JPS62500631A - human tumor necrosis factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] human tumor necrosis factor ka b1 This invention relates to the recombinant production of human protein factors. This is especially selected for tumors The production of mutin proteins that are sexually toxic and whose specific activity has been improved. Regarding construction.

1 dish of mushrooms The factor that became better known as tumor necrosis factor (TNF) was originally derived from Carsw. ell et al., Proc, Natl, Acad, Sci, (USA) (197 5) 72: Found by 3666. Bacillus Calmetschiblin (B Immune boosters such as Calmette-Gurin (BCG) Mice, rabbits, or rats that have been exposed to endotoxins in advance When syn-treated serum was injected into mice bearing implanted tumors, In some cases, a substance that induces extensive bleeding of the tumor without undesirable side effects in the recipient. It was found that it contains Therefore, this serum selectively causes necrosis to tumor cells. active and neutral regarding its reaction with normal Mlwi and therefore was predicted to contain a substance called TNF. When injected into a whole animal This selective I! The ability to cause tumor destruction is the standard that defines TNF. It has become an effective in vivo assay method.

TNF was also produced in cell culture. Matthews et al., Br1t, J. , Cancer (1981) 44: 418, BCG-injected rabbits CManne+ et al., which were able to obtain TNF activity in the culture medium of mononuclear phagocytes derived from Infect, Immun, (1980) 30: 523: Same (1981 )33:156), macro from mice infected with BCG after cell culture TNF activity obtained from phage-enriched peritoneal exudate cells is induced by endotoxin It was done.

Attempts have been made to purify factors responsible for selective cytotoxicity against neoplastic cells; Since it appears to be present in extremely small amounts in the serum of whole animals or in tissue culture media, It was not possible to perform a complete purification. Additionally, the protein(s) are clearly and two recent US patents Na4,447.355 and 11 h4,457,916 is prepared by adding albumin or carbohydrate materials, for example. It is directed toward methods of stabilizing the activity of substances.

In these disclosed methods using standard purification methods developed by others, It is possible to obtain a specific activity of the TNF tl'9 product of approximately 1 x 10 units)/mg. possible, and this unit is suitable for murine LM cells (ATCCCCCL 1.2). This was defined in an in vitro assay for cytotoxicity.

However, the in vivo (Carswell) tumor necrosis assay for TNF is active in B. and is sufficient to enable amino acid sequence information to be obtained. It was not possible to obtain material with purity.

Indeed, due to the unavailability of pure cytotoxic proteins, cancer cell It is now clear how we can obtain a large amount of protein that has a selective necrotic effect on It's not that big. The in vivo method of Carswell et al. (supra) is the standard for defining TNF. has been approved. Cross-species cross-activity of these factors sactivity), this assay is convenient and diagnostic in some respects. It is true. However, a more convenient in vitro assay for cytotoxicity is often used as an indicator of TNF activity. This method supports this in vitro Whether there is a 1:1 correlation between the assay method and the test that defines TNF. It is used despite there being considerable confusion about it. Indeed, in vitro Proteins from transformed B-cell lines that are active in the assay are was purified to homogeneity and partially sequenced ( Zenentesoku, European patent application publication 1ko100641.February 1, 1984 Released on the 5th). Lymphotoxin is classified as “T” because it is non-macrophage derived. It is predicted that the protein is different from NF''. The anti-serum prepared was purified from cytotoxic factor (TNF) from macrophages. Does not cross-react with (S tone-Wo l f f + mouth 1 etc., Muha L ha rainbow ( 1984) Dish: 828).

Clear cells capable of exerting cytotoxic effects specifically directed against tumor cells Needless to say, providing a protein sequence that has been modified is useful for the diagnosis and treatment of malignant diseases. It would bring great benefits to both the patient and the patient. Furthermore, some of these factors BCG appears to have anti-parasitic properties; A protein called TNF inhibits malaria parasites in vivo and in vitro. Plasmodium falcipari Cytotoxic to um) - 丞 Human promyelocytic leukemia cell line (HL-60, ATCC No. CCL240) was shown to produce significant amounts of tumor necrosis factor when properly induced. . This factor was purified, sequenced, and produced by recombinant techniques. subordinate First, chemically defined drugs that are selectively cytotoxic to human tumor cells There are other factors. This provides sufficient material for use in medicine of tumors in organisms, making programmed transformations to improve activity, and provides an opportunity to design appropriate diagnostic tests to detect its presence. In other words, this The availability of recombinant sources of this protein makes this useful peptide useful for therapeutic and diagnostic purposes. bring about the possibility of carrying out the operation. The above forms of INF are expressed in humans in response to tumor morphology. More likely (naturally produced), but the flexibility offered by recombinant production and without guarantees of quality and quantity, we will not have the means to exploit its properties. I can't.

