JPH07316068A - Tumor suppressor-fused protein - Google Patents

Abstract

PURPOSE: To obtain the subject protein releasing a tumor or cancer suppressor transfer factor into the cell of mammal, modifying the cell-controlling activity relevant to Rb gene and useful for suppressing the growth of tumor. CONSTITUTION: This soluble molecular conjugate contains a ligand fused to a cell membrane receptor of mammals, inducing a cancer suppressor transfer factor to the cell of mammals and reversibly bonding to the transfer factor. The transfer factor is preferably an RB agonist polypeptide (e.g. a lowmolecular derivative of P/10RB). The ligand is preferably bonded specifically to a COOH receptor, etc., of the cell membrane.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a soluble molecular complex that induces a cancer suppressor transcription factor in mammalian cells,
And a pharmaceutical composition containing the complex.

[0002]

2. Description of the Related Art Retinoblastoma sensitivity
The susceptibility gene (Rb) has been cloned and studied in detail (Brugge, J., et al., "Retinoblastoma gene: a basic model for tumor suppression," Orig.
ins of Human Cancer: A Comprehensive Review ； 1
Vol., 1st edition, pp. 413-422 (Cold Spring Harbor)). The deletion of the functional RB protein derived from Rb (or simply abbreviated as RB) seems to be involved in the development of many cancers (of human origin). These cancers include retinoblastoma,
Bladder cancer, breast cancer, chronic myelogenous leukemia, acute myelogenous leukemia,
Testicular cancer, dysplastic and cancerous ulcerative colitis, sporadic sarcoma, prostate cancer, osteosarcoma, small cell lung cancer, and synovial sarcoma are included. The diverse origins of these cancers (or neoplasms, especially what are called tumors) are due to the hyperproliferative disease (hype
rproliferative diseases), especially in human cancer,
It shows the importance and broad role of Rb.

The mechanism of RB that regulates the cell cycle is
It may explain an important role in tumorigenesis. The RB protein has been found to bind many cellular proteins, such as E2F, c-myc, N-myc, and cyclins (D class). E2F is a transcription factor that binds to many promoter parts upstream of a gene encoding a product having a function that promotes cell cycle progression. The formation of the RB protein-E2F complex can influence the transcription level of genes involved in the cell cycle,
And it can regulate cell cycle progression in the negative direction.

Many DNA tumor virus-derived transforming proteins (oncoproteins) bind or chelate with a pool of RB proteins in cells. This provides one means of deregulating cell growth.
The above proteins include adenovirus E1A, simian virus 40T antigen, and human papillomavirus E.
7 proteins are included. These transforming proteins share the ability to disrupt the interaction of the transcription factor E2F with the dephosphorylated or hypophosphorylated forms of the RB protein and to stimulate cell growth. Thus, Rb may have an important role in human papillomavirus-induced genital wart development and cervical metaplasia progression.

Rb-negative cell line WERI-Rb
27 (retinoblastoma), Saos-2 (osteosarcoma), DU
Replacement of functional Rb genes in 145 (prostatic cancer), and HTB9 (bladder cancer), with altered characteristics, including morphology, growth grade, soft agar colonization, and tumor formation in nude mice. Results in complete or partial suppression of. Defective p56 ^RB (amino acid 388 from start
Tumor cell binding pocket (domain A, positions 393-572; domain B), which is observed to retain the growth-inhibitory activity of full-length RB protein after microinjection into cells of the Saos-2 system. has a C-terminal domain that contains the position 646~772) (Goodgrich, DW et al., supra; Huang, H.-JS et al.,
"Repression of neoplastic phenotypes by replacement of the Rb gene in human cancer cells," Science , 242: 1563-66 (1988)).

However, single cell microinjection of tumor cells is not a practical method for treating cancer. Each tumor has many cells and tumor metastases are usually present. Therefore, a method that can be a systemic treatment is important. From the above viewpoint, effectively and accurately, materials and methods for delivering RB protein as described above p56 ^RB into target cells is desired.

[0007]

The present invention is to solve the above-mentioned conventional problems, and its object is to provide a soluble molecular complex for inducing a tumor or cancer suppressor transcription factor in a mammalian cell and a soluble molecular complex thereof. It is intended to provide a pharmaceutical composition comprising the same, and a method of regulating cell cycle progression of tumor cells using the complex or composition.

[0008]

The present inventors can introduce a tumor or cancer suppressor transcription factor into a tumor cell by contacting with the cell, and the introduced tumor or cancer suppressor transcription factor is a cell of the tumor cell. The present invention has been completed by creating a soluble molecular complex containing a substance that regulates cycle progression and suppresses cancer or tumor growth as a constituent element.

