WO2001064869A2

WO2001064869A2 - Tumor-related antigen

Info

Publication number: WO2001064869A2
Application number: PCT/EP2001/002353
Authority: WO
Inventors: Alberto Bartorelli; Gianni Bussolati; Ada Funaro; Alberto L. Horenstein; Daniela Concas
Original assignee: Zetesis S.P.A.
Priority date: 2000-03-03
Filing date: 2001-03-02
Publication date: 2001-09-07
Also published as: WO2001064869A3; WO2001064869A9; AU5825701A

Abstract

The present invention relates to a novel tumor related antigen named p220 which is expressed on the surface of different tumor cells.

Description

A NOVEL TUMOR-RELATED ANTIGEN

The invention concerns a new protein isolated and purified from tumor cells.

More particularly, the present invention stemmed from the availability of a group of cancer patients treated in vivo in the context of an immunotherapy trial with UK101 preparations (WO 92/10197). By analyzing the immunoglobulin repertoire of one of such patients, we selected a small panel of human antibodies specific for UK1 14 (WO 00/00591 , herein incorporated by reference), the active component of UK101. Three of these lines were stabilized by somatic fusion with a human-mouse hybrid myeloma line and three human monoclonal antibodies (HuMAb) were selected with unaltered specificity. The analysis of the reactivity of these antibodies with neoplastic and normal cells of different origin demonstrated that these reagents recognise a membrane antigen restricted to tumor tissues and cells. None of the normal cells and normal tissues surrounding the neoplastic lesions were reactive with the exception of a subset of fetal hepatocytes (see the Table).

The biochemical analysis of the target antigen was performed using different techniques and the results obtained using the 3D8 human monoclonal antibody or the P3 murine monoclonal antibody were reported as prototypic. The 3D8 huMAb, already disclosed in WO00/591 in the name of the same applicant, is produced by a human hybridoma identified as UK#1/3D8, which was deposited at ECACC under No. 9706 2409 on June 24^th, 1997.

Immunoprecipitation experiments carried out on ^12DI-surface-labeled KATO III cells showed in SDS- PAGE a predominant band of about 220 kDa (hereinafter named p220) both with 3D8 and P3 antibodies. It was also seen that when biotinylated intact LAN- 1 (neuroblastoma) cells were reacted with 3D8 HuMAb and unbound antibodies were removed, surface- expressed receptors were represented by one protein corresponding to an apparent size of about 220 kDa, in good accordance with that obtained previously with radiolabeling experiments with KATO III (gastric carcinoma) cells. The biotinylated p220 protein reacts specifically with 3D8 HuMAb after blotting.

The identity between the two proteins recognised by 3D8 and P3 was confirmed by sequential immunoprecipitation experiments which demonstrated that 3D8 and P3 react with the same molecule: extensive depletion of the ¹²⁵I-labeled cell lysate with 3D8 abrogate the reactivity with P3 antibody. Immunoprecipitation experiments performed on KATO III cells after metabolic labeling showed two major bands of 220 and 14 kDa, respectively. At the same time these experiments showed a soluble form released in the culture medium and visible after 10 min chase. The existence of a soluble form of the tumor-related antigen having a molecular weight of about 220 kDa was confirmed also in biological fluids including sera obtained from cancer patients. The human and murine antibodies also recognise the high molecular weight protein in Western blots. The p220 protein is a glycoprotein extremely sensible to trypsin or proteases digestion. In order to obtain the protein in high amounts, sufficient to perform the sequence analysis, cell cultures of different cell lines expressing the antigen at high density were scaled up and the target antigen was purified by means of affinity chromatography purification. The analysis of the amino acidic component underlined a very abundant percentage of serine (9%) resulting in many potential phosphorylation sites, with a high abundance of basic (Arg and Lys) amino acids amounting together more than 34% of the protein. The presence of over 25% of Arg content made this protein remarkably susceptible to trypsin degradation, in agreement with biochemical data.

The analysis of the biological functions exerted by p220 showed that it is involved in the growing mechanism of selected neoplastic cells. Indeed, the addition of purified 3D8 HuMAb to the in vitro culture of tumor cell lines, is followed by a significant reduction of cell proliferation evaluated in terms of 3H-thymidine incorporation.

