AU607533B2 - Methods and compositions for mimicking antigenic determinants - Google Patents

Description

:r AU-Al-21305/88 7 WORLD INTELLECTU LROP Y qRGAN ION Internonal Btfeau I

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 01337 A61K 39/00, C12P 21/00 GO1N 21/76, 33/53, 33/554 A (43) International Publication Date: 23 February 1989 (23.02.89) G01N 33/566 (21) International Application Number: PCT/US88/02342 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (22) International Filing Date: 12 July 1988 (12.07.88) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent). NL (European patent), SE (European pa- (31) Priority Application Number: 083,541 tent).

(32) Priority Date: 7 August 1987 (0i'.08,87) Published (33) Priority Country: US With international search report.

(71)Applicant: XOMA CORPORATION [US/US]; 29.0 Seventh Street, Berkeley, CA 94710 (US).

2 7 APR 1989 (72) Inventor: KNEBEL, Kathy, D, 2504 Topaz Drive, A. J.P. 2 1 Novato, CA 94945 (US).

(74) Agent: WEBER, Ellen, Lauver; Townsend and Town- Asend, One Market Plaza, 2000 Steuart Tower, San AUSTRriA Francisco, CA 94105 (US).

9 MAP 19SIO PATENT OPF1l. I (54) Title: METHODS AND COMPOSITIONS FOR MIMICKING ANTIGENIC DETERMINANTS (57) Abstract Novel methods useful in the analysis of antigen/antibody reactions and the study of antigenic determinants and immunoglobulins are provided, A novel method is provided wherein an immortal cell line capable of expressing an immunoglobulin molecule, which molecule is the anti-idicype of an antigen binding substance of interest, is substituted for the antigenic determinant nr epitope bound by the antigen binding substance, The cells of the cell line are manipulated to provide the immunoglobulin as a cell surface marker. This results in a stable, efficient source of an antigenic determinant of interest, makes accessible previously unaccessible epitopes and amplifies markers previously found on substances not amenable to FACS analysis, i -I ii WO 89/01337 PCT/US88/02342 1 METHODS AND COMPOSITIONS FOR MIMICKING ANTIGENIC DETERMINANTS BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the field of immunology and, more particularly, to the mimicry of antigenic determinants. The term antigenic determinant as used here includes any chemical structure capable of binding, complexing, or reacting with an intact or fragmented immunoglobulin molecule. Such structures include haptans, receptors, markers, epitopes, or the like.

The advent in the 1960s of the Fluorescence Activated Cell Sorter or FACS has resulted in a small revolution in the study of cell biology. (For a complete review of the principles of FACS see Flow Cytometric Applicatiok in Tumor Biology, (1979) Alan R. Liss, Inc., New York.) Used in its early years principally as a means for sorting cell populations according to the position of individual cells within the cell cycle

(G

1 S, G 2 M, or GO), the FACS has developed into a basic tool for the study of cell kinetics, identification and quantitation of cellular receptor proteins, and quantitation of cell surface antigens and antibodies for research in immunology. It is this lattnr area that the present invention is directed to.

The place of FACS in the study of cell surface antigens and the interaction of antibodies with such antigens is well established. The FACS has become an important tool for the immunologist in the study of ligand/receptor interactions. Such FACS analysis often involves a competition assay wherein a fluoresceinlabeled antibody competes with unlabeled antibody for an antigenic determinant on an antigen of interest.

waru-- 1 WO 89/01337 PCT/US88/C2342 2 Such assays can provide the researcher with valuable information about both the antigen and the competing antibodies. The antibody may be more properly referred to as immunoglobulin since reactive fragments of antibodies may be employed as well as antibodies conjugated to a therapeutic or diagnostic moiety, or the like.

There are, however, limitations with respect to the antigens that can be studied. For example, internal markers are not accessible for FACS analysis. Viral antigens are found on virions less than about 0.1 p, making them difficult candidates for FACS analysis.

