FI106129B - Processes for preparing variant CD44 surface proteins and antibodies directed against these proteins - Google Patents

Description

l 106129

Methods for making modified CD44 surface proteins and antibodies against these proteins are provided.

The present invention relates to methods for producing modified CD44 surface proteins, to DNA sequences encoding these modified protein fragments, and to methods for producing antibodies against the modified determinants of these proteins.

The very life-threatening nature of malignant tumor cells is due to their ability to form metastases. Original primary tumor cells acquire this property probably as a result of numerous changes during tumor development. As a result of these events, the cancerous cell variants which penetrate the extracellular matrix and enter the lymphatic system or the bloodstream are continuously released from the original tumor mass. These metastatic, often adherent, cancerous cells migrate into the circulatory or lymphatic system and leave the vascular system at other sites where they invade other tissues to form daughter tumors (for review, see: Hart et al., 1987); Nicolson. The formation of metastases requires a number of interactions between the tumor cells and the matrix of V. cells, or other cells. Almost all of these interactions require cell surface components, such as receptors for the matrix and the laminate. surface-bound proteolytic enzymes and "cell adhesion molecules, including those molecules that ··· V · cause organ-specific adhesion and thus result in a more sensitive formation of metastases to certain organs, in addition to growth factors and growth factors. receptors.

• t 106129

It is known that the membrane proteins of metastatic and non-metastatic tumor cells derived from BSp73 tumor in rat differ from each other, as demonstrated by the antibody response (Matzku et al., 1983 and 1989).

Subsequently, it has been found that metastatic BSp73ASML tumor cells contain a surface protein that corresponds in part to a known glycoprotein involved in lymphocyte adhesion and cell-cell and cell-matrix interaction (the human normal glycoprotein, Ppg-1 and rat, respectively (HEBF1n). However, this novel abnormal or modified CD44 surface protein differs from these known sequences by a 154 amino acid extracellular domain (SUA), which is located between amino acids 220 and 237 in the human CD44 sequence (amino acids 224 and 239, respectively). between). This novel glycoprotein appears to be very important for cell matrix or cell-cell binding in the formation of metastases. Thus, preparation of this protein region (SUA) and elucidation of its characteristic features is one of the tasks of the present invention. Spleen cells that make up the modified • »φ: ...: anti-SUA antibodies to CD44 surface protein, *. * ·; was produced by immunizing mice with membrane-1 * rotins derived from BSp73ASML. They were fused to myeloma cells by the method of Köhler (1981) using polyethylene glycol to obtain stable cultures. Antibodies which specifically target SUA against this novel protein moiety are obtained by cloning and; ·. by selecting those cultures which produced antibodies that reacted with BSp73ASML but did not react remotely. colonies with the basic form BSp73AS, as well as with other non-tumor-producing rat cells. For other studies, a monoclonal antibody was selected that stains BSp73ASML cells with a particularly potent • * · mAbl.lASML (mAb = monoclonal antibody).

in the immunofluorescence assay and is designated • · 3 106129

In the Westernblot assay, protein hydrolysis of BSp73ASML cells can be determined by mAbl.1ASML in 4 protein bands with molecular weights of 120,000, 150,000, 180,000, and 200,000, whereas rat fibroblast cells and rat metastatic tumor cells did not produce significant tumor cells. reaction. It has not yet been possible to determine whether these size differences are due to different original amino acid sequences or to post-protein modifications of varying intensity. In any case, the antibody-recognized epitope is present in all 4 protein species, in the absence of comparison with proteins derived from non-metastatic BSp73AS cells or normal rat cells.

Isolation of cDNA sequences encoding surface protein SUA

The mAbl.lASML monoclonal antibody was used to find the cDNA sequences encoding SUA in a bacterial expression library. This library was constructed with polyA + RNA derived from BSp73ASML cells and pEX vector system V (Stanley & Luzio, 1984). These cDNA sequences

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products encoded by β-galactosidase fusion protein: **. The cDNA clone, pEX34 positive for monoclonal antibody 1.1ASML isolated in this manner, termed pEX34, comprises a 167 nucleotide cDNA sequence fragment. This cDNA fragment ♦ · · was then utilized to examine the larger cDNA library present in the vector pSP65, also obtained from BSp73ASML RNA. One of these clones isolated in this way, •? * * · * PM66, was then used, the so-called "primer" extension ·: ··: (starting oligonucleotide) and polymerase chain reaction (PCR) ·; ··; using the isolation of a full-length cDNA clone pMeta-1 *. a. Full length detection was obtained by primer extension ** * (3207 nucleotides). Collinearity with BSp73ASML tumor-derived RNA 4,106,129 was found by RNase and S1 protection analysis.