Accordingly, in one aspect, the invention relates to recombinant human TNF. to other points of view This is the uninterrupted DNA sequence that encodes this protein and carries out its expression. a sequence capable of conferring the ability to express TNF to a transformed host. Transformation vectors that can be transformed, recombinant hosts thus transformed, and seeds of this invention The present invention relates to methods for obtaining various compositions.

In another aspect, the invention particularly relates to glycosylated forms or glycosylated A protein with the amino acid sequence encoded by the DNA sequence shown in Figure 1, without the Regarding quality.

This invention also contains changes in the primary structure compared to that shown in FIG. Some specific recombinant mutins of TNF, as well as methods and methods for their production. and materials.

These mutins are first in their ability to selectively kill tumor cells. The activity is comparable to or higher than that of the primary sequence TNF (mTNF) shown in the figure.

These mutins have 1 to 1 ON-terminal amino acid residues removed. protein, and 1 to 10 N-terminal amino acids are absent and cysteine is removed. Includes mutins that are present.

This invention particularly relates to the N-terminal sequence: Val-Arg-3er-Arg-Thr- Pro-Ser-Asp-Lys-Pro-Val-81a-Va1. -5er -Val-Ala-Asn-Pro-(Gln)-(Ala)-Glu-Gly and induce the production of this amino acid sequence from promyelocytic leukemia cells. and an improved method for purifying it. This invention further provides T. Pharmaceutical compositions containing NF', therapeutic methods using these compositions, and TNF An improved assay method for.

The truth is that the truth is clear. Figure 1 shows the complete nucleotide sequence of pE4 and the predicted amino acid sequence of human TNF. Indicates a column.

Figure 2 shows a restriction map of the insert in pEd.

Figure 3 shows expression plasmids and their construction for various TNF mutins. The oligomers used for this are shown.

FIG. 4 shows a comparison of the specific activities of several TNF mutins.

Figure 5 shows the S. DS gel is shown.

Figure 6 is an isoelectric focusing gel performed on purified rTNF mutin. The results are shown below.

Figures 7a and 7b show the results of an in vivo study of TNF activity in tumor-bearing mice. Show results.

, ■Pi 1 for doing... A. Definition As used herein, "tumor necrosis factor (TNF)" can be selectively cytotoxic and substantially equivalent to that shown in Figure 1. Concerning amino acid sequences. Amino acid sequences that meet this definition are contiguous as described below. In an in vitro cytotoxicity assay based on the murine connective tissue cell line L-929. must be active. This definition of TNF activity is based on Carswell et al. It is not exactly the same as that shown in the disclosure by which the term was coined by do not have. However, this in vitro cytotoxicity assay against human tumor cells More confirmed activity can be characterized as a tumor necrosis factor using this assay. Provides sufficient guarantees of utility to be justified. As shown below, for L-929 This cytotoxicity appears to generalize to other human tumors. Cytotoxicity as specified above Factors active in assays and in vivo as summarized by Carswell Expected to be substantial Oharassob between the active factor in the assay be done.

Is the TNF protein of this invention suspended or not? If it is a liquid, the p of the environment is depending on i+ or, if in solid form, the p of its environment during crystallization or precipitation. Depending on l+, it can be in the form of a pharmaceutically acceptable salt, or in the neutral form. can be. It goes without saying that the free amino groups of this protein (for example, inorganic acids, such as hydrochloric, phosphoric or sulfuric acid, or organic acids, such as acetic acid, glycolic acid , can form acid addition salts with succinic acid or mandelic acid. tt separation The carboxyl group is a base, for example an inorganic base such as thorium, potassium or potassium. lucium hydroxide, and organic acids such as piperidine, glucosamine, trimethyl Salts can be formed with amines, choline or caffein. In addition, protein By combining with other biological substances, such as libido and saccharides, or Side chain modifications such as acetylation of amino groups, phosphorylation of hydroxyl side chains, or It can be modified by oxidation of the ruhydryl group. As long as TNF activity is retained, these All modifications are within the scope of this invention.

Finally, certain variations of the -order amino acid sequence may still occur compared to the sequence shown in Figure 1. can yield a protein with substantially equivalent or enhanced activity. is understood. These transformations may be intentional, such as by site-directed mutagenesis. (or may be coincidental, such as due to mutations in the TNF-producing host) . It is not expected that a particular modification would lead to a protein with TNF activity; Although it is not possible to predict in advance which deformations will be allowed, All of these transformations are 'TNF' as long as TNF activity is maintained. ” included in the definition.

In particular, up to the first 10 amino acids at the N-terminus of the sequence shown in Figure 1 (number 10) A mutin lacking the amino acids (containing the first amino acid) is comparable to the structure of TNF shown. It was found to have a specific activity of 50% or higher, and is discussed below.