The soluble molecule complex of the present invention contains a ligand fusion for a mammalian cell membrane receptor that induces a cancer suppressor transcription factor in mammalian cells and reversibly binds to the cancer suppressor transcription factor. This achieves the above object. The term "mammalian cell" refers to cells of human or non-human mammalian origin.

The present invention also provides a pharmaceutical composition containing the above complex and a pharmaceutically acceptable carrier. The carrier includes hydrophilic polymers such as polyethylene glycol and dextran.

The method for modifying the cell regulatory activity of the present invention for modifying the cell regulatory activity of the Rb gene includes the step of bringing the soluble molecular complex into contact with cells. As a result, the cytoregulatory activity of the contacted cells may be modified,
The above object is achieved.

The present invention also uses the above pharmaceutical composition,
Methods of treating pathological conditions caused by dysfunction of the Rb gene or alteration of the Rb gene product are provided. The method comprises administering to the subject an amount of the pharmaceutical composition effective to restore the cytoregulatory function of the Rb gene or its protein product. This achieves the above object.

As used herein, the term "tumor or cancer suppressor transcription factor" refers to RB or p5
6 ^RB refers to a protein that exhibits the biological activity of soluble retinoblastoma protein and acts to suppress the growth of cancer or tumor. The term is intended to refer to both the intact native protein and fragments thereof, or to modifications or derivatives that retain the tumor suppressor activity of the native protein. The term is also intended to refer to a natural or synthetic protein that has a nuclear localization signal. For example, nuclear localization signals present on p56 ^RB well direct nuclear transport. Preferred cancer suppressor transcription factors of the present invention are RB proteins and RB agonist polypeptides, eg soluble retinoblastoma proteins. Most preferred transcription factors are p110 ^RB , p56 ^RB , and their derivatives described herein, and fusion proteins (PEA56.
RBD0, PEA56RBD1, PEA56RBD2
S, and FID56RB).

As used herein, “cytoregulatory activity”
Alternatively, the term "cell regulatory function" refers to an activity capable of regulating the cell cycle involving the above-mentioned Rb gene and its gene product RB protein in tumorigenesis, and preferably regulating the cell cycle progression of tumor tissue in the negative direction. Or means a function.

The present invention provides a ligand for a cell membrane receptor which induces endocytosis and leads to a tumor suppressor transcription factor. In order for the tumor suppressor transcription factor to escape into the cytoplasm of the cell, this ligand must be reversibly bound to the tumor suppressor transcription factor. As used herein, “reversibly bind” is intended to refer to an extracellular stable bond that is cleavable under suitable conditions intracellularly to release the tumor suppressor transcription factor intracellularly. There is. For example, bond cleavage can be pH or enzymatically regulated. Preferred ligands are Pseudomonas exotoxin A and interferon alpha. As used herein, "ligand for cell membrane receptors" refers to molecules that bind to receptors located on the cell surface. Such receptors can be tissue-specific, or ubiquitous, as present on many or all cell surfaces. Cell membrane receptors all provide -COOH binding sites for ligands. The receptor also contains an asialoglycoprotein for the ligand. Preferred asialo-glycoproteins are asialo-GM1 and asialo-GM2.
Examples of said receptors are GIP colon cell receptor, bombasin growth factor receptor on lung cells,
And ferretin.

We have tested two ligands: Pseudomonas exotoxin A (PE) and interferon alpha (Wick, MJ et al., "Analysis of Pseudomonas aeruginosa exotoxin A structure-function relationships", Mol. Micro. , 4: 527-535 (1990); Rubinstein, M. et al.,
"Interferon receptor", Crit. Rev. Biochem. ,
21: 249-275 (1986); Wileman, T., et al., "Endocytosis Involving Receptors", Biochem. , 232 (1) 1-14.
(1985)); Yonehara, S. et al., "Internalization of interferon involving cell surface receptors: Correlation of interferon with antiviral activity", J. Gen. Viro.
l. , 64: 2409-2418 (1983); Arnheiter, H. et al., "Microinjection of anti-interferon antibodies into cells does not inhibit the induction of antiviral states by interferons," J. Virol. , 52 (1): 284-07 (19
84)). Exotoxin A domain 1 is important for cell surface recognition and for receptor binding, whereas domain 2 is required for export out of endosomes (Liu, PV " The role of its various functions in the pathogenesis of Pseudomonas aeruginosa 3. The identity of lethal toxins produced in vivo and in vitro ”, Infect. Dis. , 116 (4): 481-9 (1966).