Additional experiments were carried out to determine whether intracellular proteins were associated to p220. When the cell extract was immunoprecipitated with 3D8 under conditions that preserved protein associations, the immunoprecipitation yielded two other proteins of higher molecular mass than the membrane receptor (p220). The MALDI-TOF MS technique was applied to the identification of the mentioned high molecular weight associated-proteins. The MALDI mass spectra of the peptides generated by an in-situ trypsin digestion of the bands separated by SDS- PAGE, were used as input for the computerised analysis. The analysis of the input with the PROFOUND program using a SWISS-PROT database indicated, with a probability equal to 1 , that one of these proteins is the intracytoplasmatic enzyme Fatty Acid Synthase (FASE). Extensive washes of the proteins bound to the HuMAb using high stringency conditions substantially reduced the amount of associated proteins. However, a 250 kDa band, identified by MALDI mass analysis after HPLC gel filtration chromatography as the non-erythroid α-spectrin (fodrin), was difficult to eliminate, suggesting that there may be a preferential interaction between p220 and the cytoskeleton.

The tumor-related protein p220 can be used for therapeutic and diagnostic applications. Preferably, it will be used in the preventive vaccination of subjects with susceptibility to neoplasias or in the therapeutic vaccination of neoplastic patients. The term "vaccination" as herein used is referred either to the active vaccination, i.e. the in vivo administration of the protein to activate the immune response directly in the patient, or to the passive vaccination, i.e. its use for the in vitro activation of T cytotoxic lymphocytes and their subsequent re-inoculation in the patient. The procedures for the preparation and use of vaccines are known to anyone skilled in the art; an extensive description is given for example in Paul, Fundamental Immunology, Raven Press, New York (1989) or Cryz, S. J., Immunotherapy and Vaccines, VCH Verlagsgesselschaft (1991).

Furthermore, the direct involvement of p220 in tumor cell proliferation mechanisms suggests its use as a biologic target for therapeutically active molecules.

In addition, the protein, especially its soluble form, as well as the antibodies against it, can have diagnostic applications, including the preparation of immunoassays to detect protein expression in tumors. Experimental procedures Cell surface iodination

Iodo-Gen (Pierce) was dissolved in chloroform at 0.25 mg/ml, 500 μl were dispensed into a 50 mm-glass Petri dish and the solvent was evaporated. The cells (5 x 10⁷) were added in 1 ml of PBS containing 1.85 MBq of carrier-free Nal 25I for 15 min at room temperature, then were transferred to saturated tyrosine-PBS solution and washed to remove unbound ^{I 25}I-iodide. Metabolic labeling

KATO III cells were incubated in methionine-free MEM medium (Gibco) for 30 min at 37 °C and then resuspended at 3 x 10⁷ /ml in the same medium containing 5% dialysed FCS and 1 mCi 35S-methionine/-cysteine (specific activity 29.8 TBq/mmol; Amersham) for 1 h at 37 °C. Cells and supernatants were collected at different times. The cells were successively washed twice with ice-cold RPMI- 1640 medium containing 1 mM unlabeled methionine and lysed in the presence of proteases inhibitors as previously described (Int.Immunol., 8, 1643-1650,1996). After pre-clearing post- nuclear cell lysate with GaHIgM bound to Protein G-Sepharose (GaHIgM- Protein G) (Pharmacia) the target molecules were immunoprecipitated for 12 h at 4 °C with 10 μg of the HuMAb followed by the addition of GaHIgM- Protein G for 2 h at 4 °C. The immunocomplexes were then subjected to sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis (SDS-PAGE) as described below. SDS-PAGE and Western blot

The cell lysate (5 x 10⁷ cells) was precleared as described above. The supernatant collected through centrifugation for 30 min at 14,000 g was divided into 3 aliquots. One aliquot was incubated with 10 μg of the

HuMAb; the second with 15 μg of the P3 murine MAb, and the third one without antibody for 12 h at 4 °C. Immunocomplexes were successively incubated for 2 h at 4 °C with GaHIgM-Protein G and GaMIgG-Protein G, respectively, washed 3 times and extracted by boiling in SDS sample buffer with or without 5% beta-mercaptoethanol. Proteins were analysed on SDS- PAGE (7.5% to 15%) polyacrylamide) and the gels were exposed for autoradiography to Kodak X-Omat AR film. For Western blot analysis, proteins were transferred after SDS-PAGE to a PVDF membrane according to Biochem J. 330, 1 129- 1 135, 1998 and sequentially reacted with the HuMAb and biotin-conjugated GaHIgM (Sigma) followed by Streptavidin- peroxidase (Amersham). Developing reaction was carried out by chemiluminescence using an ECL kit (Amersham), according to manufacturer's instructions. Protein sequencing

After SDS-PAGE, proteins treated with vinyl-piridine were electroblotted using 3-(cyclohexylamino)- l-aminopropanesulfonic acid onto PVDF membranes and the bands of interest were excided and used for sequencing. The MALDI-TOF spectra of the peptides generated by an in- situ trypsin digestion of the bands separated by SDS-PAGE was obtained at Bio-Industrial Park Laboratories (Ivrea, Italy). DESCRIPTION OF THE FIGURES

Figure 1. Immunoprecipitation of the target antigens from KATO III cells by means of human and murine MAb.

