Immunodetection of human tumour-associated cell-bound antigens by human monoclonal antibodies

Journal of Immunological Methads, 121 (1989)47- 52

47

Elsevier JIM 05217

Immunodetection of human tumour-associated cell-bound

antigens by human monoclonal antibodies J.R.D. Tuson 1, D.A. Jacob 2, E.W. Pascoe 2 and S.J.Y. S a x b y 2 t Department of Surgery, University of Cambridge, Cambridge, U.K, and z Quantum Biosystema Lid, Waterbeach, Cambridge, U.K.

(Received16 December1988,revisedreceived30 January 1989, accepted16 February1989)

A novel cellular enzyme-linked immunoassay has been developed to detect specific binding of human monoclonal antibodies to target turnout cells obtained by enzymatic disaggregation of surgically resected human colorectal carcinomas. Cell preparations derived from human tissues contain endogenous immunogiobulin. The method described is designed to detect specific binding of a human monoclonal antibody while minimising extraneous background signals caused by the presence of endogenous immunoglobullns in the preparation. This is achieved by first generating immune complexes, which are then incubated with the target cells. The assay is well suited for rapid screening of large numbers of tissue culture supematants and could be adapted for cells of other tumours. Small quantities of target cells and supernatant are used and the assay can be completed within 5 h. Key words: Monoclonalantibody, human; Neoplasmantigen; Surface antigen; Neoplasm; Immunoenzymetechnique; Colosectal

carcinoma

Introduction An important aspect of Mab production for tumour imaging and immunotherapy is development of assays to detect antibodies directed against TAAs and other cell surface antigens. The work of this laboratory is production, identification and isolation of human Mabs directed against human tumours - in particular colorectal carcinoma.

Correspondence to: J.ILD. Tuson, Departmentof Surgery,Addenbrooke'sHospital,Hills Road, CambridgeCB2 2QQ, U.K. Abbreviations: HBSS, Hanks' buffered saline solution; 18, immunoglobulin; Mab, monoclonal antibody; MNGG, solu. tion in PBS of 1% Marveland 10 mM N-81ycylglycine;NHS, hepurinised pooled whole normal human serum; OD, optical density; OPD, o-phenylenediamine;PBS, phosphate-buffered saline; SHRP, streptavidin-hurseradishperoxidaseconjugate; TAA, tumour-associatedantigen; TC, tissueculture.

We have devised a novel cellular enzyme-linked immunoassay to screen large numbers of TC supernatants for human antibodies which bind to cell targets obtained by enzymatic disaggregation of surgically resected human tumours. This screening strategy was chosen as it permits antibodies produced by lymphocytes from a patient beating a neoplasm to be screened against the patient's own tumour cell-hound antigens, thereby maximising the possibility of detecting antibodies directed against TAAs. A major problem encountered when attempting to detect specific binding of human antibodies to human tissues or disaggregated cells is the high non-specific background signal caused by the presence of endogenous human Igs (Imam et all., 1985). Many tumours exhibit monocyfic infiltration (loachim, 1979; Jass, 1986) and Igs may be cytophilically attached via Fc receptors to these cells. There may 'also be present antibodies bound

0022-1759/89/$03.50 © 1989 ElsevierSciencePublishersB.V. (BiomedicalDivision)

48

to the patient's own TAAs. A conventional sequential enzyme-linked immmtoassay is not possible under these circumstances since the second antibody (usually an anti-human immunoglobulin - 'antiglobulin') binds to endogenous lgs in the target cell preparation, masking any signal attributable to specific binding of the primary human Mab. Various methods are described to overcome this problem. Binding to endogenous antibody has been reduced by incubation with monomeric anti-human Fab fragments (Nielsen et al., 1987). The primary human antibody has been biotinylated and detected using an avidin-enzyme conjugate (Imam et al., 1986; Haspel et al., 1985). Alternatively, the antiglobulin may be titrated to reduce background signals (Imam et al., 1985; Kjeldsen et al., 1988). However, there are problems associated with each of these methods. Screening against established human tumour cell lines is a commonly used alternative (Houghton et al., 1983; Glassy, 1985; Kjeldsen et al., 1988) but has the disadvantage that the expression of antigen may be modified or lost (Finan et al., 1982; Durrant et al., 1986) with consequent failure to recognise valuable transformants or hybridomas. We have developed an assay which is relatively simple, quick and inexpensive and which overcomes the problem of endogenous Igs by conducting the initial incubation steps in microtitre plate wells which do not contain target antigen. Aliquots of 1 C supernatant are first incubated with biotinylated antiglobulin, to form complexes with any human lg present in the supernatant. The resultant immune complex is then incubated with NHS to block any remaining unsaturated binding sites on the antiglobulln which might otherwise bind to endogenou~ Igs on the target cells. This secondary complex is then screened for TAA specificity by a further incubation with target tumour cells.

