AU2130588A

AU2130588A - Methods and compositions for mimicking antigenic determinants

Info

Publication number: AU2130588A
Application number: AU21305/88A
Authority: AU
Inventors: Kathy D. Knebel
Original assignee: Xoma Corp
Current assignee: Xoma Corp
Priority date: 1987-08-07
Filing date: 1988-07-12
Publication date: 1989-03-09
Anticipated expiration: 2008-07-12
Also published as: AU607533B2

Description

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 struc¬ ture capable of binding, complexing, or reacting with an intact or fragmented immunoglobulin molecule. Such structures include haptans, receptors, markers, epi- topes, 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 e.g., Flow Cytometric Applications in Tumor Biology. (1979) Alan R. iss, 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-, S, G_, M, or G₀), the FACS has developed into a basic tool for the study of cell kinetics, identifica¬ tion and quantitation of cellular receptor proteins, and quantitation of cell surface antigens and antibod¬ ies for research in immunology. It is this latter 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 fluorescein- labeled antibody competes with unlabeled antibody for an antigenic determinant on an antigen of interest. 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 anti- bodies 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, inter¬ nal markers are not accessible for FACS analysis. Viral antigens are found on virions less than about 0.1 y, making them difficult candidates for FACS analysis. Further, some cells, particularly bacterial cells, bear¬ ing antigens of 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 composi¬ tions 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 par¬ ticles 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 anti¬ bodies.

Sugarbaker et _al. , Flow cytometry: General principles and applications to selected studies in tumor biology. In Flow Cyto etric Applications in Tumor Biology, pp. 125-53. New York: Alan R. Liss, Inc., 1979. The authors describe the basic principles of flow cytometry as it relates to tumor biology and spe¬ cific applications. Loken and Stall, J. I muno. Meth. (1982)50:R85- R112 describe the use of flow cytometry as an analytical and preparative tool in immunology.

Rajewsky and Takemori, Ann. Rev. Immunol. (1983)_1:569-07 describe the basic science of idiotypes including genetics, expression and function.

Beaumier et al. , J. Nucl. 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_ 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 quanti¬ fied by flow cytometry.

SUMMARY OF THE INVENTION Novel methods useful in the analysis of anti- gen/antibody reactions and the study of antigenic determ¬ inants 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-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 determi¬ nant of interest, makes accessible previously unaccessi¬ ble 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. Fig. 2 is a graphic representation of an anti¬ body/antigen interaction when target XMMME-IOl cells are incubated with increasing concentrations of XMMME-001 monoclonal antibodies, with the plateau region of the 5 binding curve defining saturation concentrations of the antibody.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides novel methods

10 for the mimicry of antigenic determinants or epitopes. These methods are particularly suited to the study and analysis of antigen/ ntibody reactions employing FACS analysis and find particular utility in FACS competi¬ tion assays.

15 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 immuno¬ globulin molecules. These cells are manipulated to 0 provide the immunoglobulin molecules as cell surface markers.

Immunoglobulin molecules of interest will usually be anti-idiotype im unoglobulins of monoclonal or polyclonal antibodies of interest. Such antibodies

25 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 immuno¬ globulin molecules are expressed by immortal cells.

³ These cells may be isolated from nature or 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 as an immunogen to produce immortal, hybrid immune cells

^{~J ~J} secreting anti-idiotype immunoglobulin molecules. For a review of a variety of techniques for producing hybri¬ doma cell lines see, Goding, J. ., Monoclonal Antibodies: Principles and Practice, (1986) Second Ed., Academic Press, which is incorporated herein by reference. These hybridoma cells may be murine-murine, murine-human, or human-human in nature. Alternatively, recombinant DMA 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 siirface 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, the immortal cells are employed as a substitute for an antigenic determinant of interest iri any of a number of assay techniques. According to the invention, the preferred assay technique is that of _a FACS competition assay 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 provide useful data on the anti- gen/antibody reactions and are an important tool in the quality control of diagnostic and therapeutic immunocon- jugates.

Less preferred assays are other forms of com¬ petition 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 cell was labeled (either directly or indirectly, e.g. with a labeled antibody or the like) with a detectable label and used in a compe- tition assay employing a support-bound antibody reac¬ tive with the cell surface immunoglobulin. In such an assay, a known quantity of labeled subject cells would 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 substituting an anti¬ genic 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 Mono- clonal Antibody (MoAb) to XMMME-001 MoAbs

Strain C57 mice were immunized with a mono- clonal anti-melanoma antibody designated XMMME-001,