Isolation of cDNA sequences by bacterial expression and antibody recognition indicates that the antibody recognizes the SUA of a primary amino acid sequence surface protein.

The messenger RNA encoding SUA is expressed in BSp73ASML cells, but not in BSp73AS cells cDNA clones were generated three different sequence probes to detect specific messenger RNA molecules by hybridization. Probe A covers the cDNA region encoded by SUA (positions 941-1108). Probe B represents sequences between positions 1403 to 1572 and probe C comprises sequences extending from pMeta-1 to position 794 (Fig. 1). The poly A + RNA of BSp73 tumor cell lines was electrophoretically separated and analyzed by RNA transfer hybridization. Four of the non-metastatic cell lines do not contain RNA homologous to probe A, while the RNA molecules derived from metastatic tumor cells react strongly * · · · * . Probe A recognizes from this RNA preparation a heterogeneous mixture of RNA molecules of varying size in the range: 2.2-3.3 kb and one larger RNA species of 4.9 kb.

: The expression of specific membrane proteins recognized by the monoclonal antibody 1.1ASML exclusively in metastatic tumor cell variants appears to be due to the specific expression of the corresponding SUA-encoding messenger RNAs. Publn. Apparently, the complete cDNA clone pMeta-1, 3.2 kb, is not able to represent all sequences of this RNA species * '' * ·. It can represent only one species from a heterogeneous mixture of RNA molecules of different sizes. Koettimil-

B and C give the same hybridization pattern as probe A

• «| In BSp73ASML RNA separation, at least as far as can be observed within this heterogeneity, i.e., 106109, these RNA species comprise sequences that complement all three probes A, B, and C. Unlike probe A, probe B and C recognize also sent RNA species to metastases in non-forming cell lines. However, these RNA sizes are clearly different from the lengths present in BSp73ASML cells, as four distinct messenger RNA species can be detected with 1.5, 2.0, 2.9, and 4.3 kb messenger RNAs. Although these RNA species may be hidden in a heterogeneous mixture of RNA molecules derived from BSp73ASML cells, it is certain that they will not be present in the same amount in BSp73ASML cells. Crucially, RNA sequences complementing probe A appear to be completely absent from cells that do not form metastases. Thus, it may be conservatively concluded that the sequences of probes A, B, and C are present in the same RNA molecules in the case of metastatic tumor, as if the sequences of probe A had been extended to BC positive RNAs, as if this alternative had occurred only forming a cell line.

S1 nuclease and RNase protection assays were performed to demonstrate the co-linearity between RNA and »·» V 'cDNA and between RNA molecules derived from BSp73AS and »· · BSp73ASML cells. to analyze difference. Protected DNA or RNA fragments can be only shorter than the full length because the 5 'ends contain vector sequences that are unable to hybridize with RNA molecules derived from tumor cells. First, look at the 3'-side transition points of the transitional sekvensseis-residues with homology to the probe A sequences, - · · · * ... having homology with the probe B. Both techniques detect a single RNA species that is co-linear with probes over a wide range of BSp73AS cells. Of which, in the 5'-; ··: direction, a cDNA or RNA probe responsible for BSp73ASML cells /. obtained RNA sequences, differs from those derived from BSp73AS cells. only RNA. RNA molecules derived from BSp73ASML cells primarily contain sequences that protect larger fragments of Experiment 106129. The largest chunks correspond to the full length of the DNA or RNA fragments provided for protection analysis. Smaller pieces can also be shown. Because RNA transfer hybridizations have revealed a heterogeneous mixture of RNA molecules of different sizes, it is possible that these smaller protected fragments show RNA species that differ from cDNA at some other site, i.e., sites located between the previously indicated deviation point and the provided probes. . Also, these RNA species cannot be detected in BSp73AS cells.

Next, the 5 'flanking point, i.e. the transition from sequences that hybridize with probe C to sequences that hybridize with probe A, a sequence encoding SUA, will be considered. Analysis yields similar results for the 5 'breakpoint. RNAs derived from BSp73AS cells can protect the provided probes in only a small region. Longer pieces are protected by messenger RNAs derived from BSp73ASML cells. Namely, they are collinear over the entire length of the probes provided. Thus, it can be concluded that the cDNA clone pMeta-1 represents sequences that are accentuated in metastatic tumor cells * · * 'BSp73ASML. The 3 'and 51 regions are also found in RNA molecules derived from BSp73AS cells. The SUA encoding sequences whose transitions can be mapped by the above techniques indicate that an alternative * ··· · * · .. second extension mechanism must occur in RNA formation.