Pseudomonas aeruginosa exotoxin A is a secreted bacterial toxin with the ability to covalently modify specific target proteins in mammalian cells. Analysis of the identified domains indicates that the amino-terminal domain (domain 1) is involved in what is recognized as a eukaryotic target cell. The central domain (domain 2) is the exotoxin A within the periplasm of E. coli.
Involved in the secretion of. Domain 2 further acts on the transport of toxin A from eukaryotic endosomes, including exotoxin A after internalization in susceptible eukaryotic cells via receptor-mediated endocytosis. The carboxy-terminal portion of Exotoxin A (domain 3) encodes the enzymatic (toxin) activity of the molecule.

Exotoxin A enters into eukaryotic cells via endocytosis involving the receptor
Internalizes in clathrin-coated pits and enters endosomes (Saelinger, CB et al.
"Intracellular movement of Pseudomonas exotoxin A" Anti
biot.Chemo. , 39: 149-59 (1987)). Exotoxin A
Receptors are species-specific, particularly glycosphingolipid-asialo-GM1 and asialo-GM2
GM2, which may function as a receptor on the cell surface,
GM3 is not functional (Krivan, HC et al., “Carbohydrate sequence found in several glycolipids, GalNAc beta 1-4Gal
Many lung pathogenic bacteria that specifically bind to'Pro
c.Natl.Acad.Sci.USA , 85 (16): 6157-61 (1988)).

The fusion protein of the present invention is constructed so as to contain the partial domain 1, complete domain 1, or both domains 1 and 2 of the above exotoxin A. Both PE and interferon alpha ligands
Some of the N-terminal constructed as fusions with deleted p56 ^RB, and was produced in E. coli. Importantly, p56 ^RB lacking a portion of the N-terminus, unlike p110 ^RB , has no activity on its own when co-incubated with tumor cells. The highly constructed fusion protein was purified and tested. Unlike the unmodified p56 ^RB , the p56 ^RB fusion protein has T antigen binding activity and the tumor growth suppressive function of full-length p110 ^RB . It is also shown in the present invention that the full length unmodified RB protein has growth inhibitory activity under pharmaceutically acceptable conditions.

Using the properties of Exotoxin A, three different fusion proteins were synthesized in Example 1 and FIG. 1 (PEA).
56RBD0 and PEA56RBD1), FIG. 2 (PE
A56RBD2S). P
Both JH8, PJH14, and Pseudomonas aeruginosa strains are from ATCC (Rockville, MD).
Plasmid 44-2 and E. coli BL21 (pLysS
Was provided by Dr. Wen Hwa Lee. Plasmids pflag (IBI, CT) and DH5α competent cells (BRL, Bethsda, MD) were obtained from the manufacturer. Figure 3 summarizes the final construction of different PEp56 ^RB fusion proteins. In the figure, aa represents an amino acid residue. Pseudomonas exotoxin (P
Domains IA, II, IB, and III of E) are each a region that binds to the membrane surface (indispensable for amino acid residues 55 to 80), a region involved in transport, a region that enhances the stability of the recombinant peptide, and an ADP ribosyl transferase. A region having (toxin) activity. P56 ^RB in the upper right of the figure is a C-terminal fragment (involved in nuclear localization) having the tumor protein binding pocket (indicated by a box in the figure). From the above both fragments, the three fusion proteins at the bottom of the figure, D0-p56 ^RB , D1-p56
^RB and D2-p56 ^RB were constructed. The characteristics of each fusion protein differ depending on the region of the fused PE.
For example, D0-p56 ^RB is cell-bound and receptor-focused endocytosis, D1-p56 ^RB is cell-binding-endocytosis, and D2-p56.
^RB is expected for cell binding-endocytosis and transport.

Another ligand tested in the present invention is
Interferon α. Also, interferon α
Uptake into cells is by receptor-mediated endocytosis. The biological activities of interferon alpha have been extensively studied and include cell growth inhibition, induction of antiviral states and modulation of immune responses.