Further, some cells, particularly bacterial cells, bearing antigens 6f interest are undesirable candidates for FACS analysis due to such factors as cost, potential health hazards, difficulty in maintaining the cells, and the like. There is, therefore, a need for compositions and methods useful in the mimicry of antigenic determinants heretofore not amenable to FACS analysis.

Desirably, such novel compositions and methods would not only provide a stable and cost effective supply of antigens of interest, but also amplify the size of particles bearing antigens previously difficult to analyze using a FACS.

Description of the Relevant Literature U.S. Patent No. 4,376,110 discloses generally immunoassay techniques and particularly improvements in two-site or sandwich assays employing monoclonal antibodies.

Sugarbaker et al., Flow cytometry: General principles and applications to selected studies in tumor biology. In .Flow Cytometric Applications in Tumor Biology, pp. 115-53. New York: Alan R, Liss, Inc., 1979, The authors describe the basic principles of flow cytometry as it relates to tumor biology and specific applications.

I

WO 89/01337 PCT/US88/02342 3 Loken and Stall, J. Immuno. Meth. (1982)50:R85- R112 describe the use of flow cytometry ao an analytical and preparative tool in immunology.

Rajewsky and Takemori, Ann. Pv. Immunol.

(1983)1:569-07 describe the basic science of idiotypes including genetics, expression and function.

Beaumier et al., J. Nuc. Med. (1986) 27:824-28 describe an assay employing a gamma scintillation counter developed to evaluate the immunoreactivity of radiolabeled monoclonal antibodies against three different human melanoma associated antigens, p97, a proteoglycan, and a GD 3 ganglioside.

Robins et al., J. Immuno. Meth. (1986)90:165-72 describe the measurement of a tumor reactive antibody and of an antibody conjugate by competition and quantified by flow cytometry.

SUMMARY OF THE INVENTION Novel methods useful in the analysis of antigen/antibody reactions and the study of antigenic determinants and immunoglobulins are provided. A n.vel method is provided wherein an immortal cell line capable of expressing an immunoglobulin molecule, which molecule is the anti-idiotype of an antigen binding substance of interest, is substituted for the antigenic determinant or epitope bound by the antigen binding substance. The cells of the cell line are manipulated to provide the immunoglobulin as a cell surface marker. This results in a stable, efficient source of an antigenic determinant of interest, makes accessible previously unaccessible epitopes and amplifies markers previously found on substances not amenable to FACS analysis.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a graphic representation of a FACS competition assay employing the methods of the present invention.

_I WO 89/01337 PCT/US83/02342 4 Fig. 2 is a graphic representation of an antibody/antigen interaction when target XMMME-I',1 cells are incubated with increasing concentrations of XMMME-001 monoclonal antibodies, wit'h the plateau region of the binding curve defining saturation concentrations of the antibody.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS The present invention provides novel methods for the mimicry of antigenic determinants or epitopes.

These methods are particularly suited to the study and analysis of antigen/antibody reactions employing FACS analysis and find particular utility in FACS competition assays.

According to the present invention, immortal cells are substituted for substances bearing antigenic determinants of interest. Immortal cells useful in the present invention are those capable of expressing immunoglobulin molecules. These cells are manipulated to provide the immunoglobulin molecules as cell surface markers, Immunoglobulin molecules of interest will usually be anti-idiotype immunoglobulins of monoclonal or polyclonal antibodies of interest. Such antibodies of interest include those binding epitopes found on bacterial or viral pathogens, tumor cell surface markers, internal cell markers, hormone markers, or the like.

According to the present invention, the immunoglobulin molecules are expressed by immortal cells.

These cells may be isolated from nature ol' produced by any of several techniques known to those skilled in the art, Particularly useful are hybridoma cells produced by techniques wherein antibodies of interest are employed a4 an inmunogen to produce immortal, hybrid immune cells secri4tig anti-idiotype immunoglobulin molecules. For a review of a variety of techniques for producing hybridoma cell lines see, Goding, Monoclonal Antibodies: WO 89/01337 PCT/US88/02342 Principles and Practice, (1986) Second Ed., Academic Press, which is incorporated herein by reference. These hybridoma cells may be nurine-murine, murine-human, or human-human in nature. Alternatively, recombinant DNA techniques may be employed to "engineer" hybridoma cells useful in the present invention.

