FIELD OF THE INVENTION This invention relates to monoclonal antibodies directed against the retinoblastoma protein pllO^. More specifically, it relates to a novel monoclonal antibody having the ability to detect pllO ^RB that has been previously bound by a naturally occurring or non-naturally occurring ligand.

BACKGROUND OF THE INVENTION The retinoblastoma gene (RB) is one of the best- studied tumor suppressor genes. Mutations which prevent the normal expression of the retinoblastoma gene have been linked to the pathogenesis of several human malignancies. These include small-cell lung carcinomas, osteosarcomas, breast carcinomas, soft tissue sarcomas, bladder carcinomas, prostate carcinomas and testicular tumors.

pllO* ³ , the protein product of the RB (retinoblastoma) gene, specifically binds and forms a complex with several DNA tumor viral oncoproteins, including adenovirus ElA, polyomavirus Tag and papillomavirus E7. It is believed that these oncoproteins bind to and inactivate an important function of pllO ^RB , thereby mimicking the loss of retinoblastoma gene function. Studies have suggested that pllO* has intrinsic DNA-binding activity and may also interact with cellular transcription factors E2F/DRTF. Improved methods of determining the absence of pllO ³ could aid clinicians in their evaluation of patients and their decision as to appropriate treatment. Recently, evidence has become available that the loss of pllO* ² expression, as measured by immunohistochemistry, may be predictive of patient prognosis in non-small lung cancer, and in bladder cancer. It is likely that additional studies of this sort will add to the list of cancers in which loss of pllO ¹³ expression will be predictive of the aggressiveness of disease. Because of this, improved methods of

immunohistochemistry are required to aid clinicians in their evaluation of patients and their decisions regarding aggressiveness or modality of therapy. In this case, increased intensity of available chemotherapy is one option, another option may be therapies derived from the tumor suppressor gene, RB, or its gene product pllO* ⁸ . In the latter case, lack of Rb protein expression would indicate that the patient is a candidate for RB-related therapy.

The currently available antibodies to pllO* are either polyclonal, and therefore, variable, or monoclonal but inappropriate because of low affinity or because they are targeted to an epitope of pllO* ³ likely to be masked by cytoplasmic proteins. If such epitopes are masked by cytoplasmic proteins, therefore giving a "false negative" reading with respect to pllO* ³ expression, then an incorrect therapeutic modality may be chosen. Thus, there exists a need for monoclonal antibodies reactive with pllO** regardless of associated cytoplasmic proteins. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

This invention provides a family of monoclonal antibodies specific for epitopes of pllO* ⁸ protein present in the nucleus. These antibodies have superior properties that prove useful for the detection of pllO ³ or its complexes with other cellular regulatory proteins in cells and in cell lysates.

This invention also provides hybridoma cell lines that produce such monoclonal antibodies and methods of using these antibodies diagnostically, prognostically and therapeutically.

Further, the invention provides a method for isolating proteins associated with pllO" proteins.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an immunoblot analysis of pllO ^u expression in various transformed cell lines with 1 μg/ml of 3C8(A), PMG3-245(B) and MuIgG (as negative control, C). The proteins were visualized by NBT/BCIP substrate.

Figure 2 shows 20 μl of A5 9 cell lysate (started from 1 mg/ml, lane 1) with 2-fold serial dilutions were immunoblotted and detected with antibody 3C8 (2 μg/ml) .

Figure 3 shows ELISA for biotinylated 3C8, 1E5 and PMG-3-245. Various concentrations of biotinylated anti-Hb antibodies were titrated in pllO" coated plates. κτ.τ?a protocol was followed as described in the Materials and Methods section iaf∑a* D3C8, Δ1E5, ® PMG-3-245

Figure 4 shows Tag/Rb association inhibits binding of 1E5 and PMG-3-245. 0.2 ug/ml of 3C8, 1 μg/ml of PMG-3-245 and 1 μg/ml of 1E5 were used to detect bound pllO ^u .