Interferon alpha has been used clinically for the treatment of hairy cell leukemia and condyle congenitalis (genital warts, or genital warts). Genital warts and cervical cancer are associated with human papillomavirus. Two viral genes, E6 and E7,
Encodes an oncoprotein that effectively immortalizes human first squamous cells (Banks, L. et al., “HPV1
The ability of the 6E7 protein to bind to RB and induce DNA synthesis is not sufficient for effective transforming activity in NIH3T3 cells. " Oncogene , 5: 1383-1389 (199).
0); Heck, DV et al., "The efficiency of binding to the retinoblastoma protein correlates with the transforming ability of the papillomavirus E7 oncoprotein" Proc. Natl. Acad. Sci USA ,
89: 4442-4446 (1992); Stirdivant, SM et al., "Human papillomavirus type 16E7 protein inhibits binding to DNA by the gene product of retinoblastoma," Mol. Cell.
Biol. , 12: 1905-1914 (1992); Huang, S. et al., "Cellular proteins that compete with the SV40 T antigen for binding to the retinoblastoma gene product," Nature , 350: 160 (1991)). The E7 protein binds to the RB protein in the C-terminal region and is required for viral replication. The interferon-RB fusion protein constructed in the present invention provides synergistic antiviral and tumor growth inhibitory effects,
And for this reason, it provides a higher therapeutic effect. Interferon α-exotoxin A-domain 2-p56
Construction of the ^RB fusion protein (FID56RB) is described in detail in Example 1, and FIG. 5 is its construction scheme. FIG. 6 summarizes the final construction of the different PEp56 ^RB fusion proteins. Aa in FIG. 6 represents an amino acid residue. Interferon-α8 in the upper left of the figure is a fragment involved in receptor binding and endocytosis. Each domain of p56 ^RB and Pseudomonas exotoxin (PE) in the drawing is the same meaning as above-described FIG. From these fragments, the fusion protein at the bottom of the figure was constructed.

The effects of therapeutically active proteins and peptides are often limited by proteolysis, solubility, and susceptibility to immunogenicity. An example of a method for stabilizing such proteins is the modification of protein covalent bonds with hydrophilic polymers such as polyethylene glycol (PEG) or dextran. Such binding has been shown to increase solubility, reduce proteolytic and immunoantigenic susceptibility, and prolong the lifespan of proteins in circulation in vivo (Beckman, JS et al . J. Biol. Chem. , 263 (14):
6884 (1988)). The fusion proteins of the invention are intended to be used with or without such modifications.

It is understood that modifications which do not substantially affect the activity of the various molecules of the invention are also included in the definition of the molecule.

The soluble molecular complex of the present invention requires a step of contacting with a target cell in order to modify the cytoregulatory activity of a dysfunctional Rb gene or an altered RB in tumor cells. This contacting means can be performed in vitro by adding the complex under various cell and tissue culture conditions that can be usually performed, and in vivo, the complex can be treated by various clinical methods. Can be delivered to tumor cells or tissues, generally by intravenous injection.

From the above detailed description, the soluble molecular complex of the present invention and a pharmaceutical composition containing the same can be applied to a subject having a pathological condition caused by dysfunction of the Rb gene or alteration of the Rb gene product (RB). It is understood that this pathological condition can be treated by administering an agent. The pathological conditions are retinoblastoma, bladder cancer, breast cancer, chronic myelogenous leukemia, acute myelogenous leukemia, testicular cancer, dysplastic and cancerous ulcerative colitis, sporadic sarcoma, prostate cancer, Examples include, but are not limited to, osteosarcoma, small cell lung cancer, synovial sarcoma, and other malignant diseases or hyperproliferative diseases characterized by dysfunction of the Rb gene or alteration of the Rb gene product. To do.

The following examples are for the purpose of specifically illustrating the present invention and are not intended to limit the present invention.

[0028]

The present invention will be described with reference to the following examples.

Example 1 Construction of Tumor Suppressor Fusion Protein The primers used to construct the plasmid for producing the fusion protein are shown in Table 1 below:

[0030]

[Table 1]

The PCR reaction was performed under the conditions shown in Table 2 below:

[0032]

[Table 2]

Plasmid 44-2Rb containing the Rb gene cut with AvaI and NcoI was separated on a 1% agarose gel and the large fragment was Genecl.
Purified by the ean (Bio101, San Diego, CA) method. A PCR fragment from 44-2Rb was generated with primers (1) and (3), and AvaI and NcoI
It was cleaved with, purified and ligated to the above large fragment to form 44-256Rb. AvaI-B
PCR NdeI- from pJH14 with the amHI56Rb fragment and primers (5) and (7).
AvaI fragment and purified pJH8 Bam
Ligated to HI-NdeI vector, pEA56RBD1
Was produced. After cutting the KpnI-KpnI fragment, pEA56RBD0 was generated by self-ligation. The above pEA56RBD1 and pEA56RBD0
The construction of is shown schematically in FIG.

Pseudomonas aeruginosa DNA (p
e) was incubated with proteinase K overnight at 55 ° C.
It was produced by digestion with and then phenol / chloroform extraction and alcohol precipitation. NdeI
-HindIII fragment (SD1D2) was prepared by PCR of pe with primers (4) and (6). The HindIII-NcoI 56Rb 5'fragment is 44-2Rb with primers (2) and (3).
Was PCR. These two fragments are Nd
3'5 generated by purifying the pEA56RBD1 large fragment cut with eI and NcoI
Ligated to a vector containing 6Rb, pEA56RBD
2S was formed. Construction of the above pEA56RBD2S
A schematic diagram is shown in FIG.

