells, smooth muscle, cerebellum, and hair follicles express Mel-CAM. It is a cell-cell adhesion molecule which may be involved in invasion and metastasis of cancer cells.
  • a Mel-CAM-ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells.
  • the ligand has an apparent molecular weight of 60 kDa and is expressed on SBcl-2 melanoma
  • a polypeptide comprising a sequence of between five and fifty contiguous amino acids of a Mel-CAM-ligand is provided.
  • the ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein
  • the ligand has an apparent
  • a method for inhibiting melanoma cell binding to endothelial cells comprises: contacting endothelial cells with a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said ligand is expressed on SBcl-2 melanoma cells, and (2) a polypeptide comprising a sequence of between five and fifty contiguous amino acids of said Mel-CAM- ligand, in an amount sufficient to inhibit the binding of melanoma cells to said endothelial cells-
  • a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells, wherein said
  • a method of inhibiting the adhesion of activated mononuclear cells to endothelial cells comprises: contacting endothelial cells with a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said ligand is expressed on SBcl-2 melanoma cells, and (2) a polypeptide comprising a sequence of between five and fifty contiguous amino acids of said Mel-CAM- ligand, in an amount sufficient to inhibit the binding of activated mononuclear cells
  • telomeres melanoma cells to endothelial cells.
  • the ligand is also involved in the adhesion of peripheral blood mononuclear cells to endothelial cells of the vasculature during inflammation and infection.
  • the ligand, or portions thereof involved in the Mel-CAM binding can be used to inhibit vascular dissemination of melanoma cells. In addition, it can be used to inhibit inflammation.
  • Mel-CAM-negative SBcl-2 melanoma cells adhere to nitro-cellulose immobilized Mel-CAM produced by baculovirus recombinants. Adhesion can be blocked with full-length Mel-CAM or with polyclonal anti-serum against recombinant Mel-CAM. However, adhesion is not affected by the presence of EDTA or truncated (extracellular domain) Mel-CAM.
  • SBcl-2 cells cluster with U937 cells (human histiocytic lymphoma; ATCC CRL 1593) which have been transfected with Mel- CAM cDNA but not with control non-transfectants. This indicates that SBcl-2 cells express a specific ligand for Mel-CAM.
  • SBcl-2 cells form heterotypic aggregates with human endothelial cells. However, formation of such aggregates is inhibited in the presence of full-length Mel-CAM or polyclonal anti-serum against recombinant Mel-CAM.
  • Mel-CAM-ligand can be co-purified with Mel-CAM, for example using an
  • the ligand can be co-immunoprecipitated using antibodies specific for Mel-CAM, in the presence of Mel-CAM.
  • the ligand has an apparent molecular weight on SDS-polyacrylamide gels of about 60 kDa.
  • Mel-CAM-ligand is expressed on most, if not all, melanoma cells but not on endothelial cells. Mel-CAM is expressed on endothelial cells. Thus Mel- CAM-ligand is involved in the heterotypic binding of melanoma cells to endothelial cells, and participates in the vascular dissemination of melanoma cells during metastasis.
  • Mel-CAM-ligand is also expressed on activated peripheral blood mononuclear cells.
  • Mel-CAM-ligand or portions thereof can be used to inhibit inflammation reactions which involve the binding of activated peripheral blood mononuclear cells to cells which express Mel-CAM, for example,
  • the amount of Mel-CAM-ligand which is expressed by tumor cells could be determined to provide a prognostic indicator. Any method known in the art for quantitating proteins could be used, including immunological or protein binding assays. Similarly, the amount of Mel-CAM-ligand which is shed into the blood stream could be determined to provide yet another prognostic indicator. Previously the expression of Mel-CAM has been found to correlate with the progression of tumors and a poor prognosis. Lehmann et al., Proc. Nat/. Acad. Sciences USA

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Mel-CAM-ligand is expressed by melanoma cells but not endothelial cells. It is a 60 kDa protein. Mel-CAM-ligand specifically binds to Mel-CAM (MUC18) a 113 kDa glycoprotein which is expressed on melanoma cells, endothelial cells, as well as on a few other types of cells. Soluble preparations of Mel-CAM-ligand can be used to inhibit interactions between Mel-CAM-ligand-expressing cells and cells expressing Mel-CAM. To the extent that such interactions are pathological, use of Mel-CAM-ligand is therapeutic.