According to the present invention, the immortal cells are manipulated to provide the immunoglobulin molecules of interest as cell surface markers on the cells. The preferred method of manipulation is to chill the cells to about 4°C at which point the secretion of expressed immunoglobulin is essentially halted, Less preferred, due to the potential for altering the binding sites on the immunoglobulin, is chemical manipulation of the cells.

Once obtained, th-e immortal cells are employed as a substitute for an antigenic determinant of interest in any of a number of assay techniques. According to the invention, the preferred assay technique is that of a FACS competition ass ay employing fluorescein-labeled antibodies that compete for binding sites on the immortal cell with other antibodies or antibody conjugates of interest. Such assays and others well known to those skilled in the art pi.ovide useful data on the antigen/antibdy reactions and are an important tool in the quality control of diagnostic and thera-, utic immunoconjugates.

Less preferred assays are other forms of competition immunoassays employing antigens of interest wherein the subject cells may be substituted for the antigen of interest. An example of such an assay would be one in which the immortal ceil vas labeled (either directly or indirectly, e.g. with a labeled antibody or the like) with a detectable label and used in a competition assay etIloying a support-bound antibody reactive with the cell surface immunoglobulin. In such an assay, a known quantity of labeled subject cells would

A

WO 89/01337 PCT/US88/0234 2 6 compete for antibody binding sites with an unknown amount of antigen of interest in a test sample.

Those skilled in the art will know that a variety of assay techniques may be employed within the scope of the present invention and that individual assays may require some modifications when substitut:ing an antigenic determinant mimic according to the invention.

The following examples are offered by way of illustration and not limitation.

EXPERIMENTAL

I. Preparation and Selection of Anti-idiotype Monoclonal Antibody (MoAb) to XMMME-001 MoAbs____ Strain C57 mice were immunized with a monoclonal anti-melanoma antibody designated XMMME-001, American Type Culture Collection Accession No. HB 8759, deposited March 26, 1985. The primary immunization was administered in complete Freund's adjuvant (200 ig/mouse) at multiple sites (including footpads). One week later, the secondary immunization (200 Vg/mouse) was administered in incomplete Freund's adjuvant, with three subsequent injections in PBS (400 vg/ mouse) given a week apart, each administered in phosphate buffered saline (PBS) at multiple sites including footpads. The final immunization was given several days before fusion and the mice were given 400 Vg of XMMME-001 antibody at multiple sites.

Spleen cells were removed and fused with the cell line SP2/O-Agl4, A.T.C.C. Accession No. CRL 1581.

Cells were plated out in 96 wells in hypoxanthine aminopterin thymidine (HAT) selective media and screened for activity in EIA using the F(ab') 2 region of the XMMME- 001 antibody. Several positive wells were cloned and retested against XMMME-001 antibody or XMMME-003 antibody, A.T.C.C. Accession No. HB 9159, deposited July 3, 1986, (control monoclonal anti-melanoma antibody which binds a different epitope than that bound by XMMME-001) WO 89/01337 PCT/US88/02342 7 for activity. Positive wells were also screened for their reactivity in varied environments. In this example, approximately 0.33 pg of XMMME-OO1-F(ab') 2 was dotted onto nitrocellulose at the 12:00 position in each of the 96 sites corresponding to 96 wells of a Hybri-dot manifold. XMMME-001 antibody, 0,6 yg, was dotted at (contacted with) the 7:00 position and XMMME- 003 antibody, 0.7 ig, at the 5:00 position, Supernatant (diluted 1:2 in tris-buffered saJine (TBS)) from antiidiotype XMMME-I01 hybridoma cultures, A.T.C.C. Accession No. HB 9160, deposited July 3, 1986, was then placed in the manifold and allowed to react with the idiotype.