The HindIII-AvaI interferon fragment was added to the primers (8) and (9) with P
Purified from PCR of cgs261. AvaI-Ava
I (XhoI) was run on a 1% agarose gel and pEA5 cut with AVaI using "Geneclean".
Purified from 6RBD2S. flag1 vector to A
Flat 1 (I cut with vaI and HindIII
BI). After joining the three pieces, FID56RB was formed. The construction of the FID56RB is shown as a schematic diagram in FIG.

Example 2 <Fusion protein PEA56RBD0, D1, D2S,
Expression and purification of FID56RB> Rosenberg BL2
1 (DE3) pLysS host organism (Rosenberg, A.H. et al.
Of the cloned DNA by T7 RNA polymerase.
Vector for Selective Expression "Gene, 56 (1): 125〜35 (19
87)) based on PEA56RBD0, D1, D2S
After transformation with rasmid, protein production
Induced by propyl-β-D-thiogalactopyranoside
Could be guided. For FID56RB, stay at DH5α
Used mainly. 100μ from the master seed bank
g / mL ampicillin and 20 μg / mL chlora
Bacteria were transferred directly to LB medium containing mufenicol.
Inoculate and prepare overnight cultures of bacteria
It was 1 at 30 ° C in New Brunswick Scientific shaker
OD between 2 and 3 after 5 hours of culture ₆₀₀Achieve the
The total volume of the flask is used to collect 3.4 L of the 5 L fermentor.
The medium was inoculated. Fermenter temperature 28 ℃, pH 6.
9, aeration 0.5 L / min, and rotation 300 rpm
Set. OD of this culture₆₀₀Reaches 7-8,
Induction of IPTG (final concentration of 0.2 mM)
Was further supplemented with LB medium and glucose. Induce
After 3 hours, collect the bacteria, pellet,
Frozen to -80 ° C until use.

Microfluidizer model M1
10 mM phosphate, pH 7.5, 10 mM,
Cell homogenates were prepared at 10,000 psig in lysis buffer containing 1 mM EDTA and 1 mM PMSF. The resulting microfluidic cell lysate is incubated at 4 ° C for 2
It was spun at 10,000 rpm for 5 minutes in a JA-10 rotor. By Western blot analysis using the fusion protein ∞-Rb monoclonal antibody 318,
It was shown that each of those proteins had the correct molecular weight and specifically reacted with the Rb moiety at their C-terminus, which is shown in FIG. Lanes M at both ends in FIG. 7 show molecular weight markers, lane 2 shows PEA56RBD1, and lane 4 shows PEA56RBD0.

(Example 3) <Purification and biological activity of D0 and D1 fusion proteins> In D2 and D1, in consideration of the wet cell weight (gram), 2 volumes (mL) in lysis buffer Resuspend the above pellet in 25 ml and re-suspend in a JA-10 rotor.
Spin for minutes. Therefore, the double volume is related to the volume of this lysis buffer. This washing procedure was repeated 3 times. The remaining pellet was agitated in 2 volumes of lysis buffer containing 0.1% Tween-80 at 4 ° C. for 1½ hours to 2 hours,
Then, it was centrifuged again with a JA-10 rotor.

After the washing step with 0.1% Tween-80, 4
The pellet was resuspended in 2 volumes of lysis buffer containing M urea. The mixture was stirred at 4 ° C. for at least one and a half hours and then centrifuged on a JA-10 rotor as above.

The pellet was resuspended in 2 volumes of lysis buffer containing 8M urea and agitated for at least 11/2 hours to 2 hours at 4 ° C. When the resuspended material was centrifuged again as above, the supernatant contained the partially purified fusion protein.

The denatured protein solution was adjusted to 10 m at pH 7.5.
Regeneration was done by dropping into a rapidly stirring flask containing 8 to 40 volumes of M phosphate + 150 mM NaCl (PBS). The resulting protein solution was concentrated and the buffer was replaced with another PBS with further reduced urea and PMSF concentrations.

The purity of the protein obtained by this method was estimated to be 80 to 85% by Coomassie blue staining of a reducing SDS-PAGE gel.
FIG. 8 (A: lane 1; molecular weight standard, lane 2; D0P)
E-RB, and B: lane 1; molecular weight standard, lane 2; D1 PE-RB) show SDS-PAGE gel analysis of purified D0 and D1 proteins. ∞
Western blot analysis with Rb Mab 3C8 supports the identity of these proteins (Figure 8,
C: Lane 1; molecular weight standard, Lane 2; D0 PE-R
B and D: Lane 1; molecular weight standard, Lane 2; D1
PE-RB).

