In vitro and in vivo regulation of tumor antigen expression by human recombinant interferons

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Nucl. Med. Bid. Vol. 18, No. 4, pp. 409-412, 1991 ht. .I. Rod&. A@. Instrum. Part B Printed in Great Britain

In Vitro and In Vivo Regulation of Tumor Antigen Expression by Human Recombinant Interferons FIORELLA

GUADAGNI*,

JEFFREY

SCHLOMf

and JOHN W. GREINER

Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A. In vitro treatment with either type I or type II interferon (IFN) can selectively enhance the expession of several tumor antigens, such as the carcinoembryonic antigen (CEA) and the tumor-associated glycoprotein-72 (TAG-72) in different human carcinoma cell lines and result in enhanced level of monoclonal antibody (MAb) binding to the cell surface. In oiuoanimal studies demonstrated that treatment of athymic mice with a type I interferon [i.e. interferon-a(A)] significantly increased the expression of a 90 kDa tumor antigen which improved the targeting of a MA\, to the carcinoma xenograft. More recent studies reported that in vitro IFN treatment of human adenocarcinoma cells isolated from human malignant serous effusions selectively increased the expression of TAG-72 and CEA. One can envision that the ability of these cytokines to upregulate the level of expression of human tumor antigens presents an important experimental model in which to study the regulation of markers often correlated with epithelial cell differentiation. In addition, the increase of selective MAb-defined antigens may also be. exploited in an adjuvant setting to localize higher amounts of MAbs to the tumor cell surface and, thereby, improve the effectiveness of a MAb for tumor diagnosis and, possibly, therapy.

Introduction An important consideration when designing clinical protocols using monoclonal antibodies (MAbs) for immunodetection and immunotherapy is the antigenie phenotype and, in particular, the extent of antigen heterogeneity in the human carcinoma lesions (Prehn, 1970; Miller and Heppner, 1979; Horan Hand et al., 1983; Kufe et al., 1983). Tumor cells which do not express or express low levels of a tumor antigen will most likely escape MAb binding and, thus, detection and therapy. One potential approach to circumvent the limitations of heterogenous tumor antigen expression is to use compounds able to alter the surface antigen expression. Therefore, it has been our interest to study those agents that can selectively enhance the level of tumor antigen expression, thereby, increasing the targeting of MAbs to human tumor lesions. Recombinant human interferons (IFNs) can induce and/or amplify the expression of class I and class II MHC antigens, Fc receptors (Heron ef al., 1978; Wallach et al., 1982), and tumor antigens on the surface of a wide variety of human cell lines (Greiner et al., 1984). Reports have demonstrated that the expression of a 90 kDa CEA-related *Present tute, Viale TAuthor

address: “Regina Elena” National Cancer InstiLaboratory of General and Clinical Pathology, Regina Elena 291, Rome 00161, Italy. for correspondence.

tumor antigen in WiDR tumor xenografts in athymic mice was increased following in viuo administration of interferon-a(A) (IFN-aA) and accompanied by a concomitant enhancement in the targeting of a MAb to human carcinoma lesions (Greiner et al., 1987a, b; Guadagni ef al., 1988). Recent studies isolated human carcinoma cells from the malignant effusions of patients diagnosed with different adenocarcinomas and subsequently treated the cells in vitro with type I [i.e. IFN-a(A), IFN-j,,] or type II (i.e. IFN-y). It was observed that both types of interferons enhanced the expression of such human tumor antigens as CEA and TAG-72 (Guadagni et al., 1989). In addition, cells isolated from non-malignant ascites and treated in vitro with interferon did not express either TAG-72 or CEA before or after treatment. These results indicate that interferon treatment does not induce de nouo expression of those human tumor antigens. Therefore, it may be possible to selectively enhance tumor antigen expression in a carcinoma lesion while the surrounding normal tissues remain tumor antigen negative. These experimental investigations have led to the design of a phase IA clinical trial to determine whether i.p. IFN-y administration could upregulate TAG-72 and CEA expression on the surface of human carcinoma cells in viuo (Greiner ef al., 1989). These results indicate the potential clinical usefulness of combining an agent able to alter the antigenic 409

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FIORELLA GUADAGNI

phenotype of a tumor cell population with conjugated MAbs, and the possibility of improving the diagnostic and therapeutic efficacy of the MAb.