; 'T'. The 5 'and 3' breakpoints of the transition sites leading to the SUA sequences are shown in Figure 1 with arrows.

• · • »• · ·

Monoclonal antibody 1.1ASML recognizes glycoprotein II »• · · *. Another Serious Form of CD44 • · ·: · *: To obtain structural information from the surface protein, the sequence of all cloned cDNAs has been determined. Full length! The nucleotide sequence of clone pMeta-1 and the deduced amino acid sequences are shown in Figure 2. The full-length cDNA-7 106129 clone comprises 3207 nucleotides. The 31 terminal has a polyA terminus, two additional polyadenylation signals located at positions 2288 and 1743. The first ATG codon is located downstream of the consensus start sequence and opens a reading frame of 1509 nucleotides corresponding to 503 amino acids. As expected for the membrane protein, the first 21 amino acids are hydrophobic and represent a signal peptide. To date, no part of these sequences has been found in the databases. However, within the scope of the invention, sequence homology was observed compared to recently published data on the Homing receptor lymphocytes CD44 (or Pgp-1, respectively) (Idzerda et al., 1989; Goldstein et al., 1989; Stamenkovic et al., 1989; Nottenburg et al., 1989; Zhou et al., 1989). These homologies are strictly limited to the 5 'and 3' portions of the cDNA, including the unreaded regions, and end at the above-mentioned divergence points between the BSp73AS and BSp73ASML RNA sequences. The entire breakpoint (indicated by color coding in Figure 2), that is, the entire snippet-specific SUA coding sequence, is not present in the Pgp1 or CD44 sequences. Apparently, the colony specific glycoprotein is a variant of the CD44 glycoprotein. Namely, it consists of an extra * * * V * extra-cellular domain of 156 amino acids and thus an extended extracellular domain (minus * ·. '··· 21%) of 410 amino acids. amino acid signal peptide) compared to unchanged CD44 glycoprotein composed of 270 amino acids (also reduced by signal peptide). However, non-colonizing BSp73AS cells contain unmodified forms of this CD44 family. The cDNA sequences of these BSp73AS RNA-Mole molecules have also been cloned, and similarity. · · · With extra clones specific to metastases • * ♦ • · · *. outside the domain is addressed.

«» · CD44 expression and metastasis; ability to correlate • · ·

Within the scope of the invention, a second series of rat isogenic tumor cell lines, namely, 13762 NF tumor cell lines of the mammary carcinoma system (Neri et al., 1982), were investigated to determine whether altered CD44-Gly co-proteinAS to form metastases or more generally to form metastases. In this study, maternal-derived cell lines, i.e., MTPa, MTC, MTF7, and MTA cells (group 1) were compared with lymph node or lung metastases, i.e., MTLγ, MTLn2, MTLn3 cells (group 2). Group 1 cells express a substantially normal CD44 pattern, similar to RNA molecules derived from BSp73AS cells, when hybridized with probe B. In contrast, probe A detects a small amount of mixed RNA having a size of about 2.5 kb. On the other hand, Group 2 cells have a completely different RNA pattern. Both probes A and B hybridize to larger RNA species. These sizes are similar to the sizes indicated for BSp73ASML cells. This similarity is also confirmed by RNase and SI protection assays. Based on these results, it is concluded that alteration of the RNA extension pattern and expression of the CD44 variant is in some way related to metastasis formation, and that the resulting figure 1 * 1V 'in these metastatic mammary carcinoma e * · cells closely approximates the metastatic · The pattern described with the BSp73ASML cell line. The high molecular * antibody-recognized proteins correspond to • · · · · * · .. each of the large-molecule species of protein indicated. *) *. BSp73ASML extracts. From this, therefore, the metastatic expression of RNA species and large molecule proteins in metastatic colonies is also noted. In fact, in Group 1, i.e., the so-called parent cell lines, there is found * * * RNA that hybridizes with the probe A, or i * ϊ''1 sequence encoding SUA, and that a protein whose molecule - i weight is 100 00 0 Daltons, slight discoloration can be observed; antibody is considered to be due to the fact that the cells of group 1 * * (!) also have a low ability to form remote colons, unlike the original tumor cell line 9 106129 BSp73AS, which did not exhibit any metastatic behavior.

Monoclonal antibody 1.1ASML inhibits the formation of metastases in the rat

In a series of experiments to form metastases on cells of the tumor cell line BSp73ASML in isogenic rats, these cells were injected subcutaneously with the monoclonal antibody 1.1ASML intraperitoneally at different times every two to three days before and after administration of the tumor cells. This immunoassay also determined how the rat immune response to the injected antibody developed. Namely, the animals developed anti-mouse immunoglobulin antibodies and anti-idiotype antibodies. As a result of this series of experiments, 1.1ASML injections significantly retarded tumor adhesion and metastasis. The kinetics of this delay can be inferred that the antibody interferes with some of the primary processes involved in metastasis formation. This experiment demonstrates that the antibody-recognized protein structure located on the surface of metastatic V * -containing cells plays some role in the formation of metastasis * and that the therapeutic and diagnostic goals are realistic.