Figure 5 shows identification of pllO" in U20S cell lysate with 3C8, PMG-3-245 and MuIςG2a (as a negative control) . Protein A-Sepharose beads (a) or protein G- sepharose beads (b) were employed to immunoprecipitate ixαmunocomplexes. The ixπmunoprecipitated proteins were analyzed by 8-16% gradient SDS-PAGE and immunoblotted. H:heavy chain of MuIgG. L:light chain of MuIgG.

Figure 6 shows im unocytostaining with 3C8, PMG-3-245 and nonspecific control antibody (MuIgG) on a 9mm x 9mm

chamber slide, containing 2 x 10 ^s formalin-fixed U20S osteosarcoma cancer cells. 0.5 μg/ml concentration of each antibody was employed as primary antibody. Diaminobenzidine was used as the final chromogen.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a family of monoclonal antibodies directed at the product of the retinoblastoma gene (pllO**) . These monoclonal antibodies have been characterized and compared with respect to their ability to target intracellular pllO* ³ by immunocytostaining, ELISA, immunoprecipitation and direct immunoblotting. Preliminary epitope analyses have been conducted by reaction with synthetic polypeptides or fragments of pllO* ³ expressed in E. coli . Localization of one of the monoclonal antibodies (1E5) to a region between amino acid residue (aa511-aa530) , within the N-terminal Tag binding domain, suggested that it may interfere in pllO* ⁸ binding to a large T antigen (Tag) . A large Tag/Rb binding assay was developed which would be suitable for screening of pllO** agonists or antagonists. This assay showed that Tag/Rb complexes prevented binding of 1E5, further supporting its epitope assignment to the Tag binding domain of pllO ¹ ^. Another monoclonal antibody, 3C8, which binds pllO** near the C-terminal end of the protein (aa886-aa905) is shown to be useful for the detection of pllO* ⁸ in cells, or in cell lysates. Because 3C8 binds outside of regions thought to be involved in pllO* ³ interactions with other cellular proteins, it may be an especially useful affinity reagent for the isolation of cellular proteins which specifically interact with pllO ¹ ^.

As used herein, the term "pllO*" refers to the expression product of the Rb gene as noted in Lee et al.. Nature 329:642-645 (1987), which is incorporated herein by reference. The numbering of the amino acid residues conforms to that of this reference.

Described herein is the initial characterization of a family of monoclonal antibodies directed against pll0 ^RB . Among these antibodies, 3C8, which recognizes a C-terminal epitope, was compared with PMG-3-245, which recognizes an epitope near or within the first Tag binding domain of pllO**. The results showed 3C8 can detect pllO ^RB with higher sensitivity than PMG-3-245 in immunoblots, immunoprecipi- tation, ELISA and immunocytostaining. Previous studies indicated that the region of pllO** needed for binding to several DNA tumor virus oncoproteins ElA, E7 and large T antigen had been mapped in the region from amino acid residues 394 to 571 and 649 to 793 as described by Huang et al., EMBO J. 9:1815-1822 (1990) and Hu et al., EMBO J. 9:1147-1155 (1990) and incorporated herein by reference. There is also evidence that cells produce one or more proteins that interact with the Tag/EIA binding domain of pllO* ⁸ in a manner resembling that employed by Tag and ElA (18-20, Kaelin et al.. Cell 64:521-532 (1991 and Lee et al., J. Virol. 38:1064-1076 (1981) and incorporated herein by reference. If so, an antibody that interacts with this region of the pllO** might, preferentially or exclusively, recognize the unbound form of the pllO* ⁸ , or, alternatively, might be expected to "miss" the bound form of pllO ¹ ^, thus resulting in a false negative conclusion regarding pll0 ^RB expression. This assumption was confirmed by using 1E5 in a Tag/pllO* ⁰ binding assay (Fig. 4). 1E5, which recognizes an epitope located within the Tag binding site which is located at, within, or near aa393-aa572, failed to recognize pllO** in the pllO∞-Tag complex. Figure 4 also reveals that PMG-3-245 failed to detect the bound form of pll0 ^RB . This may be because the Tag-Rb complex sterically blocks the epitope from antibody-combining sites in this assay format with PMB-3-245, which also binds near this domain, and 1E5.

