Antigenic regions of tumor-liberated particles (TLP) complexes a

Details Antigenic regions of tumor-liberated particles (TLP) complexes a Antigenic regions of tumor-liberated particles (TLP) complexes a

1994-12-22

1998-05-05

Feisee, Lila

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

6 to 7 amino acid residues in defined sequence

5303877, 5303888, 4242771, 435 723, A61K 3804, C07K 500, C07K 700, C07K 1600

Patent

active

057476438

DESCRIPTION:

BRIEF SUMMARY
SPECIFICATION

This invention relates to peptide regions of the human TLP proteic complexes (proteins released from tumors) with antigenic activity, as well as to antibodies reacting with such proteins, to be used for diagnostic and clinical purposes.
TLP complexes are proteic complexes present in human tumor cells, particularly in lung carcinoma cells; among the TLP proteins a 214 KDa protein is described (Tarro G., "Oncology", 40, 248-253, 1983). TLPs are isolated from tumoral tissues, as described in the EP 283 433, the description thereof is incorporated herein as reference. It is very useful to have diagnostic assays to identify such complexes, or fractions thereof, from crude lung extracts.
Author's attempts to obtain specific antibodies against whole TLP's did not succeed. Therefore it is important to identify TLP antigenic regions (epitopes) and raise antibodies thereto, in order to obtain specific reactants.
The author of the present invention has identified peptide sequences of the TLP 100 KDa protein having antigenic activity, has obtained specific antibodies and has demonstrated that said antibodies reacted specifically with TLPs from lung carcinomas.
Accordingly, an object of this invention is an antigenic peptide of Tumour liberated particles (TLP) having the amino acid sequence comprised within the sequence of the 100 KDa protein of TLP.
According to a preferred embodiment of the invention, said peptide has at least one of the amino acid sequences listed at the end of the specification as Seq ID N1, Seq ID N2 or Seq ID N3. Preferably said peptides are of synthetic origin, alternatively of natural source.
In a further embodiment of the invention, said peptide comprises a cysteine residue at either its carboxylic or aminic terminal group, and a molecule carrier, covalently bound to said residue; preferably said molecule carrier is hemocyanin, most preferably said hemocyanin is obtained from oysters.
Another object of the invention are antibodies able to specifically detect TLP proteins by recognizing an antigenic peptide of Tumour liberated particles (TLP) having the amino acid sequence comprised within the sequence of the 100 KDa protein of TLP.
Preferably said antibodies recognize a peptide having at least one of the amino acid sequences listed at the end of the specification as Seq ID N1, Seq ID N2 or Seq ID N3.
A further object of the invention are diagnostic kits to detect TLP from samples comprising the antibodies of the invention as specific reactants.
Another object of the invention is a method to detect TLP complexes from samples, comprising the steps as follows: reacting the same with a second amount of said serum anti-TLP, and with revealing means of said reaction.
This invention will be described in the following with reference to some explanatory, but not limiting examples, to which are related the annexed figures, wherein:
FIGS. 1A-1D show some graphics of RIA immunologic assays of serum samples from 4 rabbits immunized with the peptides described in Seq ID N1, N2 and N3 (A, B, C(419) and D(428);
FIG. 2A shows a direct immunoblot, using the sera 419 (1) and 428 (2), as well as previously immunized sera (4-6) on samples of lung extracts;
FIG. 2B shows an immunoblot wherein the extracts have been previously immunoprecipitated using the 419 and 428 sera; and
FIG. 2C shows an immunoblot wherein the extracts have been previously immunoprecipated, as described in the Example 3.


EXAMPLE 1

The TLP complex is isolated from lung carcinoma extracts, as described in the EP 283 443. The protein is blocked at the amino terminal group and accordingly digested by V8 protease from Staphylococcus aureus, using known techniques. One of the obtained peptide fragments shows the sequence as follows: XaaXaaArgThrAsnLysGluAlaSerIle.
Two synthetic peptides are synthetised, using the solid phase method, by means of a 430 peptide synthetizer from Applied Biosystems, as described by Bodanszky M., Springer Verlag, New York, N.Y., 1984, having the sequences as shown in the table 1 herebelow:


TABLE 1



5

REFERENCES:
Harlow et al Antibodies: A Laboratory Manual, Chapter 5, pp. 55-136, Cold Spring Harbor Laboratroy, 1988.
"Human Tumor Antigens Inducing in vivo Delayed Hypersensitivity and in vitro Mitogenic Activity" by Tarro et al.; Oncology 40, pp. 248-254 (1983) .

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