The nitrocellulose was then incubated wi,;h either control buffer (pH 7.2) or varying acid pH buffers (pH 3, 4, or Following incubation, goat antimouse IgG 1 preabsorbed with IgM and IgG2a was added to each of the wells, followed by rabbit anti-goat antiserum labeled with peroxidase.

The enzyme substrate 4-chloronapthol and hydrogen peroxide were then added, A blue dot appearing in any of the wells at the 12:00 and 7;00 but not at the 5:00 position suggested the presence of the anti-idiotope antibody. The clone line XMMME-IO1 of the IgG 1 subclass was found to react only with the XMMME-O01 idiotype and the interaction was dissociated at pH 5. XMMME-IO1 antibody also inhibited binding of XMMME-001 to a melanoma cell line (the specific target) in Fluorescence Activated Cell Sorter (FACS) analysis, confirming antiidiotype activity.

II. EAC'S Bindingl Assay Employing Anti-idiotype Hybridoma. Cells. 1. rluoresceination of XMMME-O01 monoclonal antibodies,.. Monoclonal antibody XMMME-001 was fluoresceinated according to the following technique for use as labeled antibody in the FACS competition assay: 1Lu WOg 89/01337 PCT/US88/0234 2 8 XMMME-001 antibodies were prepared in 1X phosphate buffered saline (PBS), pH 7.0, at an initial concentration of 2.8 mg/ml. A PD-10 G25M SEPHADEX column (Pharmacia Fine Chemicals) was equilibrated with bicarb/NaCl buffer M carbonate/bicarbonate, .5 M NaCI, pH and 2.5 ml of antibody was added to the top of the column, The antibody was eluted with 3.5 ml of PBS pH 7.0 buffer and chilled to approximately 4°C in a 10 ml beaker placed in an ice water bath. Fluorescein isothiocyanate (FITC) solution was prepared by adding to a 12 mm x 75 mm borosilicate tube, 200 pg of FITC and 200 pl of dimethyl sulfoxide (DMSO). The tube was then light-shielded with aluminum foil prior to use. Upon stabilization of the antibody solution at 4 0 C, 175 pl of the FITC solution was added to the antibody solution drop by 'rop under constant stirring. (FITC/antibody ratio; 25 pg FITC/mg antibody), Following addition of the FITC solution, the mixture was covered, light shielded and stirred in an ice water bath for 3 hours. A 2.5 ml aliquot of the mixture was then added to the top of a PD-10 G25M SEPHADEX column equilibrated with PBS azide buffer (10 mM NaPO 4 M NaC1, .02% sodium azide, pH7.0). FITC-labeled antibody (XMMME-OOI-FITC) was eluted with 3.5 ml of PBS azide and collected in a 15 ml centrifuge tube lightshielded with Eoil, The fluorescein antibody ratio (F/MoAb) was calculated using a LKB spectrometer. Absorbance was read at 280 nm and 496 nm and the ratio calculated according to the following formula: A F/MoAb ratio A 2.87

A

28 0 (0,35 x A 4 9 6 The F/MoAb ratio was calculate to be 2,2, The XMMME- 001-FITC solution was light-shielded and stored at 4 0

C

prior to use.

WO 89/01337 PCT/US88/0234 2 9 2. FACS competition assay.

XMMME-I01 hybridoma cells grown in Dulbecco Minimal Essential Media (DMEM), 10% fetal calf serum (FCS), 1% Na pyruvate, 1% 200 mM glutamine, wer- harvested from suspension cultures by pouring the cells and media into a 50 ml centrifuge tube and spinning at 1200 rpm for 5 minutes at 4°C. The supernatant was removed using a sterile pipette attached to a vacuum flask. This method of harvesting does not perturb the antigenic sites (including partially secreted immunogobulins) on the cells. The cells were resuspended in to 10 ml of DMEM as previously described, Viability of the cells was determined by diluting 100 pl of the cell suspension with 900 Al of 0.4% trypan blue and counting on a hemocytometer. Viability was established as 93.5%, below the >95% viability required for the assay. Fetal bovine serum (FBS) overlay was done once and viability determined to be 96%, Following viability determination, the cells were diluted to a concentration of 106 cells/Ml.