land: streptavidin-horseradish peroxidase conjugate was supplied by Zymed Laboratories, San Francisco, CA, U.S.A.; dried skimmed milk (Marvel) from Cadburys, Bournville, England; heparinised normal human serum was pooled from healthy donors. Wash buffer was PBS (pH 7.4) with 0.05% Tween 20. All reagent dilutions were carried out in the same buffer which additionally contained 0.2% bovine serum albumin.

Target cells Tumours obtained fresh from patients undergoing resection of colorectal carcinomas were disaggregated by repeated digestion with 0.01% Type IV collagenase and 0.02~ DNASe in calcium and magnesium free HBSS at 37 ° C. The suspensions were filtered through cotton gauze, w~hed ×5 in HBSS and resuspended in PBS at 2 × 10 e cells/ml.

Monoclonal antibody We have developed a human IgM Mab (HTC1/8) which recognises a TAA present on human colorectal carcinoma cells.

Assay plates (1) Target cell plates. 96 well flat bottomed Nunc Immunoplates (Gibeo, Uxbridge, England) were prepared as follows: into each well were dispensed 50 pl of PBS containing 1 × 105 disaggregated tumour cells. The plates were dried at 37 o C and stored at - 20 ° C. Before use, the cells were rehydrated by incubation with PBS at 37°C for 10 rain. Protein binding sites were then blocked by incubation with MNGG. After washing, the plates were ready for use. (2) Pre-incubation plates. In addition, a supply of 96 well plates was prepared simply by blocking protein binding sites with MNGG. The plates were washed three times and dried at 37°C. The wells of these plates formed convenient reaction vessels for the initial incubations of the assay. The contents of the wells were then transferred to target cell plates for the later stages of the assay.

Materials and methods

Assay procedure Reagents and buffers With the following exceptions, all reagents were obtained from Sigma Chemical Co., Poole, Eng-

The assay procedure comprised two initial incubation steps (A and B), and an antigen binding stage (C). Steps A and B were performed on a

pre-incubation plate and step C on a target cell plate: Step (A). 100/tl of TC supernatant were dispensed into duplicate webs of the pre-incubation plate. 50/tl of antiglobulin (affinity purified biotinylated goat anti-human IgG + A + M - heavy chain specific) containing 500 n g / n d specific antibody were added to each well and incubated for 1 h at 37°C. Step (B). After the above initial incubation, to each well was added 50 pl of NHS at a 1/100 dilution followed by incubation for a further hour at 37°C. Step (C). The contents (200 pl) of each well of the pre-incubation plate were then transferred to the corresponding web of the prepared target cell plate, using a 12 channel Titertek Multichannel pipetter (Flow Laboratories). The loaded target cell plates were incubated for 2 h at 37°C. The contents were then discarded and the target cell plate was carefully washed. 50 pl/weB of SHRP at a 1/5000 dilution were dispensed, the plate incubated at 37°C for 15 min and then washed. 5 0 / d / w e l l of OPD substrate at 2.5 m g / m l in phosphate citrate buffer (pH 5.0) containing 0.66 / t l / m l of 30~ hydrogen peroxide were dispensed. The reaction was allowed to proceed in the dark at room temperature for 20 min, and was stopped with 25 pl of 1 M sulphuric acid/well. The OD at 492 nm was measured using a Titertek Multiskan MCC/340 reader (Flow Laboratories).

o-~. 3.°00

~.o00

,~oo

o.ooo

,

1.o

,

1o.o

,

loo.o

1ooo.o

co,ce.zratton of .~cl/s ( r~/=t ) F i g . 1. T h e e f f e c t o f a n t i g l o b u l i n

concentration

on the rela-

t i o n s h i p betwecn concentration of HTC1/8 and OD. Antiglobulin was used at six ¢onc~mtrations:(e) 5000 ns/ml; (v) ng/ml; (v) 1250 ng/ml; ([3) 625 ng/ml; (O) 312.5 ng/ml; (11)156.3og/ml.

