WO1994006016A1

WO1994006016A1 - New tumor cell marker

Info

Publication number: WO1994006016A1
Application number: PCT/US1993/008162
Authority: WO
Inventors: Irwin D. Bernstein
Original assignee: Fred Hutchinson Cancer Research Center
Priority date: 1992-09-03
Filing date: 1993-08-31
Publication date: 1994-03-17
Also published as: AU5097293A

Abstract

A marker useful to identify a subset of patients of acute myelogenous leukemia (AML) with poor prognosis has been identified as a protein of approximate molecular weight 220-240 kd. The presence of this marker can be detected using an antibody corresponding to or reacting with the same antigen as antibody 7.1. Antibody 7.1 is prepared by screening immortalized antibody-secreting cells from a mammal immunized with a transformed human marrow stromal cell line for reactivity with the cell line and non-reactivity with monocytes and granulocytes.

Description

NE TUMOR CELL MARKER

Technical Field

The invention relates to the field of markers which permit the diagnosis and imaging of tumors. More specifically, the invention concerns a monoclonal antibody capable of detecting a marker unique to tumors of hematopoietic origin.

Background Art

Diagnosis and prognosis of tumor-related conditions is important in determining the proper course of treatment. Extensive use has been made of markers characteristic of a number of tumor types and the availability of these tumors has permitted both detection of their presence and localization through scintigraphic imaging. The present invention concerns a marker which is associated with tumors of hematopoietic origin, including, in particular, various leukemias . In addition to its unique presence on these leukemias, the marker permits distinction among the types of leukemias putatively present and permits prediction of the course of the disease.

Disclosure of The Invention

The invention provides monoclonal antibodies and conjugates thereof which are useful in diagnosis and localization of leukemic tumor cells of specific subtypes. The antibody of the invention recognizes a characteristic marker present on some leukemic tumor •cells in subjects that have poor prognoses and permits suitable adjustment of treatment protocols and diagnosis of the spread of such leukemias.

Accordingly, in one aspect, the invention is directed to a method to subtype a subject diagnosed with a leukemia, which method comprises detecting the presence or absence of a marker on the leukemic cells of said subject wherein the marker is specifically immunoreactive with antibody 7.1. In another aspect, the invention is directed to antibodies corresponding to the monoclonal antibody 7.1 and to materials and methods for their production, as well as to conjugates of said antibodies or fragments thereof. Further, the invention is related to immunocomplexes formed by the antibodies of the invention and to methods to purify the marker of the invention using these antibodies . In other aspects, the invention is directed to a marker or tumor associated antigen of about 220-240 kd characteristic of a subtype of leukemia cells that is specifically immunoreactive with the antibodies of the invention, and to the use of the antigen to immunize subjects against tumors bearing it on the in surfaces.

Modes of Carrying Out The Invention

A monoclonal antibody has been obtained that is specifically immunoreactive with an antigen expressed by leukemic cells of about 10% of subjects with acute myelogenous leukemia (AML) and which marker is an excellent predictor of poor outcome of the disease. Certain leukemic cells from children with acute lymphocytic leukemia (ALL) also express this marker. However, this marker is clearly not present on normal cells in vivo . It is not present on normal hematopoietic cell lines such as KG1, HEL, Jiukat, Raji, HL-60 or U937. The marker is found, however, on a number of nonhematopoietic immortalized cell lines, including the widely available Hela and COS cell lines. Thus, sources of the marker are generally available as immunogens . In addition, the marker of the invention has been found on a number of cells within solid tumors including some colon, lung and breast tumors. As described below, therefore, antibodies specific for this antigen may be useful in therapeutic and diagnostic protocols with respect of these tumors as well as leukemias.