Many factors influence the reactivity of monoclonal antibodies. These include differences in affinity.

UTE SHEET

contiguous vs. conformational epitopes as well as the other factors described above (e.g., association of the target protein with other cellular proteins) . The monoclonal antibody, 3C8, which has strong reactivity with the C- terminus of pllO* ² , should aid in the characterization of the level of pllO^ in tumor cells. As an affinity reagent it may also allow characterization of pllO* ⁸ associated proteins via the appropriate affinity chromatography or immunoprecipitation studies (8, as described by Hu et al., Mol. Cell. Biol. 11:5792-5799 (1991), and incorporated herein by reference. It is critical that an antibody such as 3C8 be used in such studies because it recognizes an epitope not thought to be critical for interaction of those domains of pllO ⁸ which interact with other cellular proteins.

In order to more fully determine whether 3C8 may have advantages for evaluation of pllO ^RB expression, four cell lines were examined for expression of the RB-encoded tumor suppressor protein. As expected, multiple forms of pllO ¹ " ³ proteins were found in A549 and U-20S, but not in SaOS2 and H128 by direct immunoblotting with 3C8 (Fig. 1). This observation is consistent with previous studies on RB-gene expression by immunoprecipitation in these four cell lines (17, as described by Yokota et al., Oncogene 3:471-475 (1988); Shew et al., supra, 1990; Hensel et al., supra 1990, and incorporated herein by reference. However, only trace amounts of RB-encoded proteins were detected with PMG-3-245 by direct immunoblotting in these assays (Fig. 1). These data show that both 3C8 and PMG-3-245 were able to recognize the native forms of pllO ^RB in cell lysates, and that monoclonal antibody 3C8 demonstrated greater sensitivity for recognition of pllO* than did PMG-3-245. The same pattern is also reported here for immunocytochemical staining. This may be due to the stronger affinity of 3C8 for pllO ³ and/or the different epitopes recognized by these monoclonal antibodies.

Previous studies have shown that functional loss of the RB gene may contribute significantly to tumor progression (11,12, as described by Cance et al., N. Eng ^\' l. J. Med. 3232:1457-1462 (1990) and incorporated herein by reference. To resolve whether RB plays a key role in carcinogenesis, an accurate and fast screening method should be established to reflect the true frequency of inactivation of RB in cancers. Molecular genetic approaches, such as Southern analysis, may not detect small molecular alterations (6,12, Figge et al., Amer. J. Pathology 139:1213-1219 (1991) and Horowitz et al.. Science 243:937-943 (1989) and incorporated herein by reference. Also, contamination of tumor tissue with normal cells may lead to underestimation of the degree of abnormalities and confound Southern, Northern, or PCR-based analysis (6,12, as described by Murakami et al.. Cancer Res. 51:5520-5525 (1991).

Immunohistochemical staining represents the most reliable method for analyzing tumor samples for pllO* ^® expression because this method allows direct visualization of tumor cells within biopsy specimens. The availability of 3C8 provides a potentially better means to screen for pllO ³ expression in cell lines and histological specimens. The use of monoclonal antibody 3C8 may lead to a more accurate estimate of pllO" ³ expression in tumor samples and therefore may allow for a more critical analysis of the prognostic value of pllO* ³ analysis in human cancers.

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

EXAMPLE I

Protein Purification: The recombinant baculovirus vector, pAcYMl/RB2.8, encoding pllO*, was expressed in AcNPV-Y4 insect cells, according to the method of Wang et al.. Cell Growth and Differentiation 1:429-437 (1990),