To determine the concentration of NHS necessary to bind and therefore block any remai,~ing unsaturated binding sites on the biotinylated goat 00 0. 7~0

Results

To determine the concentration of biotinylated goat antiglobulin producing the optimum reactivity index and sensitivity, HTC1/8 was titrated in the presence of several dilutions of antiglobulin (Fig. 1). The reactivity index for each dilution of antiglobulin was calculated (the ratio of the maximum OD obtained to the background OD in webs where an irrelevant human IBM was substituted for HTC1/8). Sensitivity was expressed as the concentration of specific antibody required to give an OD of twice the background signal (Glassy, 1985). The optimal concentration of antiglobulin was 500 ng/ml.

........

,0o

,,,0

Otlutton o~ normal huean~er~

,,, ,,

Fig. 2. The effectof dilutionof NHS in blockingthe bindingo| antiglobulin to endogenouslg, at six concentrationsof antiglobulin: (v) 5000 ng/ml; (v) 2500 og/ml; ([3) 1250 nf,/ml; (zx)625 og/ml; (A) 312.5ng/ml; (11)156.3ns/ml.

antiglobulin after the first incubation, NHS was titrated against dilutions of antiglobulin in chequerboard fashion (Fig. 2). In these experiments TC medium alone was used as a control for a situation in which supernatant would contain little or no antibody, in which case the unsaturated antiglobulin would give a false positive result by binding to endogenous Igs on the target cell plate. This artefact was overcome by incubation with excess NHS to saturate any remaining binding sites. HTC1/8 was titrated against several dilutions of SHRP (data not shown) to establish the working dilution of the SHRP conjugate. From the results of these experiments working conditions for the assay were derived; the biotinylated antiglobulin was used at 500 ng/ml, NHS at a 1/100 dilution and SHRP at a 1/5000 dilution. These dilutions were used in all subsequent assays. The performance of the assay, comparing binding of HTC1/8 to target cell plates prepared from four different colorectal carcinomas was assessed (Fig. 3). T o illustrate the extent of the problem of endogenous Ig on the target cell plates, an experiment was conducted to compare the variation of

0.0. 1.s00

o.ooo

. . . .

~. . . .

~. . . . .

~0

Concentration of HTCl/B ( ng/ml )

Fig. 4. An example of the variation of OD with concentration of HTC1/8 when background signals due to endogenous lg were (U) and were not (v) blocked with NHS. The vertical bars

represent two standard deviations about the mean of the observations(n ffi6).

OD with concentration of HTC1/8, both in the presence of NHS, where binding of antig!obulin to endogenous Ig was blocked, and in the absence of NHS, when such binding would occur unhindered if unsaturated binding sites remained on the anti~ J o b u a n (Fig. 4). When the TC supematants of cell lines were screened for antibody secretion under the proposed assay conditions, statistical analysis of the variance ratio significantly differentiated between positive and negative supernatants ( P < 0.01).

O.D

Discussion

_ti: 1.o

1o.o

1oo.o

looo.o

Concentration of HTCI/8 ( ng/ml )

Fig. 3. Appficatiun of the assay using four different turnouts as a source of target cells: (v) T87/15; (0) T87/14; (0) T87/16; (a) T87/12.

The technique described employs the generation of immune complexes which are screened against target cells in order to avoid the complications associated with endogenous Igs. The binding of the complex to target cells occurs in the tertiary incubation step and is believed to be antigenspecific, being mediated by the Mabs in the original sample.