Isolation of a human sequence homologous to the SUA coding sequence of rat cDNAt ♦ ♦ · .. Of course, in the case of cultured human derived tumor cells, there is no obvious difference. it is possible to determine, in a manner implemented in the rat system, whether the cell lymphocytes still have metastatic properties; SIA. Immunodeficiency 11a based on experiments in mice. only very limited conclusions can be drawn about the ability to * * * * form metastases in man. For this reason, the relatively large number of tumor cell lines cultured already 10 106,129 long ago somewhere in the world had to be examined for the expression of sequences that could be detected in rat metastases within the scope of the present invention. One such tumor cell line was successfully found within the scope of the invention. It is derived from human large cell lung cancer and has been designated LCLC97. From this tumor cell line, three specific RNA species (sizes: 5.5, 3.4 and 2.8 kb) can be detected that behaved completely like the RNA molecules that could have been detected in rat metastatic tumor cell lines. globally. Namely, they hybridize to probe A as well as probes B and C, which means that these human RNA species are also broadly similar to the cDNA pMeta-1 clone (85%).

However, the monoclonal antibody 1.1ASML did not react with this tumor cell, i.e., the protein portion recognized by the antibody must distinguish, in terms of the antigenic determinants region, from the proteins present on the surface of the human tumor cell. Even the smallest deviations due to the high specificity of the antibodies are sufficient for non-response. This human tumor cell LCLC97 was used. ···. now just to build a cDNA library. Rat and Human • «·. ·. : Due to the high degree of sequence alignment, one cDNA clone having homology to probe A could be isolated.

"** The sequence of the human cDNA was determined. Figure 3 (a, b) shows the primary chain and the deduced amino acid sequence.

• · ·

It can be noted that rat and human sequences are similar over large areas. This human sequence, as well as the deduced amino acid sequence, are also the subject of the following patents.

. Performance Examples • »

Cells and Antibodies »·

The following cloned Bsp cell lines were used for the studies: BSp73 14ASML-1 and 10AS-7 maintained in cultures as described in Matzku et al., (1983); in addition to Neri. et al., (1982) mammary cancer cell lines.

BSp73 monoclonal antibodies directed against ASML membrane proteins were made by immunizing Balb / c mice. After the spleen cells from the immunized mouse were isolated, they were fused to Ag8 myeloma cells to obtain them in immortal form, using the method of Köhler (1981) to produce monoclonal antibodies. The hybridoma cells obtained in this manner were screened to find cells whose cell-produced antibody specifically targeted BSp73ASML cells, but not BSp73AS cells or normal rat fibroblast cells. The exact procedure is described in Matzku et al. (1989).

As desired, hybridoma cells producing specific monoclonal antibody (mAk) were allowed to proliferate in tissue culture and mAk secreted from the culture area was heavily concentrated by ammonium sulphate precipitation and assisted by column chromatography (protein A-Sepharose and Mon.). was found in this form in the accompanying studies. One of these antibodies is mAkl.LASML.

• · · ** ·· Immune luorescence • · i • ·

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··· *. * * To detect the modified CD44 molecule on the surface of various tumor cells, these cells were transferred to culture, washed with phosphate-buffered saline (PBS) and incubated with 1.lASML for 30 minutes at 40 ° C. temperature. As a secondary antibody to demonstrate binding. rabbit-linked rabbit anti-mouse IgG was used and observations were made under a fluorescence microscope.

Construction and Immuno Screening of 106129 cDNA Expression Libraries Oligo (dT) and hexanucleotides of different composition were used as primers for polyA + RNA from BSp73ASML cells, and the first strand of cDNA was synthesized by reverse transcription from AMV. The second strand of cDNA was prepared with E. coli DNA polymerase I RNase H and E. coli ligase, after which the ends of the double-stranded cDNA were repaired with T4 DNA polymerase. Vectors pEX1, 2 and 3 (Stanley and Luzio, 1984), which allow fusion of cDNA into three different reading frames, were opened.

SmaI restriction endonuclease and ligated with cDNA (T4 DNA ligase). Capable E. coli DH5 bacteria (pCI857) that produce a temperature-sensitive repressor are transfected with pEX cDNA and cultured on nylon filters.