which is incorporated herein by reference. Cells were treated with lysis buffer (25mM Tris-HCl, pH8.0/150mM NaCl/0.5% NP40/50mM NaF/10 ug/ml/aprotinin/10 ug/ml leupeptin) on ice for 1 hour. The pllO ^RB was purified by sequential chromatography on CM Sepharose CL6B and DEAE Sepharose CL6B. Column fractions were analyzed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The fractions that contained pure pllO* ⁸ were pooled and concentrated. For preparation of TrpE-RB fusion proteins, RB cDNA fragments Avall/Bglll/Hindlll (exon 19-27) expression plasmids were transfected into E. coli as described in Wang et al.. Cell Growth and Differentiation 1:233-239 (1990), and incorporated herein by reference. The transfected cells were treated with lysis buffer and the fusion proteins (pRBOl-10, pRBlO-19, pRB23-27 and pRB 19-27) were purified by continuous-elution electrophoresis (Model 491 Prep Cell, BioRad, Richmond, California) .

Hvbridoma Production: Hybridomas were generated as described in Godwing, Monoclonal Antibodies: Principles and Practice, Academic Press Inc. 1983, which is incorporated herein by reference. Briefly, purified pllO* ³ , pRB 01-10 (aa44-aa330) and pRB 19-27 (aa612-aa928) were used to immunize Balb/c mice by intraperitoneal injection. After high titers of antibodies specific for pllO* ³ , pRB 01- 10 and pRB 19-27 were detected, a representative mouse was sacrificed and hybridomas were prepared by fusing mouse spleen cells to Sp2/0 cells. Positive clones were identified by the enzyme linked immunoadsorbent assay and immunoblotting. All hybridomas were subcloned and propagated in Dulbecco\'s modified Eagle\'s medium supplemented with 10% fetal calf serum. Single cell cloning was done by limiting dilution. Antibody isotypes were determined using a kit provided by Zymed Laboratories Inc., San Francisco, California.

SUBSTITUTE SHEET

Immunoblotting: Purified pllO* ⁸ fragments were prepared as fusion proteins from E. coli lysates. These included aa41-aa928, aa41-aa330, aa612-aa928 and aa776- aa928. Each fusion protein was employed for initial epitope screening and monoclonal antibody characterization. Cells were solubilized in 50mM Tris-HCl pH 7.5/250mM NaCl/0.1% NP40/50mM NaF/5mM EDTA/lOug/ml aprotinin/lOug/ml leupeptin on ice for 2-20 minutes and protein was quantitated (BioRad, Richmond, California) was employed for hybridoma screening. The protein was then electro- transferred to an Imobilon P membrane (Millipore, South San Francisco, California) . After primary antibody incubation. The membranes were washed three times, 10 minutes each, with 0.05% Tween-20 in PBS. Goat anti-mouse IgG conjugated to alkaline phosphatase (Promega, Madison, Wisconsin) , diluted 1:7500 in 0.5% BSA/PBS was added to immunoblots for 1 hour at 24°C. Blots were again washed in 0.5% BSA/PBS three times for 10 minutes each. Alkaline phosphatase substrate, NBT/BCIP (nitro blue tetrazolium/5-bromo-4- chloro-3-indolyl-phosphate; Promega) was then added and incubated for 15-30 minutes.

Immunoprecipitation: A previously described protocol was employed as described in Lee et al., J. Virol. 38:1064- 1076 (1981), and incorporated herein by reference. Purified monoclonal antibody (10 μg) was added into 1 mg of U20S total cell lysate. After a 30 minute incubation on ice, protein A-sepharose 4B beads or protein G-sepharose 4B beads (Pharmacia, Milwaukee, Wisconsin) were added to precipitate the immune complex. The immunoprecipitates then were analyzed by 8-16% SDS-PAGE and immunoblotting.

Immunocvtochemical staining: Immunocytochemical analysis was performed using monoclonal antibodies directed against pllO ¹ " ³ and nonspecific control antibody (MuIgG) on formalin-fixed cells. The avidin-biotin complex- im unoperoxidase technique as described in Cordon-Cardo et

al., Amer. J. Pathol. 126:269-284 (1987), and incorporated herein by reference; (Vector Laboratories, Burlingame, CA) was used to visualize antibody binding. Diamiriobenzidine was employed as the final chromogen.