(Example 4) <T antigen binding property of PEA56RBD0> The ability of the purified fusion protein to bind to the purified viral tumor protein SV40 T antigen (Tag) was determined by the modified ELI of Wen et al.
It measured according to SA method. Briefly, each well of a 96-well microtiter plate was
Coated with g of purified Tag overnight. (10m
After blocking with BSA (in g / ml PBS), various concentrations (2 fold dilution from 2 μg / ml to 0.016 μg / ml) of purified PEA56RBD0 fusion protein were added to each well. Wells were washed and Tag bound by standard dye reaction with biotinylated Mab 3C8, 1: 2500 dilution of Streptavidin-POD, (Boehringer Mannheim GmbH), and ABTS (Sigma, St. Louis, MO) substrate. RB protein was detected (Fig. 9). Dye expression was measured by absorbance at 405 nm on a Coulter microplate autoreader (Molecular Devices). FIG. 9 is a graph showing the binding ability of the purified fusion protein to the purified viral oncoprotein SV40 T-antigen, where the vertical axis is the absorbance and the horizontal axis is the dilution ratio. The curves in the figure show that purified samples of D0 and D1 PE-RB fusion proteins have reduced T antigen binding with dilution. The control curve represents the dilution of E. coli lysate treated in the same way as the purified protein.

Example 5 Inhibition of growth of p110 ^RB- deficient lung tumor cell line in vitro by fusion protein and full-length RB (p110 ^RB ) Tumor cells used include the following: Rb
Negative non-small cell lung carcinoma (NSCLC) cell line (H5
96) (Harbour, JW et al., "Abnormalities in structure and expression of the human retinoblastoma gene in SCLC", Science ,
241: 353-7 (1988)) and small cell lung carcinoma (SCLC)
Cell lines (H69 and H128) ((Hensel, CH et al.
"Changes in structure and expression of human retinoblastoma susceptibility gene in small cell lung cancer", Canc. Res. , 50: 3067-3072 (199
0)); and Rb positive non-small cell lung carcinoma line (A5
49) (Yokota, J. et al., "Changes in the expression of the retinoblastoma (RB) gene in small cell carcinoma of the lung", Oncogene , 3: 471-5.
(1988)). Here, as the tumor protein, p56
^RB and p110 ^RB , and D1 as a fusion protein
-P56Rb was used to produce these in baculovirus and Escherichia coli by a conventional method. In FIG. 10A, R
p11 in b-negative H128SCLC tumor cells
3 shows the structure of p56 ^RB in 0 ^RB and Rb positive A549 NSCLC tumor cells. These cell lines are
Obtained from TCC (Rockville, MD). Approximately 5 × 10 ³ cells were seeded in each well in a 96-well plate,
And it equilibrated at 37 degreeC in RPMI culture medium overnight. The medium was aspirated and 100 μl of fresh RPMI supplemented with 5% fetal bovine serum was added to each well. Furthermore, the fusion protein was added to the medium to a final concentration of 25 μg / m 2.
The range was from L to 0.4 μg / mL. A control with no analyte was also run. Each incubation was done in triplicate. Cells were incubated for 2 hours at 37 ° C in the presence of analyte. Subsequently, the medium was aspirated and an equal volume of new RPM supplemented with 5% fetal calf serum.
Replaced with I. Cells were incubated for 4 hours in analyte-depleted medium. The second 2 hour treatment with analyte was
The same concentration was used in each well described above. Then 2
A fourth 4 hour incubation lacking analyte was performed. A third 2 hour treatment was performed, followed by another 4 hour incubation lacking analyte. Therefore, the total exposure time to the analyte was 6 hours.

Growth by RB protein (DNA synthesis)
To study inhibition in vitro, a ³ H-thymidine incorporation assay was performed. ^{For 3} H-thymidine incorporation studies, the medium in each well was removed and replaced with fresh RPMI supplemented with 5% fetal calf serum and 5 μCi / mL ³ H-thymidine. The cells were incubated in this environment for 16 hours. The cells were then harvested and ³ H-thymidine incorporation was counted. p110 ^RB
Inhibition of DNA synthesis by p110 ^RB in a deletion tumor cell line is shown in FIGS. 10B and 11.