The temperature-sensitive repressor RCI857 gene is located in plasmid pCI857, which is compatible with pEX plasmids. The β 1 promoter, which directs the synthesis of fusion proteins, does not operate at 28 ° C. When the temperature is raised to 42 ° C, the CI repressor is inactivated and the synthesis of β-galactosidase / ASML fusion proteins becomes very potent. Then the filters .'j *. heat-induced bacterial colonies on top of the dena- ···. turbo, then incubated in PBS solution containing 3% dry milk powder, lysozyme, · · * and DNase. Bacterial fusion proteins attached to these nylon filters and now incubated with mAkl.LASML • ·: **, the non-specifically bound mAk is washed away, whereupon binding is demonstrated using a secondary antibody. 125I-labeled rabbit anti-mouse IgG. After auto-: * ·· radiography, positive clones were isolated from the original bacterial filter and analyzed for their outline. One clone synthesized by a fusion protein • *. reacted specifically with l.LASML, was pEX34. The pEX plasmid contained in the bacterial clone comprises 167 nucleotides in length *. * · «CDNA encoded e.g. epitope (or antigenic ·; ··: minant) with which mAkl. 1ASML is specific.

106129 Isolation of full length cDNA mMeta-1 was then performed according to standard procedures.

Immunization of rats with mAkl.1ASML to BDX rats that are syngeneic to BSp73 tumor cells were injected subcutaneously or intraperitoneally with mAkl.LASML (linked to keyhole limpet mollusc hemocyanin) in complete Freund's excipient. The first injection (to the paw fat layer) was given 10, 7 and 3 days before injection of BSpASML cells, then the subsequent injections were given 3, 7, 11, 14 and 21 days thereafter. After 28 days, the rats are sacrificed, the various lymph nodes are separated, weighed and the macroscopically visible metastases in the lungs are counted.

Relationship between Expression of Modified CD44 Surface Proteins and Metabolic Rate of Metastasis

A second set of rat tumor cell lines derived from 13762NF mammary carcinoma (Neri et al., 1982) sought to determine whether expression of altered CD44 glucoproteins in the BSp73ASML cell line alone was studied. : a feature of whether this expression can be considered to be related to the ability to form metastases. In addition, cell lines derived from primary tumors V. "[MTPa, MTC, MTF7, and MTA (group 1)] were compared to cell lines derived from lymph node and • · · * · 'lung metastases [MTLy, MTLn2, MTLn3 (group 2). )] .The pattern of RNA from CD44 is shown in Fig. • '·· 4, with probes A, B and D corresponding to page 4 of this application and with the probes depicted in Fig. 1. All cells in Group 1 provide a probe. B using the normal CD44 image> By contrast, the cells in Group 2 produce a pattern different from this · · · RNA is larger than Group 1 RNA and it ·, * ·: corresponds to BSp73ASML RNA. are lost upon hybridization to probe D. The remainder of the figure shows similarity between both rat tumor systems.

14 106129

Also, the Group 2 RNA pattern obtained using probe A corresponds to that of BSp73ASML. Probe A does not hybridize to BSp73ASML-derived RNA, whereas Group 1 cells produce a small confused 2.5 kb RNA band. The protection assays performed with RNase and S1 nuclease also show structural similarity. Based on these results, the cleavage pattern and expression of modified CD44 RNA molecules undergoes a change in the formation of metastases.

Transfer of metastasis formation metastasis to non-metastatic BSp73AS cells by intermediate expression of pMeta-1

The relationship between expression of altered CD44 species and metastatic capacity in two rat tumor series suggests that these glycoproteins have a causal relationship to metastasis formation. To investigate this, pMeta-1 was transferred to BSp73AS cells, and then examined whether this alters the behavior of these cells. The complete coding region of pMeta-1 (Figure 2) was placed under the SV40 promoter, and this construct (Figure 5 in Figure 5) was co-seeded with PSV2neo in BSp73AS cells. At this time ···. single G418 resistant and pMe were obtained; colonies expressing ta-l. Figure 5 shows an RNA pattern obtained from two colonies. Hybridization of the · · · ** ·· probe A specific for modified CD44 shows a dominant copy of about 2.2 kb • · •] ', which corresponds to the smallest *. *' Size of the most common RNA copied in BSp73ASML cells. (Figure 5).

However, transformed cells contain approximately R * 10 times this RNA than BSp73ASML.