The pattern of specific activity shows optimal activity when the 6-8 N-terminal amino acids are removed. appears to follow a bell-shaped curve.

Therefore, the definition of TNF specifically encompasses these truncated forms. clearly, Removal of up to 10 amino acids from the N-terminus does not destroy biological activity, but In fact, it often enhances it.

Therefore, in this specification, the definition of TNF is specifically defined as substantially the same as that shown in FIG. have the same amino acid sequence, but one in the N-terminal sequence shown in the figure. Includes proteins lacking ~10 amino acids. 5hirai, T, etc., Na ture (1985) 313:803-806 was obtained from the Human Genome Bank. It has been reported that recombinant TNF was produced using an expression vector constructed from this DNA. Get noticed. In this construct, the encoded protein is N - lacks the first two amino acids of the terminal sequence. Not obvious but Rabbit For reasons likely related to the TNF genome structure, 5hirai et al. is expected to start at position 3 as indicated in this specification, and this The above vector was constructed for this purpose. As a result, TN manufactured by 5hirai F has the N-terminal sequence Ser-3er-3er-Arg-Thr, etc. 5hi rai-produced TNF was shown to have in vivo activity. This egg White matter activity and that of recombinantly produced mTNF and TNF mutin of this invention. No direct comparison is currently available.

Furthermore, removal from the C-terminus of TNF as shown in Figure 1 is also expected to be harmless.

Genes encoding the removal of up to 17 amino acids have also been created.

U.S. patent m4.518.584 removes cysteine from biologically active proteins. It describes the mutins that were created. In the case of TNF, Sisti ranked 69th. Replacement of the protein with a neutral amino acid results in an active TNF protein. 101st place Cysteine also appears to be unnecessary, and an alternative neutral amino acid at this position Mutin with and both cysteines 69 and 101 were replaced Mutin was also prepared. These mutins can also be modified according to the method of this invention. therefore, it is transformed and retains TNF activity, and in vivo and in vitro. A truncated form can be obtained which will have enhanced specific activity. this Their mutin consists of 1 to 10 amino acids at the N-terminus and an amino acid at the C-terminus. lacking the acid sequence, or both. These mutins are also characteristically T It belongs to the definition of NF.

Finally, a gene was created using TNF purified from natural sources as a model. Ta. The code encodes a mutin in which two serine residues at positions 3 and 4 have been removed. The gene and the his-val couplet at positions 15 and 16 become val-ser. A gene encoding a more substituted mutin was prepared.

Regarding symbols, proteins having amino acid sequences numbered 1 to 157 in Figure 1 for convenience. Using white matter as a reference and perhaps arbitrarily calling it mTNF (mature TNF) will be done. Has homology to mTNF and exhibits the biological activity of TNF All other amino acid sequences are referred to as mutins of m, TNF, and Differences from mTNF are indicated using the residue numbers shown in the figure. cormorant. For example, a mutin with a cysteine substitution at position 69 has a substitution of the substituted residue and position number, e.g., Seri instead of cysteine at position 69. The peptide with the 5erbqTNF will be designated as 5erbqTNF. If the residue is simply missing If it is missing, it is named as a des-residue, thus e.g. and the mutin in which serine at position 4 is removed is des-ser3des-s It will be called ernTNF. Removal of the N-terminus and subsequent deletion The number of deletions will be used to indicate that the corresponding number of amino acids are missing. example For example, a muti protein lacking one N-terminal amino acid compared to the protein shown in Figure 1 The component will be named as ITNF. For C-terminal deletions, the next most The remaining residues after will be numbered and marked with a minus sign.

Therefore, for a mutin with 7 amino acids removed from the C-terminus, The name would be 150-TNF. If a combination of the above changes is made, the name shows all of them, for example △LdeS-3er3deS-5eraSerhq 150-TNF.

Not all mTNF mutins are recombinantly or intentionally produced. do not have. Indeed, D.1. TN secreted by Ht, -60 described later in h. The sequence obtained for the 22 N-terminal amino acids of F was combined with the predicted sequence shown in Figure 1. By comparing with the corresponding part of the sequence, both white matter showed TNF activity. Nevertheless, it can be seen that there are slight changes in the primary structure. in particular, The deduced sequence is the 6th position of the predicted sequence from position 4 to 12 of the protein derived from HL-60. An additional pair of serines next to serine at position 3 before restoring the homology shown between positions It has a serine residue. Furthermore, positions 13 and 14 of the HL-60-derived protein are va r-set, and the corresponding positions 15 and 16 of the deduced sequence are his-va It is r.

“Operably linked” means that the components are in such a manner that they perform their normal functions. It means the juxtaposition of the

Thus, control sequences operably linked to a coding sequence control the expression of the coding sequence. It can be performed.