Description

ME- CAM LIGAND AND ITS USE
This invention was made using funds from U.S. government grant
CA25874 from the National Cancer Institute.. The government therefore retains certain rights in the invention.
BACKGROUND OF THE INVENTION
Mel-CAM (also known as MUC18 and A32 antigen) is a member of the
immunoglobulin supergene family. Lehmann et al., APTOC. Nail. Acad. Sciences USA 86:9891-9895 (1989); Shih et al., Cancer Research 54:2514-2520 (1994). It is an integral membrane glycoprotein expressed by most human malignant melanomas and nevi, but not on normal epidermal melanocytes. Of the normal tissues tested, only endothelial cells, smooth muscle, cerebellum, and hair follicles express Mel-CAM. It is a cell-cell adhesion molecule which may be involved in invasion and metastasis of cancer cells.
It has been suggested by Lehmann et al. , supra, that dissemination of tumor cells to distant sites may involve the adhesion of cell types which each express Mel-CAM, i.e., by means of a homotypic interaction. There is a need in the art for clarification of the role and mechanism of action of Mel-CAM in human disease.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a preparation of a ligand involved in cell-cell adhesion.
It is another object of the invention to provide a polypeptide which can
inhibit cell-cell adhesion.
It is yet another object of the invention to provide a method of inhibiting the interaction of melanoma cells with endothelial cells.
It is still another object of the invention to provide a method of inhibiting the interaction of activated peripheral blood mononuclear cells with endothelial
cells.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment a cell-free preparation of
a Mel-CAM-ligand is provided. The ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells. The ligand has an apparent molecular weight of 60 kDa and is expressed on SBcl-2 melanoma
cells.
According to another embodiment of the invention a polypeptide comprising a sequence of between five and fifty contiguous amino acids of a Mel-CAM-ligand is provided. The ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein
expressed on melanoma and endothelial cells. The ligand has an apparent
molecular weight of 60 kDa and is expressed on SBcl-2 melanoma cells. In another embodiment a method is provided for inhibiting melanoma cell binding to endothelial cells. The method comprises: contacting endothelial cells with a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said ligand is expressed on SBcl-2 melanoma cells, and (2) a polypeptide comprising a sequence of between five and fifty contiguous amino acids of said Mel-CAM- ligand, in an amount sufficient to inhibit the binding of melanoma cells to said endothelial cells-
According to yet another embodiment a method of inhibiting the adhesion of activated mononuclear cells to endothelial cells is provided. The method comprises: contacting endothelial cells with a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said ligand is expressed on SBcl-2 melanoma cells, and (2) a polypeptide comprising a sequence of between five and fifty contiguous amino acids of said Mel-CAM- ligand, in an amount sufficient to inhibit the binding of activated mononuclear cells
to said endothelial cells.
Thus the present invention provides the art with the methods and tools for inhibition of pathological cell-cell interactions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It is a discovery of the present invention that most melanoma cells express a ligand, called here Mel-CAM-ligand, which specifically binds to the cell surface adhesion molecule Mel-CAM. This ligand is involved in the adhesion of
melanoma cells to endothelial cells. In addition, the ligand is also involved in the adhesion of peripheral blood mononuclear cells to endothelial cells of the vasculature during inflammation and infection. The ligand, or portions thereof involved in the Mel-CAM binding, can be used to inhibit vascular dissemination of melanoma cells. In addition, it can be used to inhibit inflammation.
Mel-CAM-negative SBcl-2 melanoma cells adhere to nitro-cellulose immobilized Mel-CAM produced by baculovirus recombinants. Adhesion can be blocked with full-length Mel-CAM or with polyclonal anti-serum against recombinant Mel-CAM. However, adhesion is not affected by the presence of EDTA or truncated (extracellular domain) Mel-CAM.
In cell aggregation assays, SBcl-2 cells cluster with U937 cells (human histiocytic lymphoma; ATCC CRL 1593) which have been transfected with Mel- CAM cDNA but not with control non-transfectants. This indicates that SBcl-2 cells express a specific ligand for Mel-CAM.
SBcl-2 cells form heterotypic aggregates with human endothelial cells. However, formation of such aggregates is inhibited in the presence of full-length Mel-CAM or polyclonal anti-serum against recombinant Mel-CAM.
Mel-CAM-ligand can be co-purified with Mel-CAM, for example using an
immunoaffinity column which employs polyclonal anti-serum against recombinant Mel-CAM. See Shih et al., Cancer Research 54:2514-2520 (1994). In addition, the ligand can be co-immunoprecipitated using antibodies specific for Mel-CAM, in the presence of Mel-CAM. The ligand has an apparent molecular weight on SDS-polyacrylamide gels of about 60 kDa.
Mel-CAM-ligand is expressed on most, if not all, melanoma cells but not on endothelial cells. Mel-CAM is expressed on endothelial cells. Thus Mel- CAM-ligand is involved in the heterotypic binding of melanoma cells to endothelial cells, and participates in the vascular dissemination of melanoma cells during metastasis.
Mel-CAM-ligand is also expressed on activated peripheral blood mononuclear cells. Thus Mel-CAM-ligand or portions thereof can be used to inhibit inflammation reactions which involve the binding of activated peripheral blood mononuclear cells to cells which express Mel-CAM, for example,
endothelial cells.
It is also contemplated as part of the present invention that the amount of Mel-CAM-ligand which is expressed by tumor cells could be determined to provide a prognostic indicator. Any method known in the art for quantitating proteins could be used, including immunological or protein binding assays. Similarly, the amount of Mel-CAM-ligand which is shed into the blood stream could be determined to provide yet another prognostic indicator. Previously the expression of Mel-CAM has been found to correlate with the progression of tumors and a poor prognosis. Lehmann et al., Proc. Nat/. Acad. Sciences USA
S6.-9891-9895 (1989).