• · · • ♦ · • · · 't. Other orders of magnitude are found in one transfected. cell (BSp73ASML-pSVMetal-14), which may depend on the plasmid-d integration site. The pSV2neo simulation transfer clone (not shown) and the receiving BSp73AS cells do not contain ·; ·: RNA that would be complementary to probe A. To detect endogenous normal CD44 copies (without the extra is 106129 domain of pSVMeta-1) from the transfectants, the filter was exposed and hybridized to probe D. This portion of the unreaded 31 sequence is not present in the expression clone (see Figure 5). Probe D shows two major copies of 2.9 and 4.9 kb in the RNA of each transfectant (Fig. 5 right column), both in comparison with BSp73AS and also in BSp73AS-pSVneo, not shown.

Approximate quantification of the various compatible hybridizations shows that these transfectants express about 5-fold more modified CD44 RNA copied by the expression plasmid than endogenous gene copies.

Overexpressed cDNA is considered a protein. Each of the transfectants shown in Figure 5 synthesizes, by mAbl.1ASML, immunostainable proteins of apparently the same size, namely a 150 kDa major product and a 100 kDa weaker band. Because this cDNA sequence encodes a primary protein product of only 503 amino acids (corresponding to about 60,000 Daltons), all visible bands must present modified shapes. The 150 kDa band coincides with a modification of the modified CD44 ···. a stunted shape expressed by metastases; cell BSp73ASML. BSp73AS or simulated transplanted BSp73ASpSVneo cells do not comprise this protein. As with BSp73ASML cells, the epitope of cells expressed by transfectants is exposed on the cell surface.

• »· • · ·

To demonstrate that the expression of modified CD44 is sufficient to induce metastasis in BSp73AS cells, transfectants were injected into syngeneic BDX rats (spontaneous metastasis method). In earlier experiments, metastases formed tumor cells. BSp73ASML rested rapidly at the injection site and was completely dispersed about 10 days after injection (Matzku, 1984). All local tumors were therefore removed by dissection on day 10. All 16,106,129 BSp73ASML cell carriers and all animals injected with overexpressing transfectants had metastases to their lungs (Table 1). The formation of metastases occurred relatively quickly, 5 to 8 weeks after injection. Animals receiving BSp73AS cells or simulation transfectants after this time were completely healthy (except for surgery) and no metastases could be detected even after 5 months.

Despite the surprising similarity observed in the formation of intense metastases, some interesting differences are also observed. BSp73ASML cells reach lymph nodes in all animals and cause marked enlargement of the various nodes in the groin area and adjacent to the aorta (Table 1). The transfectant (BSp73AS-pSVMetal-14) causes lymph node enlargement in 3 out of 8 animals, although numerous metastases develop in the lungs of all animals (Table 1). The second transfectant (BSp73AS-pSV-metal-15) does not detect lymph node enlargement. Thus, these transfectants appear to be able to form colonies in the lungs without the obligatory growth phase in lymph nodes.

«· · '; The experiment of Table 1 further refers to another difference between BSp73AS transfectants and BSp73ASML cells. Individual lung metastases in the lung are macroscopically visible, whereas the metastases caused by BSp73ASML are small and numerous, but in a larger series of experiments on BSp73ASML cells (Reber et al., 1990) in 11 animals • * ·· 20 develop 5-20 larger colonies per lung compared to trans::: fectants.

«·. In order to verify that the generated far-flap fragments are due to the injected transfectants, a spontaneous: / ·· mutation that gives rise to metastasis, v: an unlikely possibility, thus excluding, epitope-positive proteins was determined from whole extracts from cells. The 150 kDa glycoprotein can be detected in whole lung extract as well as in specific pulmonary extracts of animal that had received BSpAS-pSVMetal-15 transfectants. The G418-resistant strain expresses a protein having this same molecular weight during in vitro growth.

Diagnosis and Treatment 1. Human tumor material is analyzed by in situ hybridization to existing human pMeta-1 sequences. These experiments are intended as pre-tests before the human SUA recognizes the antibody.

2. Preparation of Antibodies to Human SUA. Human pMeta-1 sequences are cloned into bacterial expression vectors to generate fusions of β-galactosidase or tryptophan E product. Rabbits are immunized with these fusion proteins or synthesized peptides derived from SUA (linked to carrier molecules:). Polyvalent or monospecific antibodies ···. isolated.

Uses: • Immunohistological examination of clinical tumor material • · * * (diagnostics); - detection of soluble SUA in patient serum by ELISA assays (diagnostics); : - generation of toxin-coupled antibodies to deliver toxin by tumor or metastasis. keen area (treatment); . - generation of antibodies with two specific antigen binding sites. This dual specificity is intended to trigger cytotoxic reactions in the metastatic region (e.g., anti-CD2 or CD3 coupling) (treatment).

106129 3. Preparation of hMeta-1 by transfecting human or rat cells with an expression vector comprising the complete hMeta-1 cDNA sequence; or purification from LCLC97 cells.