American Type Culture Collection (A.T.C.C.) Accession No. HB 8759, deposited March 26, 1985. The primary immunization was administered in complete Freund's ad¬ juvant (200 μg/mouse) at multiple sites (including foot- pads). One week later, the secondary immunization (200 μg/mouse) was administered in incomplete Freund's adju¬ vant, with three subsequent injections in PBS (400 μg/ mouse) given a week apart, each administered in phos¬ phate 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 SP2/0-Agl4, A.T.C.C. Accession No. CRL 1581. Cells were plated out in 96 wells in hypoxanthine aminop- terin thymidine (HAT) selective media and screened for activity in EIA using the F(ab')_ region of the XMMME- 001 antibody. Several positive wells were cloned and retested against XMMME-001 antibody or XMMME-003 anti¬ body, 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) for activity. Positive wells were also screened for their reactivity in varied environments. In this exam¬ ple, approximately 0.33 yg of XMMME-001-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 yg, at the 5:00 position. Supernatant (diluted 1:2 in tris-buffered saline (TBS)) from anti- idiotype XMMME-IOl 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 with either control buffer (pH 7.2) or varying acid pH buffers (pH 3, 4, 5, or 6). Following incubation, goat antimouse IgG₁ pre- absorbed with IgM and IgG_ was added to each of the wells, followed by rabbit anti-goat antiseru labeled with peroxidase.

The enzyme substrate 4-chloronapthol and hydro- gen 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-IOl of the IgG., subclass was found to react only with the XMMME-001 idiotype and the interaction was dissociated at pH 5. XMMME-IOl antibody also inhibited binding of XMMME-001 to a mela¬ noma cell line (the specific target) in Fluorescence Activated Cell Sorter (FACS) analysis, confirming anti- idiotype activity.

II. FACS Binding Assay Employing Anti-idiotype

Hybridoma Cells

1. Fluoresceination of XMMME-001 monoclonal antibodies. Monoclonal antibody XMMME-001 was fluorescein- ated according to the following technique for use as labeled antibody in the FACS competition assay: XMMME-001 antibodies were prepared in IX phos¬ phate buffered saline (PBS), pH 7.0, at an initial concen¬ tration of 2.8 mg/ml. A PD-10 G25M SEPHADEX column (Pharmacia Fine Chemicals) was equilibrated with bicarb/NaCl buffer (.1 M carbonate/bicarbonate, .5 M NaCl, pH 9.0) 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 boro- silicate tube, 200 yg of FITC and 200 yl of dimethyl sulfoxide (DMSO) . The tube was then light-shielded with aluminum foil prior to use. Upon stabilization of the antibody solution at 4°C, 175 yl of the FITC solution was added to the antibody solution drop by drop under constant stirring. (FITC/antibody ratio: 25 yg 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 biffer (10 mM NaPO., .15 M NaCl, .02% sodium azide, pH7.0). FITC-labeled antibody (XMMME-001-FITC) was eluted with 3.5 ml of PBS azide and collected in a 15 ml centrifuge tube light- shielded with foil. The fluorescein antibody ratio

(F/MoAb) was calculated using a LKB spectrometer. Ab- sorbance was read at 280 nm and 496 nm and the ratio calculated according to the following formula:

A ₆

F/MoAb ratio = _ ,_Q 35 „ , x 2.87

^A280 ^(U-^ ^{x A} 96^}

The F/MoAb ratio was calculated to be 2.2. The XMMME-

001-FITC solution was light-shielded and stored at 4°C prior to use. ^ 2. FACS competition assay.

XMMME-IOl hybridoma cells grown in Dulbecco Minimal Essential Media (DMEM) , 10% fetal calf serum (FCS), 1% Na pyruvate, 1% 200 M glutamine, were har- 5 vested 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

10 antigenic sites (including partially secreted immuno- gobulins) on the cells. The cells were resuspended in 5 to 10 ml of DMEM as previously described. Viability of the cells was determined by diluting 100 yl of the cell suspension with 900 yl of 0.4% trypan blue and

15 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 viabili¬ ty determination, the cells were diluted to a concen-

20 tration of 10 cells/ml.

All samples for FACS analysis were prepared in an ice bath under a laminer flow hood. All reagents were kept refrigerated. A 1 ml aliquot of the cell suspension was placed in each 12 x 75 mm tube (Falcon

25 #2054 or equivalent) used for the assay. The tubes were centrifuged for 5 minutes at 4°C 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 wash¬ ing, the tubes were centrifuged, the supernatant de- 5 canted and the excess blotted as described above. Ex¬ actly 1 ml of PBS-BSA was added to each sample and the cells resuspended. Competing antibodies and immunoconjugates consisted of XMMME-001 #51102 (standard), XMMME-001-RTA #60407, XMMME-001-RTA #60505, and XMMME-001 #50513 with azide. The XMMME-001-RTA immunoconjugates were pre- pared as disclosed in U.S. Patent No. 4,590,071, the disclosures of which are hereby incorporated by refer¬ ence. Each competitor was initially diluted to a con¬ centration of 4 yg / 20 yl (0.2 yg/μl) and serial dilu¬ tions prepared of 2 yg / 20 yl, 1 yg / 20 yl, and 0.5 yg / 20 yl of each. Each was prepared in sufficient quantity to prepare duplicate samples. The fluorochrome labeled antibody, XMMME-001-FITC, prepared as described above, was diluted to saturating concentration in 20 yl of Dulbecco's PBS with 1% BSA (Sigma) (1 yg / 20 yl) . Aliquots of competing and labeled antibodies and/or conjugates were added to each sample tube except the autofluorescence controls. All samples were incubated ori ice in the dark for 1 hour.