An illustrative form of the antibody of the invention, designated 7.1 herein, was deposited with the American Type Culture Collection on 3 September 1992 under the conditions of the Budapest Treaty and has ATCC Deposit No. HB11110. The ATCC, located in

Rockville, Maryland 20852, is a recognized depository under the treaty. Furthermore, all conditions on distribution of the deposit will be irrevocably removed upon issuance of a U.S. patent thereon. As used herein, an antibody "corresponding to" a referent antibody, such as antibody 7.1, refers to an antibody which is capable of binding the same epitope as the referent. This capability can readily be determined by use of a simple inhibition assay wherein, for example, in one method for conducting such an assay, the labeled referent antibody and the candidate corresponding antibody are allowed to compete for the shared antigen. When the antibodies correspond, the binding of the labeled antibody will be decreased by the presence of the corresponding antibody.

An antibody which reacts with the same antigen as antibody 7.1 (although not necessarily with the same epitope) may also be obtained by the methods described herein, and the required reactivity verified by a simple sequential precipitation assay. The shared antigen (i.e., the 220-240 kd marker) is first contacted with either the candidate antibody or antibody 7.1, and any complex formed is removed. The remaining sample is then contacted with the other antibody and residual complexation determined. Antibodies that react with the same antigen as antibody 7.1 will result in substantial abolition of the residual complexation. Further, as used herein, the term antibody includes not only intact immunoglobulin molecules of any subtype, e.g., IgG, IgM, etc., but also the immunologically reactive portions thereof, such as, for example, the Fab, Fab', or F(ab')₂ portions. The antibodies reacting with the antigen specific for antibody 7.1, including those corresponding to antibody 7.1, may be produced by immunizing a suitable subject with the appropriate antigen (e.g. the tumor marker, purified as described below, or relevant portion thereof) . In addition to the use of purified antigen, cells having the antigen at their surface may also be used. Such cells include a number of nonhematopoietic cell lines such as the widely available Hela and COS cell lines. Suitable immunization protocols may be optimized according to standard practices and conventional methods . In some instances a suitable preparation may be obtained simply by harvesting the polyclonal antisera. Monoclonal antibodies may be prepared by culturing of immortalized cell lines prepared from cells from immunized subjects that secrete monoclonal forms of the antibody and, optionally, screening for the correct specificity. Corresponding antibodies may also be prepared by recombinant methods, thus permitting the production of modified forms of the antibody, which may have advantageous properties with respect to immunogenicity.

The corresponding antibodies of the invention or those that react with the same antigen can thus be prepared in a manner analogous to that described below by using, as an antigen, an SV40 virus transformed marrow stromal cell line, and obtaining antibody secreting cells from the immunized animal . The cells are then immortalized using standard techniques, typically by hybridomal formation with a tumor cell line and the resulting hybridomas or other immortalized cells are screened for the production of suitable antibody by their reactivity with the antigen and nonreactivity with monocytes or granulocytes. The status of these antibodies produced as corresponding antibodies can be verified by the above- described competition reaction with authentic 7.1 antibody, or as antibodies which react with the same antigen by the sequential precipitation assay described above.

The antibody of the invention, the 7.1 antibody, and antigen-reactive analogs, including its corresponding immunoglobulins or analogs, are useful in detecting subtypes of acute leukemias which have poor prognoses. The mechanism of detection is through the recognition of a marker characteristic of these cells, wherein the marker is a proteinaceous substance of about 220-240 kd. By "proteinaceous substance" is meant a material which has at least a portion of its mass accounted for by amino acid sequence; of course, the amino acid sequence may be coupled with other materials such as carbohydrates or lipid, which may account for a portion of the total molecular weight. In addition to the use of the antibody to detect the subset of leukemic cells in si tu, the antibodies corresponding to 7.1, those which react with the same antigen, and antibody 7.1 per se, are useful for affinity purification of the marker substance. The marker in purified and isolated form is useful as a standard in immunoassays for the detection or quantitation of the leukemic or other cells that contain the marker substance, as well as an immunogen, as further described below.