Applications: - Injection of a protein or portion thereof to block tissue binding sites of tumor cells; After characterization of the binding sites, it would also be conceivable to use in therapeutic applications based on the injection of large amounts of binding protein to block mobile tumor cells.

Bibliography:

Goldstein, L.A., Zhou, D.F.H., Picker, L.J., and Minty, C.N.

Bargatze, R.F., Ding, J.F. and Butcher, E.C. (1989) The human lymphocyte homing receptor, the Hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell 56:. ··. 1063-1072.

««

«4 I

,:. Hart, I. R., Goode, N. T. and Wilson, R. E. (1989) Aspects of the Molecular '.I' * of the Metastatic Cascade. Biochim. Biophys. Acta 989: h. '65-84.

• · · • · ·

Drink, R. L., Carter, W. G., Nottenburg, C., Wayner, E. A., • ·

! *** Gallatin, W. M. and St. John, T. (1989) Isolation and DNA

• · · · Sequence of a cDNA Clone encoding a lymphocyte adhesion receptor for high endothelium. Proc. Natl. Acad. Sci. U.S.

t »,,,, r 86: 4659-4663.

t Köhler, G. (1981) In: I. Lefkovits and B. Pernis (eds).

'> "Immunological Methods. Vol. 2 pp. 285. N.Y. Academic Press.

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Matzku, S., Komitowski, Mildenberger and Zöller, M. (1983) Characterization of Bsp 73, a spontaneous rat tumor and its in vivo selected variant showing different metastatic capacities. Inv. Met. 3: 109-123.

Matzku, S., Wenzel, A., Liu, S. and Zöller, M. (1989). Antigenic differences between metastatic and nonmetastatic BSp73 rat tumor variant characterized by Monoclonal antibodies. Cancer Res. 49: 1294-1299.

Neri, A., Welch, D., Kawaguchi, T. and Nicolson, G. L. (1982) Development and biological properties of malignant cell sublines and clones of spontaneously metastatic rat mammary adenocarcinoma. J. Natl. Cancer Inst. 68: 507-517.

Nicolson, G. L. (1987) Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res. 47: 1473-1487.

Nottenburg, C., Rees, G. and St. John, T. (1989) Isolation of mouse CD44 c DNA: structural features are distinct from the • · ·. ···. primate cDNA. Proc. Natl. Acad. Sci. U.S. 86: 8521-8525.

4 4 · • 4 4

Stamenkovic, I., Amiot, M., Pesando, J. M. and Seed, B. (1989) • · · ♦ **** A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell 56:; 1057-1062.

• · • '·· Stanley K.K. and Luzio, J.P. (1984), Construction of a new • · *: /: family of high efficiency bacterial expression vectors: ^ *, 5 identification of cDNA clones coding for human liver proteins.

• · i {_. EMBO J. 3: 1429-1434.

'·, <; Wenzel, A. (1986) Characterization of Dif ferenzierungs- (Antigenic Auf dem Rattentumor Bsp 73 mit Hilfe Monoclonal Antibody), Master's Thesis, University of Karlsruhe.

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Zhou, DFH, Ding, JF, Picker, LF, Bargatze, RF, Butcher, EC and Goeddel, DV (1989) Molecular Cloning and Expression of Pgp-1 - The Mouse Homolog of the Human H-CAM (Hermes) Lymphocyte Homing Receptor . J. Immunol. 143: 3390-3395.

table 1

Metastatic proliferation of BSp73AS cells expressing modified CD44 cDNA pMeta-1 ***

Local Dispersion in Finding Remote Tumor Colonies by Autopsy Clone_tvminen_LN * inq_LN * par_Lung BSp73ASML 0/8 8/8 8/8 8/8 φ 1.5-2.5 ** φ 2.2-5.0 Granular BSp73AS- 0/8 3/8 3/8 8/8 pSVMetal-14 φ 0.3-1.2 φ 1.0-4.5 multiples.

φ 0.3-5.0 BSp73AS- 0/8 0/8 0/8 8/8, 5-20 pSVMetal-15 φ 0.3-10.0 BSp73AS 1/8 0/8 0/8 0/8 • «« * ·. *. ' BSp73AS - 0/8 0/8 0/8 0/8 ^ / pSVneo • LN = Lymph Nodes ** Average Diameter in mm ·: After injection of cells.