Following incubation, the samples were fil¬ tered by pipetting each sample from its tube and re- aliquoting through a 37 y 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 using green FLUORESBRITE mic- robeads (Polysciences, orrington, PA) at PMT voltages of 650 and 750. Samples were analyzed at 750v and gated on forward angle scatter and gating on the middle distri- bution to eliminate dead cells from the analysis. The results of this analysis are presented in Fig. 1. Fig. 1 was obtained by calculating the means of the fluores¬ cence distribution (F) , taking the mean of the duplicate means, inverting, subtracting autofluorescence, and plotting the ratio of competing (C) antibody or conju¬ gate to labeled antibody (L) concentrations. The-ratio of equilibrium constants of L (K_r ) and C (K_,) was used to derive the equation:

1/F_C = K_C/K_L x (1/F_Q x [L]) x [C] x 1/F_Q, where: F and F_ are the values of F for each [C] and in the absence of C, respectively, which is linear, and the slope s = K_,/ _L x (1/F_Q x [L]).

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

^(sl ^s2 ^{= K}C1^/KC2⁾'

The results presented in Fig. 1 indicate that the XMMME-IOl 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 prior art in that antigenic determinants previ¬ ously unamenable to FACS analysis may now be studied. The present invention also provides a stable and effi¬ cient source of antigenic determinants of interest for immunological analysis and study. Although the foregoing invention has been described in some detail through illustrations and ex¬ amples for purposes of clarity and understanding, it will be apparent to one skilled in the art that certain modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. In a method for determining the presence or concentration of an antigen binding substance employ- ing the antigen bound to said substance, the improvement comprising substituting for the antigen an immortal cell capable of expressing an immunoglobulin molecule which molecule is the anti-idiotype of said substance, said cell providing said immunoglobulin as a cell sur- face marker wherein said marker comprises the binding site of said immunoglobulin.

2. The method according to Claim 1, wherein said method employs a Fluorescence Activated Cell Sorter.

3. The method according to Claim 1, wherein said immortal cell is a hybridoma cell.

4. The method according to Claim 3, wherein said hybridoma cell is a murine/murine hybridoma cell.

5. The method according to Claim 4, wherein said murine/murine hybridoma cell is an XMMME-IOl hybri¬ doma cell having A.T.C.C. Accession No. HB 9160.

6. The method according to Claim 1, wherein said antigen binding substance is an immunoglobulin.

7. The method according to Claim 6, wherein said immunoglobulin is produced by hybridoma cell line XMMME-001 having A.T.C.C. Accession No. HB 8759.

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 imn moglob- ulin.

9. The method according to Claim 1, wherein said immunoglobulin is labeled with a detectable label.

10. The method according to Claim 9, wherein said label is a fluorescent label.

11. The method according to Claim 10, wherein said fluorescent label is fluorescein isothiocyanate.

12. In a method for determining 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 comprises said binding site.

13. The method according to Claim 12, wherein said immortal cell is a hybridoma cell.

14. The method according to Claim 13, wherein said hybridoma cell is a murine/murine hybridoma cell.

15. 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.

.

16. In a method for determining the binding activity of an immunoconjugate for a target antigen comprising measuring the binding of said immunoconjugate to said antigen in competition with a labeled, uncon- jugated first immunoglobulin molecule, the improvement comprising substituting for said antigen an immortal 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.

17. The method according to Claim 16, wherein said method employs a Fluorescence Activated Cell Sorter.

18. The method according to Claim 16, wherein said immortal cell is a hybridoma cell.

19. The method according to Claim 18, wherein said hybridoma cell is a murine/murine hybridoma cell.

20. The method according to Claim 19, wherein said murine/murine hybridoma cell is an XMMME-IOl hybri¬ doma cell having A.T.C.C. Accession No. HB 9160.

21. The method according to Claim 16, wherein said immunoconjugate is an immunotoxin.

22. The method according to Claim 21, wherein said immunotoxin is XMMME-001-RTA.

23. The method according to Claim 16, further comprising maintaining said immortal cell at a tempera¬ ture of about 4°C to halt secretion of said second immu- noglobulin.

24. The method according to Claim 16, wherein said labeled first immunoglobulin is labeled with a fluorescent label.

25. The method according to Claim 24, wherein said fluorescent label is fluorescein isothiocyanate.

26. The method according to Claim 25, wherein said first immunoglobulin is produced by hybridoma cell line XMMME-001 having A.T.C.C. HB 8759.