Enzyme linked immunoadsorbent assay (ELISA) : Purified pllO" ³ , or purified fusion proteins, were used to develop an ELISA for hybridoma screening and monoclonal antibody characterization. 50 μl of coating buffer (25mmol/l carbonate buffer, pH 9.6) containing 50 ng of purified pllO* ⁸ or fusion proteins were coated on microtiter plates (Costar, Pleasanton, California) overnight at 4°C. An optimized ELISA protocol was followed as described in Campbell et al.. Monoclonal Antibody Technology; Laboratory Techniques in Biochemistry and Molecular Biology (1987) and incorporated herein by reference.

Tag/Rb binding assay: A modified ELISA protocol was employed. 50 μl of 1 μg/ml purified Tag in coating buffer was bound to wells in microtiter plates at 4°C overnight. After blocking with BSA (10 mg/ml in PBS) various concentrations (2 μg/ml to 0.016 μg/ml) of purified pllO ⁸ were added into each well. The plate was incubated for 1 hour at room temperature. After washing four times with PBS/Tween-20, biotinylated 3C8, 1E5 or PMG-3-245 was added to the appropriate wells and the plate was incubated for an additional 1 hour at room temperature. The plate was then washed four times as described above. 1:2500 dilution of Streptavidin-POD (Boehringer Mannheim GmbH, Chicago, Illinois) was added to the wells and was incubated for 1 hour at room temperature. ABTS(2.2-Azino-bis(3-ethylbenz- thiazoline-6-sulfonic acid) ) was used as chromogen. Color development was measured after 20-30 minutes incubation and the absorbance (405 nm) was determined by microtiter plate autoreader (Coulter microplate reader. Molecular Devices, Menlo Park, California).

Affinity measurements: The Kd values for 3C8 and PMG- 3-245 were determined by ELISA as described by Friguet et al., J. Immuno. Methods 77 (1985), and incorporated herein by reference. First, a constant concentration of 3C8 or PMG-3-245 was incubated overnight in solution with various concentrations of pllO* . After equilibrium was reached, the unbound antibody was determined by ELISA protocol as described above.

EXAMPLE II

Generation and initial characterization of monoclonal antibodies directed against pllO* ⁸

Purified pllO ^RB was used to immunize Balb/c mice. After the third boost, the most responsive mouse, as determined by ELISA, was chosen for the preparation of hybridomas. All of the monoclonal antibodies generated by purified pllO ^RB were found to recognize only sequences within pllO ^RB amino acid residues 330 to 612, determined by standard immunoblot techniques. In order to obtain antibodies that would recognize different regions of pllO ¹ ^, E. coli TrpE-RB fusion proteins encompassing amino acid residues 41 to 330 and 612 to 928 respectively, were used as immunogens. This provided a means for focusing antibody production to regions of pllO* ³ which is important for prognostic applications. The initial screening procedures required positive ELISA and immunoblotting results. After single cell cloning of positive hybridomas the locations of the respective epitopes on pllO* ³ were determined by competition ELISA and immunoprecipitation with fusion proteins and/or with synthetic polypeptides as described in Table 1. Six independent clones were selected as representative clones recognizing different regions of pllO* ³ . To further characterize these six antibodies, each was tested for its ability to recognize pllO by immunoblotting, ELISA, immunoprecipitation and

immunocytochemical staining assays. All of the tested antibodies were able to recognize native pllO** as judged by their activity in immunoprecipitation assays (summarized in

Table 1). One of the antibodies, 3C8, recognized an epitope contained within amino acid residues 886 to 905 of pllO ^ω , and demonstrated very strong immunoreactivity with pllO* ³ (summarized in Table 1). To further characterize

3C8, U-20S and A549 which express a normal pllO∞ (17,29); and SaOS2 and H128 which have previously been reported to express low levels of an altered RB gene product as described in Shew et al., Proc. Natl. Acad. Sci. USA 87:6-

10 (1990); Hensel et al.. Cancer Res. 50:3067-3072 (1990) and incorporated herein by reference, were immunoblotted directly with 3C8 (Fig.l). The results showed a tight cluster of bands consistent with the presence of phosphorylated and unphosphorylated pllO" ³ proteins with apparent molecular weights between 110 and 116 kDa in both