All samples for FACS analysis were prepared in an ice bath under a laidner flow hood. All reagnts were kept refrigerated. A 1 ml aliquot of the cell suspension was placed in each 12 x 75 mm tube (Falcon #2054 or equivalent) used for the aasay, The tubes were centrifuged for 5 minutes at 4QC and 1200 rpm and the supernatant decanted, The tubes were then placed inverted on absorbant material in a test tube rack to absorb as much supernatant as possible. Any remaining supernatant was removed by blotting with gauze squares.

The cells were washed by adding 1 ml of PBS-BSA/tUbe and gently flicking or swirling to resuspend the cells while avoiding air bubble formation. Following washing, the tubes were centrifuged, the super'latant cdecanted and the excess blotted as described above, ERactly 1 ml of SPB-BSA was added to each sample and the cells resuspended.

c_ -u I- .'-ICUE_; WO 89/01337 PCT/US88/02342 Competing antibodies and immunoconjugates consisted of XMMME-001 #51102 (standard), XMMME-001-RTA #60407, XMMME-001-RTA #60505, and XMMME-001 450513 with azide. The XMMME-001-RTA immunoconjugates were prepared as disclosed in U.S. Patent No. 4,53n0,071, the disclosures of which are hereby incorporated by reference, Each competitor was initially diluted to a concentration of 4 pg 20 p1 (0.2 pg/pl) and serial dilutions prepared of 2 pq 20 pl1, 1 pg 20 p1, and pg 20 p1 of each. Each was prepared in sufficient quantity to prepare duplicate samples. The fluorochrome labeled antibody, XMMME-O01-FITC, prepared as described above, was diluted to saturating concentration in 20 l of Dulbecco's PBS with 1% BSA (Sigma) (1 Pg 20 pl).

Aliquots of competing and labeled antibodies ,and/or conjugates were added to each sample tube except the autoafluorescence controls, All samples were incubated or ice in the dark for I hour.

Following incubation, the samples were filtered by pipetting each sanple from its tube and realiquoting through a 37 p nitex filter by pipetting down the side of the tube through the mesh immediately prior to FACS analysis to avoid clumping and insure a single cell suspension. Each sample was then analyzed using a dual laser FACS IV (Becton-Dickenson, Mt. View, CA) following calibration usitng green MLUORESBRITS microbeads (Polysciences, Worrington, PA) at PMT voltages of 650 and 750, Samples were analyzed at 750v and gated on forward angle scatter and gating on the middle distribution to eliminate dead cells from the analysis, The results of this analysis are presented in Fig. 1. Fig.

I was obtained by calculating the means of the fluorescence distribution taking the mean of the duplicate means, inverting, subtracting autofluorescence, and plotting the ratio of competing antibody or conjugate to labeled antibody concentrations,. The ratio Co WO 89/01337 PCT/US88/02342 11 of equilibrium constants of L (X<r and C (Kc) was used to derive the equation: 1/FC Kc/K L x (I/F O x x x I/F

O

where: FC and F O are the values of F for each and in the absence of C, respectively, which is linear, and the slope s Kc/Kr x (1/F O x The relative binding of the antibodies or conjugates (Cl) to the standard antibody (STD)(C2) was determined from the ratio of the slopes of their binding constants, which equals the ratio of the slopes of these plots (S1/ 2 KcI/Kc2).

The results presented in Fig, 1 indicate tr.t the XMMME-IOI cell line proved a suitable substitute for the normal target Minor cells employed previously, Those skilled in the art will appreciate that the present invention provides novel methods for the mimicry of antigenic determinants of interest for study and analysis. The subject methods are an improvement in the prio- art in that antigenic determinants previously unamenable to FACS analysis may now be studied, The present invention also provides a stable and effic3ient source of antigenic determinants of interest for immunological analysis and study, Although the foregoing invention is i e described in some detail through illustrations and examples for purposes of clarity anld understanding, it will be apparent to one skilled in the a.t that certain modifications may be practiced within the scope of the appended claims.