A) SDS-PAGE profiles of antigens precipitated with the 3D8 HuMAb and the P3 MAb. KATO III cells were surface-labeled with ¹²⁵I and the target antigens were precipitated with the 3D8 HuMAb (lane a) or with the P3 MAb (lane b), as described in the Materials and Methods section. After immunodepletion of the 220 kDa protein with 3D8

HuMAb (lane c), the lysate does not provide any structure reactive with P3 MAb (lane d). Molecular mass of protein standards are indicated in kDa.

B) SDS-PAGE profile of antigens immunoprecipitated with the 3D8 HuMAb from KATO III cells after metabolic labeling with ³⁵S- methionine/-cysteine. Immunoprecipitation was carried out as described in the Materials and Methods section. Molecular mass of protein standards are indicated in kDa.

Figure 2. Western blot analysis. KATO III cell lysates were immunoprecipitated with the 3D8 HuMAb and proteins separated by SDS-

PAGE were blotted onto nitrocellulose. The membranes were probed with the 3D8 HuMAb (lane a) or with the P3 MAb (lane c) and the reaction revealed using an ECL kit. The 220 kDa antigen was not visualised when irrelevant primary antibodies were used (lane b and d). The position of the heavy chains (arrows) of antibodies used for immunoprecipitation and visualised by the anti-Ig developing reagent and molecular mass of protein standards (kDa) are indicated. Figure 3. Effect of trypsin digestion on p220 expression on KATO III cells.

5 x 10⁶ cell/ml were incubated for 30 min at 37 °C in PBS supplemented or not with 0.250 mg/ml of trypsin. After washing in the presence of 10% fetal calf serum at 4 °C the cells were labeled with human (3D8, 2G1 1 , 1A1 1) and murine (P3D1D1 1) antibodies and analysed by indirect immunofluorescence.

Figure 4. Effect of biological fluids on anti-UK1 14 human antibodies binding to KATO III cells.

5 x 10⁶/ml cells were incubated with normal serum, cord blood serum, human ascites and serum from a UK1 14-treated cancer patient for 1 h at 37°C.

After washing, cells were labeled with 3D8 human antibodies followed by an anti-human immunoglobulin-FITC and the results were analysed by cytofluorimeter. Figure 5. Amino acid analysis of purified p220 obtained by acid hydrolysis.

Figure 6. Immunoprecipitation of the target antigen from LAN-1 cells by means of 3D8 human monoclonal antibody.

Biotinylated intact LAN-1 (nueoblastoma) cells (lane 3) were reacted with 3D8 HuMAb or control IgM antibody (lane 2) and unbound antibodies were removed. Immunoprecipitated surface-expressed receptors were represented by one protein corresponding to an apparent size of about 220 kDa (lane 4). After blotting the bitinylated p20 protein reacts specifically with 3D8 HuMAb (lane 1). Molecular mass of protein standards are indicated in kDa.

Figure 7. Inhibitory effects of the anti-p220 human monoclonal antibody 3D8 on tumor cell growth evaluated in terms of 3H-thymidine incorporation.

Table - Reactivity of the 2G3, 3D8 and 1 Al 1 HuMAb on normale and neoplastic cells.

Claims

1. A protein having the following features: a) it has an apparent molecular weight of 220kDa as determined by SDS- PAGE (7.5% to 15% polyacrylamide); b) it is specifically bound by the human monoclonal antibody produced by hybridoma UK#1/3D8 deposited at ECACC under No. 9706 2409; c) it is expressed on the surface of KATOIII and LAN- 1 cell lines;

2. Pharmaceutical or diagnostic composition containing the protein of claim 1.

3. Pharmaceutical composition according to claim 1 , in form of vaccine.

4. The use of the protein of claim 1 in immunoassays.

5. The use of the protein of claim 1 as a biological target for the identification of therapeutically active molecules.