"Control sequence" is one or more DNA sequences that can act on the desired coding sequence. which, when operably linked, facilitates the expression of the coding sequence in a host compatible with the DNA sequence. means something that can be done. Such control sequences are found in prokaryotes and eukaryotes. promoters in both hosts and, in prokaryotes, also ribosome binding. Contains binding site sequences and, in eukaryotes, termination signals. manifestation Additional factors that may be necessary or preferred to accomplish this will also be identified later.

As used herein, "control sequences" refers simply to the particular host used. Contains all DNA sequences required for expression.

“Cell,” “recombinant host,” or “host cell” are often used interchangeably; It will become clear from the context.

These terms encompass the immediate subject cells, as well as their progeny.

Due to chance mutations and changes in the environment, all progeny are exactly identical to the parent cell. It is understood that this is not the case. However, when the above term is used, This includes descendants with such changes.

B, two-pronged blood ju The method set forth below for obtaining a DNA sequence encoding human TNF is exemplary. It is very typical. Other methods can also be used. Perfect NA arrangement Since the sequences were obtained in an intron-free form, and as disclosed herein, Needless to say, it is necessary to repeat the same method to obtain the desired DNA sequence. There isn't. as well as using the same systems exemplified herein for expression. There's no need. As shown in more detail in Section 0, the production of the desired TNF is A variety of host and control sequences are available in the art that could be used to perform It is possible.

In the exemplary method, B. secretes TNF as described in Section 3. , the human promyelocytic leukemia cell line I (L-60) was induced by an improved induction method. M=’li was done. This protein is a novel protein as described in Section B.21. The resulting purified protein is purified to homogeneity using a purification method, and the resulting purified protein has an amino acid sequence. The decision was made. This allows the creation of a probe mixture, which is then was used to obtain the appropriate coding sequence as described in Section B, Section 39. child The coding sequence of contains appropriate prokaryotic control systems and for expression in complement mammalian cells. In this example, by ligating into a vector using a viral promoter, was used for expression in

Generate mutins in DNA sequences already obtained from cDNA sequence searches. The code sequence was modified to make it easier.

Such transformations are performed by primer-directed mutagenesis and result in enhanced activity. A shortened form of TNF has been provided.

And knee improvement i: tlJ,:'1 tota The cell line HL-60 human promyelocytic leukocyte cell line subjected to gaff is itself is relatively undifferentiated. If allowed to differentiate, this thing becomes even more specific. Form colonies of defined cell types. Depending on subsequent events, this cells mature into granulocytes or monocytes, which then further differentiate into macrophages. can become an agent. Macrophage fraction is responsible for in vivo production of TNE It is expected that On the other hand, the apparently closely related lymphotoxin is a B-lymph. It is thought to be produced by balls.

The level of TNF produced by target cells can be increased 10-20 times by improving the induction method. It was discovered that it can be added. Known method devised by Gifford, G., et al. (Personal communication) horbo at 10 Mg/m11 for 30 minutes at 37°C in 20% serum. phorbol ester 12-0-tetradecanoylphorbol 13-a Treatment with acetate (TPA) is used. Next, the cell culture is spun down and its bloodless cells supplemented with insulin and transferrin (10 μg/mJ each) Treated with 10 IJg/ml endotoxin in clear medium. first incubation The concentration of phorbol ester was adjusted to 100 μg/day using serum-free medium in the 10 by reducing mβ and in the endotoxin treatment step. By adding MM calcium ionophore A23817, endotoxification There is a substantial increase in the content of TNF in the supernatant after incubation with the supernatant.

B, 2.1 Bendou Fuu Blind Rip Additionally, improved methods for purifying TNF from induced cell cultures have been developed. shown. In this method, the TNF-containing supernatant is prepared under conditions that do not allow adsorption of TNF. The TN’F-containing fraction was treated with an anion exchange resin under to obtain an active fraction, which is then subjected to 5OS-PAGE. Ru. The TNF-containing fractions from 5DS-PAGE are further purified by HPLC.

In the first step, the supernatant containing TNF is removed before application to the anion exchange resin. In some cases, for example, commercially available concentration filters, such as Amicon hollow fiber or Micon Lipoapericone Ultra is concentrated by treatment with perunit. Then the concentrate a suitable anion exchange resin, such as DEAE agarose, DEAE cellulose, or treated with QAE agarose, preferably DEAE agarose. processing conditions, i.e. pH is about 7-9 and total salt? A solution with a concentration of about 0.01 to 0.07M is T The NF activity is such that it does not adsorb to the support.