Claims (19)

1. 3. The method according to Claim 1, wherein said immortal cell is a hybridoma cell.
2. 4. The method according to Claim 3, wherein said hybridoma cell is a murine/murina hybridoma cell. The method according to Claim 4, wherein said murine/murine hybridoma cell is an XtMMME-101 hybri- doma cell having A.T.C.C. Accession No, HB 9160.
3. 6. The method according to Claim 1, wherein said antigen binding substance is an immunoglobulin.
4. 7. The method according to Claim 6t wherain said immunoglobulin is produced by hybridoma cell line XMMME-001 having A.T.CC. Accession No. HB 8759.
5. 8. The method according to Claim 1, further comprising maintaining said immortal cell at a tempera- ture of about 4°C to halt secretion of said irnmunoglob- ulin. ^U WO 89/0132'7 PCT/US88/02342 13
6. 9. The method according to Claim 1, wherein said immunoglobulin is label-Bd with a detectable label. The method according to Claim 9, wherein said label is a fluorescent label.
7. 11. The method according to Claim 10, wherein said fluorescent la.el is fluorescein isothiocyanate.
8. 12. In a method for deternining the presence or amount of an antigen in a sample comprising employ- ing a known amount of labeled antigen and detecting the amount of said labeled antigen bound to an immunoglobulin, the improvement comprising substituting for said labeled antigen a labeled, immortal cell capable of expressing an immunoglobulin molecule which molecule mimics a bind- ing site of said antigen, said cell providing said immuno- globulin as a cell surface marker wherein said marker compriss said binding site.
9. 13. The method according to Claim 12, wherein said immortal cell is a hybridoma cell,
10. 14. The method according to Claim 13, wherein said hybridoma cell is a murine/murine hybridcma cell. The method according to Claim 12, further comprising maintaining said immortal cell at a tempera- ture of about 4 C to halt secretion of said immunoglob- Ulin.
11. 16. In a method for determining the binding activity of an immunoconjugate for a target antigen comprising neasuring the binding of sa'd immunoonjugate to said ancigen in competition with a labeled, uncon- jugated first immunoglobulin molecule, the improvement comprising substituting for said ar.tigen an immortal 'II I ri. WO 89/01337 PCT/US88/02342 14 cell capable of expressing a second immunoglobulin mol- ecule which molecule is the anti-idiotype of said immuno- conjugate's antibody moiety, said cell providing said second immunoglobulin as a cell surface marker wherein said marker comprises the binding site of said second immunoglobulin.
12. 17. The method according to Claim 16, wherein said method employs a Fluorescence Activated Cell Sorter.
13. 18. The method according to Claim 16, wherein said immortal cell is a hybridoma cell.
14. 19. The method according to Claim 18, wherein said hybridoma cell is a murine/murine hybridoma cell. The method according to Claim 19, wherein said murine/murine 'ybridoma cell is an XMMME-101 hybri- doma cell having A.T.C.C. Accession No. HB 9160.
15. 21. The method according to Claim 16, wherein said immunoconjugate is an immunotoxin.
16. 22. The method according to Claim 21, wherein said immunotoxin is XMMME-001-RTA.
17. 23. The method according to Claim 16, further comprising maintaining said immortal cell at a tempera- ture of about 4 0 C to halt secretion of said second immu- noglobulin.
18. 24. The method according to Claim 16, wherein said labeled first immunoglobulin is labeled with a fluorescent label. The method according to Claim 24, wherein said fluorescent label is fluorescein isothiocyanate, r. II. WO 89/0 1337 PCT/L TS88/02 342
19. 26. The method according to Claim 25, wherein said first immunoglobulin is produced by hybridoma. cell line XMt4'E-O0l having A.T.C.C. H-B 8759.