FIG. 10B shows uptake of ³ H thymidine by the combination of tumor cell line and tumor protein, where □ is H128 + p110 ^RB , and ◆ is A549 + p110.
^RB , ■ is H128 + p56 ^RB , and ◇ is A549 + p
56 ^RB is shown. The vertical axis is the control% of ³ H thymidine incorporation, and the horizontal axis is the protein concentration (μg / ml)
Is. . As a result, normal p110 ^RB produced by baculovirus was transformed into RB negative H128SC.
It is shown to inhibit the growth of LC tumor cells. Figure 1
1 is Rb negative SCLC tumor cells, Rb negative NSCLC tumor cells, and Rb positive NSCL
³ depicts uptake of ³ H thymidine of p110 ^RB in C tumor cells. The vertical axis is% of control. Black bars indicate p110 ^RB produced by baculovirus, open bars indicate p110 ^RB produced by E. coli, and hatched bars indicate addition of BSA (bovine serum albumin) only. This gives p1
It has been shown that 10 ^RB directly inhibits the growth of cell lines lacking expression of p110 ^RB .

According to FIG. 12, the ligand fusion protein D
It is shown that 1-p56Rb inhibits the growth of non-small cell lung carcinoma NCl-H596. ■ in the figure is Rb110,
◯ indicates D1-p56Rb, and ▼ indicates p56Rb. The vertical axis is% of control, and the horizontal axis is protein concentration (μg
/ Ml).

FIG. 13 shows the cell fraction of the lung carcinoma cell line H596 dominated by ¹²⁵ I-labeled full length Rb protein (Radke, K. et al., “Avian sarcoma virus-transformed chicken embryo fibers. 3 of tyrosine phosphorylation of blast cells
Membrane Association of 6,000 Dalton Substrates ", Cell Biol. , 97:16.
01-1611 (1983)). In the figure, the solid black bars indicate the nucleus, the open bars indicate the membrane, and the shaded bars indicate the cytoplasm. The vertical axis represents ¹²⁵ I CPM taken up into cells, and the horizontal axis represents elapsed time. p110 ^RB is a manufacturer (Pierce, Rockfold, IL)
Radioiodine was used with the rhodogen reagent according to the recommendations of J. et al. And added to cultures of H596 tumor cells as described in the text. Uptake and subcellular fraction polarization assays were performed by standard methods. As a result, p110 ^RB −
¹²⁵ I appears to be time-dependently localized in the nucleus of H596 cells.

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, a soluble molecular complex that induces a tumor or cancer suppressor transcription factor in mammalian cells is provided. This compound releases the above transcription factor into mammalian cells by the method of the present invention,
It is useful for suppressing tumor growth by modifying the cell regulatory activity involving genes.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing in detail the construction of a plasmid for producing the fusion proteins PEA56RBD0 and PEA56RBD1 of the present invention.

FIG. 2 Fusion protein PEA56RBD2S of the invention
It is the schematic diagram which showed the construction of the plasmid for production in detail.

FIG. 3: Various Pseudomonas exotoxin p56 ^RB
It is the figure which briefly showed the final structure of a fusion protein.

FIG. 4 is a diagram showing a mechanism of interaction between the fusion protein of the present invention and a cell.

FIG. 5 is a schematic diagram showing in detail the construction of a plasmid for producing the fusion protein FID56RB of the present invention.

FIG. 6 is a schematic diagram briefly showing the construction of the interferon-RB fusion protein of the present invention.

FIG. 7 shows the results of Western blot analysis of fusion proteins of the invention.

FIG. 8A and B show SDS-PAGE gel analysis of purified D0 and D1 proteins. C and D show Western blot analysis with ∞Rb Mab 3C8 of purified D0 and D1 proteins.

FIG. 9: Purified viral oncoprotein SV40 T-
It is a graph which shows the binding ability of the purified fusion protein with respect to an antigen.

FIG. 10A: p110 ^RB and Rb positive A549 in Rb negative H128SCLC tumor cells.
It is a diagram showing the structure of p56 ^RB in NSCLC tumor cells. B is a graph depicting ³ H thymidine incorporation by a combination of tumor cell line and tumor protein.

FIG. 11. Rb negative SCLC tumor cells, Rb negative NSCLC tumor cells, and Rb positive NS.
FIG. 6 is a graph showing ³ H thymidine incorporation of p110RB in CLC tumor cells.

FIG. 12: Tumor protein and fusion protein D ₁ P
It is a graph showing the cell growth inhibitory ability of E-RB.

FIG. 13 is a graph showing the intracellular distribution of the lung carcinoma cell line H596 with respect to the time-dependent subcellular localization of ¹²⁵ I-labeled full-length Rb protein.