The unbound fraction is then transferred to, e.g., any commercially available size fractionating gel, e.g. Sephadex G-75 superfine (with a molecular weight exclusion of 00 daltons) Further purification is performed by gel filtration using a 100% polypropylene (Pharmacia). T after treatment with gel The NF-containing fractions were then subjected to 5O3-PAGE under appropriate conditions and the TNF activity Collect the gel slice containing the . 5DS-PAGE is 1, aemmli etc. It was carried out according to the method of Nature (1970) 227:680 and this This is a technique well known in the art.

Variations in specific conditions within reasonable limits are possible and well understood.

The activity-containing fractions from 5DS-PAGE were then subjected to reverse phase HPLC and 0. Elute with a 0-60% acetonitrile gradient in 1% TFA. use A possible gradient elution system includes acetic acid and n-propanol.

The human TNF thus obtained is sufficient to allow sequencing of the N-terminal amino acid. It's so pure.

B.3. 2nλ Kamitane mRNA prepared from HL-60 cells was added to a standard rabbit cell translation system. production of active TNF factor in the L-929 cytotoxicity assay. I can do it. The relative efficiency of the oligomer probe was determined using this mRNA preparation and the previous ink. Those that have a negative effect on the production of TNF in this translation system when incubated (“bibrid capture”). Images in the oocyte system If the desired TNF code mRNA has already been identified by translation, of the mRNA gradient hybridizing to the kinased probe. Radioautography allows further refinement of the above criteria. Ru. 4 elements encoding the sequence Asp-Lys-Pro-Val-81a in the protein contains 16 14-mers designed to be complementary to the Boolean mRNA of It was possible to determine which of the following contained the correct sequence. 16 pieces This mixture of oligomers hybridizes only to the TNF-encoding mRNA. It can be shown by the Northern plot that the We therefore repeated these “bibrid capture” experiments using eight pairs of 14-mers. Ta. One pair gave good results in specifically hybridizing mRNA.

Once a sufficiently specific probe has been identified, the mRNA encoding the TNF of interest This was used to search the cDNA library formed from the fractions. 2 Eight successful hybridizing colonies were picked and plasmid DNA was isolated. and several inserts were sequenced. Plasmid containing the complete coding sequence The preparation was named pE4 and was used as a source of coding sequences. additional plasmid The hard preparation pB11 was used for mammalian expression.

B, 4. Engineering Ndan Shaoguli Nucleotide sequencing of the pE4 insert will, of course, determine the location of the coding sequence. and allowed analysis of available restriction sites within the insert. Homology is not perfect However, it was easy to make the correct replacement. For ease of operation, in phase with and immediately preceding the codon for the N-terminal amino acid of the mature protein. Place the ATG initiation codon and immediately upstream of this ATG contain the old ndI[r site. It was desirable to have it. This was achieved by site-directed mutagenesis. the Details will be provided later. Next, add two coding sequences with this appropriate initiation signal. , namely the HindI[I/Pstl fragment and PstT/Ba cut into mtl I fragments, and insert these fragments into host plants containing control sequences. It was possible to insert it into the current vector. Alternatively, you can convert the complete array to the old n dII [could be cut out as a fragment. Specific host expression vector used is the trp promoter upstream of the 11indlIr site and the downstream BamHI site. pTRP3 containing pTRP3, as well as a similarly arranged PL promoter. pFC54, and ppt, op.

These expression vectors were transformed into a suitable E. coli host, The resulting transformant was then cultured under conditions that lead to the production of TNF. Sonic treatment and then sonicated with a chaotropic agent. TNF is removed from cells by solubilizing the desired TNF by treatment with or the sonicate can be assayed directly.

Initial expression was achieved in E. coli. However, in term C,1, it is very As described in detail, coding sequences from pE4 or pBll can be transferred to other prokaryotes. , yeast, Mi tissue culture, or plant cells. I was able to do it too. Expression in mammalian cells is actually caused by the SV40 promoter of pBll. was achieved using. Selection of an appropriate host depends on the potential for secretion, post-translational processing, etc. the possibility to perform multiplication and produce high levels of the protein of interest under appropriate growth conditions. It will depend on many factors including capacity.

B.5. assay B.5. a, 1 Assay method The L-929 assay system offers improved convenience that allows rapid measurement of TNF activity. This is a unique in vitro assay method. Carsivell's In Vivo Tumor Necrosis Assay The extent of the correlation with A is currently unknown. However, this is especially Since Zumi tumor cells are used, the correlation is expected to be high. EPO public release 1’ho The protein called lymphotoxin in No. 100641 (cited above) was also tested using this assay. It gives activity in A. This assay is conceptually based on murine LM cells and disclosed in U.S. Patent Nf 14,457,916 using methylene blue staining. similar to what is being The L-929 assay method is effective for human tumor cell line cytotoxicity. (for HL-60-derived TNF) (D, see section 1.b). Teru).