N H S is required to overcome the problem of false positive rssults should the supematmit contain no human lg. However, if the NHS is added in great excess then competition between Igs present in the sample and lgs in the NHS, for antiglobulin binding sites, reduces the specific signal. Therefore the amount of NHS added is just sufficient to saturate the antiglobulin. Possible sources of interference in this assay include (i) the specific binding of Igs in the N H S to target cells; (ii) non-specific binding mediated by Fc receptors in the target cell pooulation; (iii) endogenous peroxidase activity; and (iv) nonspecific binding of the SHRP conjugate to target cells. Appropriate controls were incorporated which indicated that, of these four possibilities, only the last materially affected the results to an extent which varied from tumour to tumour. The biotin-streptavidin system was chosen since it increased reactivity and sensitivity compared to an antiglobulin-enzyme conjugate. These parameters remained constant through a wide range of dilutions of SHRP and economical considerations therefore influenced the selection of the working dilution. The reactivity index of this assay is relatively low (3.10-5.d5) when compared to simple sandwich assays using in vitro cultured cell lines or non-cellular antigens. However, the assay is sensifive, with a linear dynamic range of 10-100 n g / m l of specific IgM antibody. This degree of sensitivity is sufficient for the detection of potentially useful antibodies in TC supernatants. Analysis of the behaviour of the assay using four different tumours sugvests 'that assay sensitivity and reactivity vary, presumably" reflecting differences in cell membrane epitope density. The quantitative data in this work were obtained using 100/tl aliquots of TC superuatant but in practice smaller volumes (50-75 ~1) may be used if the quantity of supernatant is limited. The method may be adapted for immunohistological screening and this is the subject of a further pubfie~ation. In conclusion, this assay has potential applications for the screening of human monoclonal antibodies with specificities against cellular antigens. We have effectively exploited it to detect human Mabs, present in TC superuatants of transformed

B lymphocytes, which show reactivity against a¢tologous and allogeneic tumours.

Acknowledgements We would like to acknowledge the assistance of the following: Addenbrooke's Hospital Trust F u n d for financial support; Dr. Hugh Davies of the Department of Surgery, University of Cambridge, for reviewing the text; Mr. W.G. Everett and Mr. W.A.B. Smellie, Consultant Surgeons at Addenbrooke's Hospital, Cambridge, for providing surgical specimens; Bruce Wright, Joanna Hutchings, Sue Martin and A n n i Tatham of Quantum Biosystems Ltd.

References Durrant, L.G.. Robins, fLA., Pimm, M.V., Pezkin~, A.C., Armitage, N.C., Hardcastle,J.D. z~ndBaldwin,ILW. (19g~) Antigenicityof newly established colorectal carcinomacell lines. Br. J. Cancer 53, 37. 7inan, PJ., Grant, ILM., De Mattos, C., Takei, F., iierry, P.L, Lennox, E.S. and Bleehen, N.M. (1982) Immunohistochemical techniquesin the early screening of monoclonalantibodiesto humancolonicepithelium.Br. J. Cancer 46,9. Glassy, M.C. and Surh, C.D. (198S) Immunodetectionof cellbound antigens using both mouse and human monoclonal antibodies.J. lmmunol. Methods 81,115. Haspel, M.V., McCabe, R.P., Pomato, N., Janesch, N.J., Knowlton,J.V., Peters, L.C., Hoover,Jr., H.C. and Hanna, Jr., M.G. (1985) Generation of tumour cell-reactivehuman monoclonalantibodiesusing peripheral blood lymphocytes from actively immunised colorectal carcinoma patients. Cancer Res. 45, 3951. Houghton, A.N., Brooks, H., Cote, R.J., Taormina, M.C., Oettgen, H.F. and Old, LJ. (1983)Detectionof cell surface and intracellulurantigens by human monoclonalantibodies. J. Exp. Med. 158, 53. Imam, A., Drushella, M.M., Taylor, C.IL and Tokes, Z.A. (1985) Generation end immunohistologicalchuracterisation of human monoclonalantibodies to mammary carcinoma cells. Cancer Res. 45,163. Imam, A., Drushella, M.M. and Taylor, C.R. (1986) A novel immunoperoxidaseprocedure of using human monoclonal antibodies for the detection of cellular antigens in tissue sections. J. Immunol.Methods 86,17. loachim, H.L. (1976) The stromal reaction of turnouts: an expressionof immunesurveillance.J. Natl. Cancer Inst. 57, 465.

Jass, J.R. (1986) Lymphocytic infiltration and survival in rectal cancer. J. Clin. Pathol. 39, 585. Kjeldsen, T.B., Rasmussen, B.B, Rose, C. and Zeuthen, J. (1988) Human-human hybridomas and human monoclonal antibodies obtained by fusion of lymph node lymphocytes from breast cancer patients. Cancer Res. 48, 3208.

Nie]se~ B., Borup-Christensen, P., Erb, K., Jensenius, J.C. and Husby, S. (1987) A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screenin8 of human hybridoma antibody in culture s u ~ a t a n t s . Hybridoma 6, t03.