For use in the detection of the relevant subtypes of leukemic cells, the antibodies of the invention can be used in an in vi tro or in vivo assay. For an in vi tro assay, leukemic cells are placed into standard immunoassay formats either for direct or competitive measurement. Preparation of the samples either from whole blood or plasma follows conventional methodology. Unpurified whole blood or plasma may be usable in the assay or gradient purification, for example, using a Ficoll-Hypaque gradient may employed. In any event, the suitably prepared sample is assessed in the standard formats of immunoassay. In such formats, complexes are formed between the detecting antibody and the cells and the complexes are detected using standard labeling techniques, either by directly providing label to the invention antibody or by secondary labeling through additional specific reaction with the complex. Such assays are useful both in characterizing the subtype of leukemia present and in monitoring treatment by detection of residual cells of the malignancy. The antibody may also be used in the treatment of the relevant subtypes of leukemias by coupling the antibody or fragment thereof to a drug, toxin or source of radiation which can interact with the target tumor cell. For use in this method, suitable coupling techniques are employed to prepare conjugates of the antibody with the desired toxin, such as ricin-A, diphtheria toxin, pertussis toxin, or other tumor-specific agent, such as methyltrexate, prednisone and the like. The antibody then delivers the active molecule or radioisotope to the target tumor cell with enhanced specificity. The target tumor cells may include not only leukemias having the required marker, but also solid tumors which contain cells bearing this tumor-associated antigen. In addition to toxins, the antibodies of the invention may be coupled to radioisotopes such as iodine-131, which emit radiation, then, in the vicinity of the tumor cells. Thus, additional specificity of radiation treatment may be obtained. The methods to conjugate both radioisotopes and other organic materials to antibodies or fragments thereof are by now conventional and well understood in the art. The conjugates may be made by direct coupling or may include the use of linkers such as those obtained from Pierce Chemical Company, Rockford, Illinois .

In addition to the therapeutic use of antibody conjugates, the antibodies or their fragments may be provided alone to confer passive immunity. The marker typical of the leukemic subtypes can also be prepared and further characterized by purification using the 7.1 or corresponding antibody as an affinity reagent or by obtaining the relevant encoding DNA and producing the material recombinantly, preferably in a host that is capable of post- translational modifications which would correspond to those experienced by authentic marker. Such hosts would include eucaryotic host cells in general, especially mammalian cells. The DNA encoding the marker is obtained by expressing a cDNA library prepared using standard techniques from the above-referenced nonhematopoietic cell lines found to express the antigen and screening the expression library with the antibodies of the invention. The clones found to express the marker substance are then isolated and the DNA recovered and sequenced. The amino acid sequence of the proteinaceous portion of the marker can then be deduced from the DNA sequence. Post-translational changes can be ascertained by ligating the retrieved DNA into an expression system and effecting the production of the marker in hosts which are capable of such processes. The recovered marker is readily purified, especially if produced in secreted form. Recovery is aided by the availability of the antibodies of the invention as affinity reagents.

The purified marker is useful as an immunogen to stimulate the production of an immune response, including an antibody or T-cell response with respect to tumors carrying the marker. As explained above, these tumors comprise a subset of leukemias as well as certain solid tumors containing marker-bearing cells. For use in such a regimen, the antigen is formulated for administration in conventional ways, such as those set forth in

Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA. Generally, immunization using administration by injection is preferred although alternatives such as transmucosal administration may also be employed.

The following examples are intended to illustrate but not to limit the invention.

Example 1 Preparation of Antibody 7.1 Two BALB/c mice were immunized with a SV40 transformed marrow stromal cell line obtained from Dr. J. Singer. The mice received 0.6-3 x 10⁶ cells intraperitoneally over a time period of two months. One of the mice received five injections and the other four. One week later, both received 1.5 x 10⁶ cells intravenously and three days later the spleen cells were obtained and fused with SP2/0 cells as described in Bernstein, I.D., et al . J Immunol (1982) 126:876- 881. Briefly, 5.6 x 10⁸ spleen cells and 5.6 x 10⁷ SP2/0 cells in serum-free RPMI 1640 were pelleted in six 40-ml round-bottomed tubes, resuspended in 40% polyethylene glycol, and centrifuged for 12 minutes at 330 x G. The pellets were resuspended in RPMI 1640 supplemented with 15% FCS, 2mM glycine and 1 mM pyruvate (complete medium) , washed once more, resuspended in complete medium which was, however, modified to correspond to HAT medium. The cells were cultured in 96-well tissue culture plates in a volume of 200 μl at a density of 5 x 10⁵ cells/ml. On days 2, 3, 5 and 7, one-half the medium was removed and replaced with fresh HAT medium. On day 9, supernatant fluid from each well was tested for complemented dependent cytotoxicity against the cells used for immunization. Hybrid cells from wells containing specifically cytotoxic for leukemic cells were passaged at a density (5-10 cells/well) with 6 x 10⁵ BALB/c thymocytes/well as feeder layer. Hybrids specifically reactive with the immunizing stromal cells were again mini-cloned and then finally cloned twice by limiting dilution.