In the L-929 assay system of this invention, L~929! II cysts are microtied Prepared overnight as a monolayer in a tarplate. Spread the test sample across the plate. diluted 2x, UV irradiated, and then applied onto the prepared cell monolayer. next, Adjust the medium in the well to 1 μg/ml actinomycin D. 3 plates Incubate for 18 hours at 7°C and visually scan the plate under a microscope. I'll do it. 25%, 50%, 75% and 100% indicating the degree of cell death in the well Label each well. Causes 50% killing of 1 unit of TNF activity It is defined as the reciprocal of the dilution.

Additionally, more aggressive variations of this assay have been developed.

This method tests the test sample and when treated with actinomycin D. The release of 3SS-labeled peptide from labeled cells is monitored.

This variant of the assay is used to quantify titers e.g. It can be used to assess the relative potency of substances. In summary, actively increasing Growing cultures of L-929 were supplemented with 2% dialyzed fetal calf serum. 3 with 358-methionine (200 μCi/ml) in methionine-free medium. Label the time. Cells were then washed and plated into 96-well plates. , and - incubate overnight, and the next day a 2-fold dilution of the test sample and Treat with 1 μg/ml actinomycin D. Next, the culture was brought to 37°C. Incubate for 18 hours. Next, aliquot 100 μl of supernatant from each well. The plate was transferred to another 96-well plate, acid (TCA) precipitated, and glass plated. Collect on a fiber filter. Wash the filter with 95% ethanol and dry. , and count. NP for all assays. Release from cells, including detergent controls. Measure the maximum radiation release. Next, compare the 153 release (%) between treated cells and untreated controls. NP, the difference in counts between. Divided by the difference in cantons between treated and untreated cells. The ratio is calculated using the ratio expressed by . TNF titer is high This ratio increases as the temperature increases. The above assay method uses human cells as target cells. Conveniently modified for use with tumor cell lines. The above L-929 cell field Define the unit and calculate the release (%) in the same way as for the case.

B.5. b　-±2　　-±2　　　-±2　　　-±2　　　-±2ヱヱ-&-(-Preparations also kill tumors or the ability of the substance to inhibit tumor growth and protect the tumor-bearing animal from death. can be used to test for TNF activity. Various Ba1h/c mice Localized tumors were formed by subcutaneous injection of tumor cells of this type.

The tumor cell line is a MethA mouse fibrous tissue obtained as a cell suspension from ascites. and MCF-7 human breast cancer administered as 1 mm3 cell clumps.

For the assay, female Ba1h/c mice (19-22 g) were placed with a 26-gauge needle. ri 0.1m 7! A suspension containing 5 x 105 fibrosarcoma cells in the medium of or MCF-7 mass was injected subcutaneously (fibrosarcoma suspension was obtained by counting cells from ascites of the white of the 8th eye). (prepared by number and dilution with serum-free medium). After 9-10 days, the tumor is palpable. When possible, inject the amount of TNFO to be tested (in the range of 1 μg per mouse); TNF administration was then repeated as desired on subsequent days. Measuring tumor volume Results were evaluated by survival rate.

B, 6. TNF mutin stop 1 This invention contemplates numerous variations of mTNF that may be advantageous in order to obtain a protein of optimal activity. do. Said B, 5. Comparable or increased specific activity in the cytotoxicity assay of section a Several specific changes in the amino acid sequence were obtained that conferred a higher sex. Sequence of mTNF Some of the removal of the first 10 or fewer amino acids from This results in a protein with a specific activity equal to or several times higher than that of the recombinant protein. m The cysteine-removed mutins of TNF also contain the N-terminus of these mutins. It exhibits TNF activity, as does the truncated form.

Some productions of these mutins contain the coding sequence for TNF The expression vector is subjected to site-directed mutagenesis using primers corresponding to the desired coding sequence. This is done by applying a Thus, having appropriate changes in the coding sequence A modified expression vector is obtained. The resulting modified vector is transferred to a suitable host. and then transform this under conditions that result in the production of the encoded mutin. Cultivate. Next, these mutins, like “natural proteins,” can be derived from bacterial cultures. and have been shown to have undiminished or enhanced activity. be done.

In particular, 4TNF mutin and 6-10 TNF mutin have two points. It is clearly superior to mTNF.

That is, they have higher specific activities, and they are "clean" products. It is produced as. Some of these preferred embodiments are shown in more detail below. The activity of the purified protein was determined by mTNF in a cytotoxicity assay. is several times higher than that shown. Additionally, due to its behavior on isoelectric focusing gels, As demonstrated, purified recombinant mTNF appears to have side chain modifications. proteins, whereas these mutins are Gives one qualitative band.