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[Procedure amendment]

[Submission date] October 13, 1994

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 7

[Correction method] Change

[Correction content]

FIG. 7 is an electrophoretic photograph showing the result of Western blot analysis of the fusion protein of the present invention.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] Figure 8

[Correction method] Change

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FIG. 8A and B are electrophoretic photographs showing SDS-PAGE gel analysis of purified D0 and D1 proteins.
It C and D are ∞ of purified D0 and D1 proteins
It is an electrophoretic photograph which shows Western blot analysis using Rb Mab 3C8. ─────────────────────────────────────────────────── ───

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[Submission date] October 13, 1994

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[Document name to be corrected] Drawing

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[Figure 7]

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[Figure 8]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ross E. Davidson United States California 92126, Penara Street, San Diego 10718 (72) Inventor Doane E. Johnson USA CA 92024, Encinitas, Truscroad 1546 (72) Inventor H. Michael Shepherd United States California 92067, Rancho Santa Fe, Pea. Oh. Box 8333 (no address)

Claims (24)

[Claims] 1. A ligand fusion for a mammalian cell membrane receptor that is a soluble molecular complex that induces a cancer suppressor transcription factor in a mammalian cell, wherein the complex reversibly binds to the cancer suppressor transcription factor. A soluble molecular complex containing. 2. The soluble molecular complex of claim 1, wherein the cancer suppressor transcription factor is an RB agonist polypeptide. 3. The RB agonist polypeptide is p1
The soluble molecular complex according to claim 2, which contains a low molecular weight derivative of 10 ^RB . 4. The soluble molecular complex of claim 3, wherein the low molecular weight derivative of p110 ^RB exhibits the biological activity of full-length soluble retinoblastoma protein. 5. The soluble molecular complex of claim 4, wherein the biological activity comprises a nuclear localization signal. 6. The soluble molecular complex of claim 4, wherein the biological activity comprises tumor growth inhibition. 7. The soluble molecular complex of claim 3, wherein the low molecular weight derivative is p56 ^RB , and fragments thereof. 8. A soluble molecular complex for inducing a cancer suppressor transcription factor to the mammalian cell according to claim 1, wherein the ligand specifically binds to a -COOH receptor of a cell membrane. 9. A soluble molecular complex that induces a cancer suppressor transcription factor in the mammalian cell according to claim 1, wherein the ligand specifically binds to asialoglycoprotein. 10. The soluble molecular complex that induces a cancer suppressor transcription factor in the mammalian cell according to claim 9, wherein the asialoglycoprotein is asialo-GM.
1 or asialo-GM2. 11. A soluble molecular complex for inducing a cancer suppressor transcription factor to the mammalian cell according to claim 1, wherein the ligand is Pseudomonas exotoxin A. 12. A soluble molecular complex that induces a cancer suppressor transcription factor in the mammalian cell according to claim 1, wherein the ligand is interferon α.
Complex. 13. PEA56RBD0, PEA56RB
A soluble molecular complex that induces a cancer suppressor transcription factor in a mammalian cell selected from the group consisting of D1, PEA56RBD2S, and FID56RB. 14. The soluble molecular complex according to claim 1,
A pharmaceutical composition comprising: and a pharmaceutically acceptable carrier. 15. A pharmaceutical composition comprising the soluble molecular complex according to claim 2 or 13, and a pharmaceutically acceptable carrier. 16. A pharmaceutical composition comprising purified soluble p110 ^RB and a pharmaceutically acceptable carrier. 17. A method of modifying the cell regulatory activity of the Rb gene, comprising the step of contacting the cell with the soluble molecular complex of claim 2 so that the cell regulatory activity is modified, Method. 18. The method of claim 17, wherein the ligand releases the RB agonist into the cytoplasm of mammalian cells. 19. The method of claim 17, wherein the contacting step is performed in vitro. 20. The method of claim 17, wherein the contacting step is performed in vivo. 21. The method of claim 17, wherein the regulatory activity comprises tumor growth inhibition. 22. A method for treating a pathological condition caused by dysfunction of the Rb gene or alteration of the Rb gene product, which is effective for restoring the cell regulatory function of the Rb gene or its protein product. Quantity of claim 1
14. A method comprising the step of administering to a subject a pharmaceutical composition according to either 2 or 13. 23. The pathological condition is retinoblastoma,
Bladder cancer, breast cancer, chronic myelogenous leukemia, acute myelogenous leukemia,
Characterized by testicular cancer, dysplastic and cancerous ulcerative colitis, sporadic sarcoma, prostate cancer, osteosarcoma, small cell lung cancer, synovial sarcoma, and dysfunction of Rb gene or alteration of Rb gene product 23. The method of claim 22, wherein the malignancy is another malignant disease or hyperproliferative disease. 24. The subject of claim 22, wherein the subject is a human.
The method described in.