Results Pre -clinical in vitro and in vivo studies IFN-regulation of human tumor antigens

A series of anti-CEA MAbs (designed COL-I through 15) have been generated in our laboratory using extracts or membrane-enriched fractions of primary or metastatic colon lesions (Muraro et al., 1985). Immunohistochemical and RIA analyses demonstrated that COLs-1, -4, -12 are able to recognize distinct CEA epitopes (Kuroki et al., 1989). These MAbs, along with MAb B6.2 which recognizes a different antigen of 90 kDa glycoprotein normal crossing reacting antigen (NCA-related to CEA), were used to study tumor antigen regulation by recombinant human IFN-a A. The results of these studies showed that IFNs can significantly enhance the level of expression of a variety of human tumorassociated antigens (Greiner et al., 1984, 1987a, b; Guadagni et al., 1988). These results are partially reported in Table 1. As shown, the addition of 1000 U/mL of IFN-aA to a human colorectal carcinoma cell line (WiDR) can increase the level of binding of MAbs (i.e. COL-4 and B6.2) specific for tumor antigens (from 135 to 280%). Likewise, the IFN-aA-administration of 250,000 units daily for 6 days, to athymic mice bearing WiDR grown as subcutaneous tumors resulted in a substantial enhancement of MAb tumor binding. Subsequent studies have shown that the increase in MAb binding can be explained, for the most part, by concomitant increases in tumor antigen content. Furthermore, another important observation obtained from the studies was that a human melanoma cell line (A375), which does not constitutively express either TAG-72 or CEA, remains negative for the expression of these tumor antigens after either in vitro or in vivo interferon treatment. The results also established that interferon treatment can enhance the localization of a radioconjugated MAb to a human tumor xenograft grown in athymic mice. Additional studies have

et al.

shown that IFN-/J,, and IFN-7 were also capable of inducing significant alterations in the antigenic phenotype of human tumor cells (Kantor et al., 1989; Greiner et al., 1990; Guadagni et al., 1990a). Moreover, those findings revealed that the increase in CEA expression associated with treatment with IFN-y is accompanied by an increase in the level of CEArelated mRNA transcripts (Kantor et al., 1989; Guadagni et al., 1990b). These findings suggest that IFN-y treatment regulates CEA expression at transcriptional and/or posttranscriptional sites which is reminiscent of previous observations for IFN-7 regulation of class II HLA antigens (Rosa and Fellous, 1988). Human tumor antigen termed TAG-72 (tumorassociated glycoprotein-72) has been extensively studied in our laboratory (Colcher et al., 1981; Johnson et al., 1986; Thor et al., 1986). TAG-72 is a high molecular weight glycoprotein (> lo6 Da) that is found in a wide variety of human adenocarcinomas, including breast, colon, ovary, pancreas, stomach, endometrial and non-small cell lung carcinoma (Thor et al., 1986). It is not expressed in most adult tissues with the exception of secretory endometrium (Thor et al., 1987) and transitional colonic mucosa (Wolf et al., 1989). MAb B72.3 which reacts with the TAG-72 antigen has been successfully used in clinical trials for the localization of metastatic colorectal and ovarian carcinoma lesions (Colcher et al., 1987a, b; Lastoria et al., 1988; Schlom et al., 1989). In addition, immunohistochemical studies have reported that the expression of the TAG-72 antigen in most human carcinomas is highly heterogeneous (Thor et al., 1986). Therefore, we were interested to determine whether treatment of human tumor cell types which constitutively express TAG-72 resulted in an increased amount of the antigen expressed. As stated previously, this could, therefore, be exploited to increase MAb binding both in vitro and in vivo. Nevertheless, the in vitro study of the TAG-72 regulation by the interferons has been difficult because few established human cell lines constitutively express this antigen (Horan Hand et al., 1985). In fact the only adherent human cell lines that constitutively express TAG-72 are the MCF-7 (breast carcinoma)

Table 1. Chanaes in antiaenic uhenotyoe of human colorectal carcinoma cell after IFN-aA treatment MAb binding Cell surface in vitro

Tumor extract

(cpm/5 x 10” cells)

(cpm/lO pg protein)

in vim

Cell line

MAb

Untreated

+IFN

Untreated

+IFN

WiDR

COL-4 86.2 W6/32 MOPC-21

3210 5880 3880 negf

9970 (211%). 13,810(135%) I 1,690 (201%) neg

5640 4860 NDt neg

17,990(219%) 18,460 (280%) ND neg

A375

COL-4 06.2 W6132 MOPC-2

neg neg 3810 nea

neg neg 5420 (42%) nea

neg neg 2440 mg

neg “eg 6220 (155%) nag

*( )-%

I

increase above untmated values. tND-not determined. (< 500 cpm). Taken in part from Guadagni et al. (1988).

tneg-no

significant binding

411

Regulation of tumor antigen Table 2. Ability of interferon treatment to enhance TAG-72and CEA expression

ConstitutiveMAC binding’ (No. samples antigcn positive/No. tested)

EnhancedMAC binding*t (No. samples IFN mponsive/No.tested)

TAG-72

35/43(81.4%)

27135(77.1%)