C3 theft quasi-crying Transform cells, construct a vector, extract Metsenger RNA, and convert cDNA Most of the techniques used to prepare A libraries, etc. are well known in the art. is widely practiced, and most practitioners do not describe specific conditions and methods. Familiar with standard methods for However, for convenience, the following sections serve as guidelines. It will be helpful.

C, 1,, Cloth'' Ji8hada M Tobi p Prokaryotes are most often represented by various strains of E. coli. however , other microbial strains, such as Bacillus, such as Bacillus subtilis. us 5ubtilis), Pseudomonas color Various species or other bacterial strains can also be used. In such prokaryotic systems a plasmid containing an origin of replication and control sequences derived from a species compatible with the host; vector is used. For example, E, Cori, Boliver, etc., Gene (1977) 2:95, pBR, a plasmid derived from E. coli strain. 322 derivatives. pBR322 contains ampicillin and tet Construction of a vector containing the gene for lacycline resistance and therefore desired provides additional markers that can be maintained or destroyed.

In this specification, promoters and optionally operators for transcription initiation are used. A commonly used Prokaryotic control sequences include -211 uttamase (penicillinase) and lactose. lac promoter system (Chang et al., Nature (1977) 1981056), and tryptophan (trp) promoter system (Goed del et al., Nucleic Ac1ds Res, (1980) 8: 4 057), and lambda-derived PL promoter and N-gene ribosome binding Part CS Shimatake et al., Nature (1981) 292: 1 28) (This was filed on February 8, 1984 and has been succeeded by the same successor. As described in pending application No. 11h 578,133, Commonly used promoters, such as those made useful as control cassettes - is included. However, any available promoter compatible with prokaryotes - system can be used.

In addition to bacteria, eukaryotic microorganisms such as yeast can also be used as hosts.

Baker's yeast Satsukaromyces selehisi I - (5; Laboratory strains of 111 types are most commonly used. However, many other strains are also commonly available. It is possible. Vectors using a 2 micron origin of replication are exemplified (Broac h.

J. R., Meth, Enz, (1983) 101: 307) Other vectors suitable for maternal expression are known [e.g. Stinch comb et al., Nature (1979) 282: 39; mpe et al., Gene (1980) Uni 157; and C1arke, L. et al. See Meth, Enz, (1983) 101:300. fermentation Control sequences for the mother vector include promoters for the synthesis of glycolytic enzymes. (lless et al., J, ^dv.

(1968) Knee 149;) Iolland et al., Bioch emistr (1978) 17: 4900), known in the art. Other promoters include the 3-phosphoglycerate kinase promoter ( Litzeman et al., J. Biol, Chem, (1980) 255 : 2073), as well as other glycolytic enzymes, such as glyceraldehyde-3-phosphate. Sphate dehydrogenase, hexokinase, pyruvate decarboxylase , phosphofructokinase, glucose-6-phosphate isomerase, 3- Phosphoglycerate mutase, pyruvate kinase, triphosphate isomera Contains promoters of enzyme, phosphoglucose isomerase, and glucokinase. be caught. Other promoters have the added advantage that transcription is controlled by growth conditions. alcohol dehydrogenase 2, isocytochrome CX acid phosphata degradative enzymes related to nitrogen metabolism, and assimilation of maltose and galactose (Illoland, supra). Furthermore, the 3′ of the coding sequence It is believed that a terminator sequence at the end is desirable. It's like this - Mine The promoter is found in the 3' untranslated region following the coding sequence in yeast-derived sequences. illustration The enolase gene-containing plasmid pono46 C11 Holland, M., J., et al., J., Biol, Chem. (1981) 256:1385) or the LEIJ2 gene obtained from YEp13 (B reach.

J0 etc. 1 gun (1978) main: 121), but yeast compatible promoter Any vector containing an origin of replication, an origin of replication, and other control sequences is suitable.

Needless to say, it is possible to encode polypeptides in eukaryotic host cell cultures derived from multicellular organisms. It is also possible to express a gene that encodes a gene. For example, Ti5sue Cu1 tures, Academic Press, Cruz and Pattersonlig ( (1973). Useful host cell lines include Vero cells, HeLa cells, and and Chinese hamster ovary (CHO) cells. For such cells Expression vectors generally contain promoters and control sequences compatible with mammalian cells, e.g. Commonly used early and late strains derived from e.g. Simian Virus 40 (SV40) Promoter (Fiers et al., Nature (1978) 273:113) , and other viral promoters such as polyoma, adenovirus 2, bovine Contains promoters derived from papillomavirus or coracoid sarcoma virus.