• · * · • • • • • • • • • * • ♦ • «« «« • «« • <«iti I«

Claims (8)

106129 A method for preparing a polypeptide comprising amino acid sequences. . . .-R .. i protein in IATTPWVSAHTKQNQERTQWNPIHSNPEVL LQTTTRMTDIDRNSTSAHGENWTQEPQPPF NNHEYQDEEETPHATSTTWADPNSTTEEAA TQKSKWFENEWQGKNPPTPSEDSHVTEGTT AS A HNNHPSQR M TTQSQZDVS λ TDFFDP I S F F M Q G G H Q S H E Γ and h-protein: I p I S S T T T? R A F D H T K Q N Q D W T Q W N? S r. S N? E V L L Q T T T R M T D V D R N! G T T A Y Ξ G N W N? Ξ A J ·] * H? ? L I H H E H H E E E E T? H S? S T I Q A T? S S T T \ .. Ξ Ξ T A T Q K E Q vv F G N R W H I G Y R Q Γ? R E G S H S T T G T A A S A H T S H? M Q G R T T? Ξ P Ξ D S Ξ W 7 G F F N F I S H F M G R G H Q A G R ?. as well as allelic variants thereof, and glycosylation products, which are part of metastatic tumor cells:: a surface protein, characterized in that the DNA fragment encoding this h_ polypeptide is selected from 22 1 06 1 29 a) nucleotide sequences ... rMeta- 1 ... ATT GCA ACT ACT CCA TGG GTT TCT GCC CAC ACA AAA CAG AAC CAG GAA CGG ACC CAG TGG AAC CCG ATC CAT TCA AAC CCA GAA GTA CTA CTT CAG ACA ACC ACC AGG ATG ACT GAT ATA GAC AGA AAC AGC ACC AGT GCT CAT GGA GAA AAC TGG ACC CAG GAA CCA CAG CCT CCT TTC AAT CAT GAG TAT CAG GAT GAA GAG GAG ACC CCA CAT GCT ACA AGC ACA ACC TGG GCA GAT CCT AAT AGC ACA ACA GAA GAA GCA GCT ACC CAG AAG TG TG TTT GAG AAT GAA TGG CAG GGG AAG AAC CCA CCC ACC CCA AGT GAA GAC TCC CAT GTG ACA GAA GGG ACA ACT - GCC TCA GCC CAC AAC AAC CAT CCA AGT CAA AGA AGT ACA ACA CAG AGT CAA GAG GAT GTT TCA TGG ACC GAT T TTC GAC CCA ATC TCA CAT CCA ATG GGA CAA and hMe ta-1. . ; AAC CCA AGC CATTCA AAT CCG GAA GTG CTA CTT CAG ACA ACC ACA AGG ATG CAT GAT GTA GAC AGA AAT GGC ACC ACT GCT TAT • «. ··. GAA GGA AAC TGG AAC CCA GAA GCA CAC CCT CCC CTC ATT CAC CAT GAG CAT CAT GAG GAA GAA GAG. ACC CCA CAT TCT ACA AGC '/ ACA ATC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ;; · ACC CAG AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG- GGA «·: *' TAT CGC CAA ACA CCC; AGA GAA GAC TCC CAT TCG ACA ACA GGG • · · * AL.-. OW 1 U'w.-. VJV..W. wA OL * AL, AGv_ C-Λ- LCA Λ-VJ LA. ···. b) nucleotide sequences which are degenerate in the kchdas) ·· .β a) and / or are allelic variants of • · *! * and are inserted under the promoter. .J. genes of viable cells which are cultured and propagated in a known manner and the polypeptide is isolated from cultured and / or proliferated cells. 106129 2. A method according to claim 1, characterized in that nucleotide sequences are selected which hybridize to the nucleotide sequence of any one of claim 1 a) and encode a complete surface glycoprotein of metastatic tumor cells. A method according to claim 2, characterized in that the sequence hybridizing to the nucleotide sequence of claim 1 is at least 85% homologous to the reaction partner. 4. The method of claims 1-3, wherein the DNA fragment of claim 1, 2 or 3 is inserted into a vector-derived nucleotide sequence to form a recombinant DNA molecule. 5. The method of claim 4, wherein the recombinant DNA molecule is an expression vector. A method according to claims 4-5, characterized in that the polypeptide is expressed in a host cell which: is transformed with or contains the recombinant DNA molecule of claims 4 and 5. 1, 2 or 3. * · · * • «•« «· · 7. The method produces multivalent and monovalent antibodies which are reactive with the epitope of the polypeptides produced by the method of claims 1-5, characterized in that they are obtained by immunization. suitable host animals with an epitope of any of the polypeptides of claims 1-6, and the antibody formed in the blood of the animals is isolated or the antibodies are produced by a hybridoma technique known per se. Use of an antibody produced by the method of claim 7 for the identification, preparation or isolation of the polypeptide of claims 1-6. • • * «» »· t t t • • • • f ((f (((((((((((((((((((((((((((((((((((((((((((((((((((((((((((