The cloned cells were deposited at the ATCC as set forth above.

Example 2 Distribution of the Marker

The antibody 7.1 secreted by the cells of Example 1 was used to detect a tumor-associated antigen (TAA) by immunoperoxidase staining. The TAA was shown to have a molecular weight of 220-240 kd on SDS-PAGE.

The TAA was found on leukemia cells from patients with AML or ALL who had poor outcomes from the disease. It was also found on cells present in certain solid tumors, including sarcomas, small cell lung carcinoma, and tumors of the breast and colon. It was not detectable in the immunoperoxidase assay in normal heart, kidney, skin, spleen, small or large intestine, bladder, thymus, lung , liver or pancreas.

Claims

1. A composition consisting essentially of antibodies or immunologically reactive fragments thereof which react with the same antigen as antibody 7.1 or of antibodies corresponding to antibody 7.1.

2. An immortalized cell line which secretes the antibodies of claim 1.

3. A method to produce an antibody composition consisting of antibodies corresponding to or reacting with the same antigen as antibody 7.1 which method comprises culturing the cell line of claim 2 under conditions wherein said antibodies are secreted, and recovering the antibodies from the culture.

4. A conjugate comprising an antibody or immunologically reactive fragment thereof corresponding to or reacting with the same antigen as antibody 7.1 coupled with an additional moiety.

5. The conjugate of claim 4 wherein said additional moiety is a biologically active moiety, a solid support, or a label.

6. An immunocomplex which comprises an antibody or immunologically reactive fragment thereof corresponding to or reacting with the same antigen as antibody 7.1 coupled with a tumor cell bearing a marker specifically immunoreactive with said antibody.

7. A method to purify a marker for a subset of acute myelogenous leukemic cells which method comprises contacting a sample containing said marker with a solid support to which is coupled antibodies of claim 1 under conditions wherein said marker is adsorbed to the solid support, removing unabsorbed components, and eluting the marker from the solid support.

8. A marker prepared by the method of claim 7 which is a proteinaceous substance of approximate molecular weight 220-240 kd.

9. A method to prepare a composition which comprises antibodies immunoreactive with the same antigen as antibody 7.1 or corresponding to antibody 7.1 which comprises immunizing a vertebrate subject with the marker of claim 8.

10. A method to subtype a subject diagnosed with acute leukemia which method comprises detecting the presence or absence of a marker on the leukemic cells of said subject, which marker is specifically immunoreactive with antibody 7.1.

11. The method of claim 10 which comprises contacting the leukemic cells of said patient with an antibody or immunologically reactive fragment thereof that reacts with the same antigen as or which corresponds to the antibody 7.1 under conditions wherein said antibody or fragment is bound to the cells bearing the marker; and detecting the antibody or fragment bound to cells.

12. A method to treat an acute leukemia in a subject which method comprises administering to a subject in need of such treatment an effective amount of the conjugate of claim 5 wherein said biologically active moiety is toxic to said tumor, and waiting for sufficient time for the antibody to target said tumor.

13. A method to immunize a subject against tumors bearing an antigen specifically immunoreactive with antibody 7.1 which method comprises administering to said subject an amount of said antigen sufficient to effect an immune response in said subject to said antigen.