Claims

CLAJMS
1. A cell-free preparation of a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma
and endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said ligand is expressed on SBcl-2 melanoma cells.
2. A polypeptide comprising a sequence of between five and fifty
contiguous amino acids of a Mel-CAM-ligand, wherein said ligand specifically binds to Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and
endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said ligand is expressed on SBcl-2 melanoma cells.
3. A method of inhibiting melanoma cell binding to endothelial cells,
comprising: contacting endothelial cells with a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to
Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells,
wherein said ligand has an apparent molecular weight of 60 kDa and wherein said
ligand is expressed on SBcl-2 melanoma cells, and (2) a polypeptide comprising a sequence of between five and fifty contiguous amino acids of said Mel-CAM-
ligand, in an amount sufficient to inhibit the binding of melanoma cells to said endothelial cells.
4. The method of claim 3 wherein the endothelial cells are in a human body which has a melanoma.
5. A method of inhibiting the adhesion of activated mononuclear cells to endothelial cells, comprising:
contacting endothelial cells with a protein selected from the group consisting of: (1) a Mel-CAM-ligand, wherein said ligand specifically binds to
Mel-CAM, a 113 kDa glycoprotein expressed on melanoma and endothelial cells, wherein said ligand has an apparent molecular weight of 60 kDa and wherein said
ligand is expressed on SBcl-2 melanoma cells, and (2) a polypeptide comprising a sequence of between five and fifty contiguous amino acids of said Mel-CAM- ligand, in an amount sufficient to inhibit the binding of activated mononuclear cells to said endothelial cells.
6. The method of claim 5 wherein the endothelial cells are in a human body and the body is undergoing an inflammation reaction.
7. The method of claim 5 wherein the endothelial cells are in a human body and the body has a bacterial or viral infection.
PCT/US1995/015812 1994-12-01 1995-11-16 Mel-cam ligand and its use WO1996016986A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU44162/96A AU4416296A (en) 1994-12-01 1995-11-16 Mel-cam ligand and its use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35216394A 1994-12-01 1994-12-01
US08/352,163 1994-12-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150218266A1 (en) * 2009-06-10 2015-08-06 Val-Chum, Limited Partnership Inhibition of th17 cells migration to inflamed tissues using antibodies directed against mcam
US11653668B2 (en) 2016-09-30 2023-05-23 Wm. Wrigley Jr. Company Gum bases incorporating polymers derived from Russian Dandelion

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANCER AND METASTASIS REVIEWS, Volume 10, issued 1991, JOHNSON, "Cell Adhesion Molecules of the Immunoglobulin Supergene Family and Their Role in Malignant Transformation and Progression to Metastatic Disease", pages 11-22. *
CANCER RESEARCH, Volume 54, issued 01 May 1992, SHIH et al., "Isolation and Functional Characterization of the A32 Melanoma-Associated Antigen", pages 2514-2520. *
CELL GROWTH AND DIFFERENTIATION, Volume 1, issued 1990, ZHOU et al., "Specificity of Intercellular Adhesion Mediated by Various Members of the Immunoglobulin Supergene Family", pages 209-215. *
CURRENT OPINION IN ONCOLOGY, Volume 7, issued 1995, EDWARD, "Integrins and Other Adhesion Molecules Involved in Melanocytic Tumor Progression", pages 185-191. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150218266A1 (en) * 2009-06-10 2015-08-06 Val-Chum, Limited Partnership Inhibition of th17 cells migration to inflamed tissues using antibodies directed against mcam
US11653668B2 (en) 2016-09-30 2023-05-23 Wm. Wrigley Jr. Company Gum bases incorporating polymers derived from Russian Dandelion

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