CEA TAG-72 and/or CEA

22/43(51.1%) 42143 (97.7%)

13/22(59.1%) 36142 (85.7%)

Antigen

‘Values in parentheses represent the percentage of total samples that showed constitutive MACbinding or the percentage of samples in which MA\, binding was increased ~49% after IFN-treatment. tSamples in which a 48-72 h treatment with 500-2000 U mL of IFNs increased the binding of B72.3 or COL-4 by at least 50% above that in untreated cells. Taken from Guadagni er al. (1989).

and the highly differentiated LS-174T colorectal cell line (Greiner et al., 1987a, b). Studies have shown, however, that the LS-174T cells are unresponsive to the ability of either type I or type II IFNs to regulate normal HLA as well as tumor antigen expression (Guadagni et al., 1990b). Previous studies have shown that in Vitro treatment of MCF-7 cells with recombinant interferon can enhance TAG-72 expression (Greiner et al., 1984), and other reports (Johnston et al., 1985) have shown that a high percentage of human malignant effusions constitutively express TAG-72. Therefore, we decided to design a study which would isolate a variety of human adenocarcinoma cell populations from serous effusions of patients diagnosed with adenocarcinoma or other non-malignant diseases. The antigen phenotype of each cell population would be determined by the binding of various MAbs which recognize normal membrane antigens (i.e. HLA, cytokeratins, etc.) as well as those which react with human tumor antigens (TAG-72, CEA, etc.). The cells would subsequently be treated in vitro with IFN-a(A), IFN-fl,, or IFN-y and the changes in the level of expression of the various cell surface antigens analyzed by various immunological assays (i.e. radio-immunoassay, flow cytometry, etc.). In particular, this study presented an opportunity to investigate the ability of recombinant interferons to regulate TAG-72 expression in vitro on different human carcinoma cell populations. A large variety of serous effusions of patients diagnosed with adenocarcinoma (i.e. ovarian, pancreas, breast, unknown, etc.) non-epithelial tumors (i.e. melanoma, lymphoma, sarcoma) and benign diseases (i.e. reactive mesothelium) were analyzed for the constitutive expression of TAG-72 and CEA as well as changes in the level of antigen expression (i.e. MAb binding) as a result of interferon treatment (Guadagni et al., 1989). Table 2 summarizes these results and compares the ability of the type I and type II Hu-IFNs to increase the expression of constitutive cell surface TAG-72 and/or CEA on human tumor cell populations. 35 of the 43 effusions cytologically diagnosed as adenocarcinoma constitutively expressed TAG-72. Treatment with either IFN-a(A), IFN-fl,, or IFN-y increased the level of binding of B72.3 by at least 50% above that measured on the untreated, control cells in 27 of 35 (77.1%) of the samples. Furthermore, of the 22 effusions that were CEA positive, interferon treatment increased the binding of the anti-CEA

MAb COL-4 in 59.1% of the cases. Of the 43 effusions from primary adenocarcinoma patients studied, 42 (97.7%) constitutively expressed either TAG-72 or CEA on their cell surface, and interferon treatment increased B72.3 and/or COL-4 cell surface binding in 36 of the 42 antigen-positive cases (85.7%). Moreover, those effusions obtained from patients with non-epithelial tumors or benign diseases, failed to express either tumor antigen (TAG-72 or CEA) before or after interferon treatment. These studies provided the framework to design a clinical trial to determine whether the intraperitoneal (i.p.) administration of IFN-y in patients with ascites secondary to progressive ovarian or gastrointestinal carcinoma could enhance the expression of TAG-72 and/or CEA in vivo (Greiner et al., 1989). Patients diagnosed with adenocarcinoma with secondary malignant ascites were given weekly escalating doses (0.1-100 MU) of IFN-y i.p. Aliquots of ascites were removed from each patient 3 times per weekpre-IFN-y and 24 and 48 h after treatment. The tumor cells were isolated by the same procedure used for the in vitro study of interferon effects on tumor antigen expression on the surface of human tumor cells isolated from malignant effusions (Guadagni et al., 1989). Results of the study showed that IFN-y treatment was well tolerated by all patients. Moreover, the i.p. IFN-y administration increased the level of TAG-72 expression on tumor cells isolated from seven of eight patients all of whom had constitutive expression of the antigen. The results of the experimental as well as the phase 1A clinical trial provide substantial argument that the administration of a cytokine, such as IFN-y, in an adjuvant setting might facilitate the localization of a MAb to a human tumor cell population. The ability of the interferon to increase MAb targeting is directly related to the accompanied enhanced expression of cell surface human tumor antigens. Additional studies are needed to investigate whether interferon treatment can, indeed, improve the use of a MAb in a diagnostic and, possibly, a therapeutic setting.

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