U-20S and A549 cell lysates. In contrast, none of these bands was detected in SaOS2 and H128 cells. Under these conditions, PMG-3-245 failed to detect significant pllO" ³ in

U-20S or A549 cells (Fig. 1). This indicated that the 3C8 antibody may be more sensitive than PMG-3-245 for immunoblotting analyses. To test this possibility further,

A549 cell lysate (1 mg/ml 2-fold serial dilutions) was immunoblotted with antibodies 3C8 or PMG-3-245 (Fig. 2). pllO* ³ proteins were readily detected by 3C8 even when total lysate protein loaded per lane was as low as 0.625 μg, while PMG-3-245 only slightly revealed a signal of pllO** even in the undiluted total lysate (20 μg loaded). ELISA

(Fig. 3) data also suggested that 3C8 may show a higher affinity for pllO* ⁵ than PMG-3-245. These results were confirmed by direct measurements of the dissociation constant of antigen-antibody equilibria. The Kd values were calculated as described by Friguet et al., supra 1985, and incorporated herein by reference. A Kd value of 2.9x10"

⁹ M for 3C8 and 6.2xl0" ⁹ M for PMG-3-245 were determined which supported other data that 3C8 appears to have

TABLE I Characterization of Anti RB Monoclonal Antibodies

Elisa: pll0 ^RB coated on plates IB: Immunoblotting (Baculovirus lysate or purified pllO∞)

IP/IB: Immunoprecipitation-Immunoblotting (Baculovirus lysate or purified pllO* ⁵ ) detection by 3C8

IS: Immunocytochemical staining (U20S cell line) ND: Not done

This monoclonal antibody recognized Tag binding region of pllO* ⁸ , which is located at, within or near aa393-aa572.

improved binding for pllO ^∞ than PMG-3-245. However, it is not claims that affinity alone is important, as the physical properties of the monoclonal antibody as well as its epitope, are probably important contributing factors.

The Tag/Rb binding assay (Fig. 4) demonstrates that 3C8 can recognize pllO** in a Tag-pllO ^RB complex. However, the binding of PMG-3-245 and 1E5 to pllO* ⁰ were abrogated by the Tag-pllO* ⁸ association. The results support the proposal that 3C8 may be superior for detection of pllO"* in cell lysates -. 1E5 or PMG-3-245 because it reacts outside the regions of pllO** which interact with other regulatory proteins. The relative affinity for pllO ¹ * of 3C8 may also be an important determining factor.

The characterization of 3C8 was also investigated by immunoprecipitation and immunocytochemical staining. As shown in Figure 5a, nonradiolabeled immunoprecipitation/ immunoblotting assays indicate that the efficiency of 3C8 in immunoprecipitation studies of native pllO"* from U20S tumor cells apparently was higher than with PMG-3-245 (Fig. 5) . Protein G-sepharose 4B was used to exclude the possibility of the different affinity of protein A- sepharose RB to different subclasses of the antibodies. (3C8 is IgG _2a , and PMG-3-245 is IgG,). A band of pllO* ⁸ was easily observed when protein-A-sepharose 4B was used (Fig. 5a,b) . These results suggest that 3C8 can immunoprecipitate pllO** more effectively than PMG-3-245 and that the efficiency of immunoprecipitation is unrelated to the isotype of 3C8.

Because tumor cell pllO ^RB immunostaining is useful as a prognostic/diagnostic method, the monoclonal antibodies 3C8 and PMG-3-245 were compared for their immunocytochemical staining abilities (Fig. 6). U-20S tumor cells were immunostained with 3C8 and PMG-3-245 monoclonal antibodies. While there is no obvious

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difference in immunocytochemical staining while using high concentrations (4 μg/ml) of PMG-3-245 or 3C8, staining with 3C8 gave more intense staining when both monoclonal antibodies were compared at 0.5 μg/ml (Fig. 6).

Although the invention has been described with reference to the presently-preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.