A general overview of mammalian cell host system transformation is given in A., August 16, 1983. Xel et al., US Pat. No. 4,399,216. Now even more It seems that the “enhancer” area is important for optimizing the current Non-code DN A? Found upstream or downstream of the promoter region in the J region It is an array. If necessary, origins of replication can be obtained from viral sources. death However, chromosomal integration is the general mechanism of DNA replication in eukaryotes. It is. Plant cells can also now be used as hosts, and are compatible with plant cells sexual control sequences, such as the tubalin synthase promoter and polyazonylage tion signal sequence [Depicker, A. et al., J. Mo1. A1. Gen .

(1982) 1:561 can be used.

C121 anti-red exchange depending on the host cell used, using standard techniques appropriate for such cells. A transformation takes place. Cohen, S.N., Proc.

Natl, Acad, Sci, (LISA) (1972) 69: 211 Calcium treatment using calcium chloride as described by Man. iatis etc., Mo1ecular C1onin: Eight Laborator Manual (198) Cold Spring Harbor Press, page 254 RbCjl being done! 2. Prokaryotic cells, or others containing a substantial cell wall barrier used for cells. Agrobacterium tumefaciens Infection by O- (Shaw, C, H, etc., Gene ( 1983) 23:315) for certain plant cells. This way For mammalian cells without cell walls, see Graham and van der Calcium phosphate precipitation method of Eb, Watahara ■■ (1978) 52: 546 is preferred. Transformation into yeast was performed by VanSolingen, P. et al., J. Bac. t, (1977) 130:946 and l1siao, C,L.

et al., Proc, Natl, Acad, Sci, (USA) (1979) 76 :3829.

C, 3, CD-NA and library hole c DNA or genome library Libraries are screened using colony hybridization methods. each Microtiter plates were filtered using two nitrocellulose filter papers (SAS type BA-8). 5) Replicate on top and incubate colonies at 37°C for 14-16 hours with 50μg/ Contain ml of Amp and grow on agar. Colonies are lysed and then By treating with 500mM Na011.1.5M NaCl for 5 minutes. The DNA was fixed on the filter and 5× product standard salt citrate (SSC’) Wash twice for 5 minutes each. Air dry the filter and Heat at ℃ for 2 hours. Filter two filters at 42°C for 6-8 hours. - 10 ml of DNA hybridization buffer (5X5sc, pH 7, 0.5x Denhardt solution (polyvinylpyrrolidone + Ficoll and bovine serum alcohol) Bumin; 1x - 0.02% each), 50mM sodium phosphate buffer, pH 7.0 , 0.2% SDS, 2 (lcrg/mj! polyU, and 50 μg/mβ modified salmon hybridize with sperm DNA).

The sample is incubated with the kinased probe under conditions that depend on the desired stringency. Ibridize. Typical moderate-severity conditions include probes DNA hybridization buffer 1-5 mβ/24-36 with filter A temperature of 42° C. is used. For higher severity higher temperature & shorter time A pause is used. Filters were incubated at 37°C for 4 times for 30 min each in 2XSSC, Washed with 0.2% SDS and 50mM sodium phosphate buffer (pH 7), Then washed twice with 2xssc and 0.2% SDS, air dried and -7 Autoradiograph for 2-3 days at 0°C.

C04, Samata Mata 2q bottom Construction of suitable vectors containing the desired coding and control sequences is within the skill of the art. (Use understood standard ligation and restriction techniques.Isolated plasmid , DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and and then reconnected into the desired shape.

Site-specific DNA cleavage can be achieved by treatment with appropriate restriction enzyme(s). under conditions generally understood in the industry, and the specific conditions are as specified by the manufacturer of commercially available restriction enzymes. For example, New England Please refer to the BioLapse product catalog. Typically about 1 μg of plasmid or DNA The A sequence is performed with 1 unit of enzyme in approximately 20 μl of buffer. An example of this invention In this case, an excess of restriction enzyme is typically used to ensure complete digestion of the DNA substrate. Ru. Incubation times of approximately 1 to 2 hours at 37°C can be performed; It can also be different from that. After each incubation, phenol/chloroform Proteins can also be removed by extraction using Nucleic acids are then recovered from the aqueous fraction by precipitation with ethanol, and then Pass through a Sephadex G-50 spin column. Optionally, the cleaved fragments Size separation can be performed on polyacrylamide gels or agarose gels using standard techniques. This can be done by pneumothoresis. General techniques for size separation are Methods Enz molo (1980) 65:499-560.

The restriction enzyme cleaved fragment is E. coli DNA polymerase large fragment (Kleno w) using four types of deoxynucleotide triphosphates (dNTPs) Using an incubation time of approximately 15-25 minutes at 20-25 °C in the presence of 50mM Tris (pH 7,6), 50mM NaCl, 6mM MgCl! z, treated in 6mM DTT and 5-10pM dNTP In particular, blunt ends can be obtained. The Klenow fragment connects the 5′ cohesive end. However, even in the presence of four types of dNTPs, the protruding 3′ single strand is chewed back. do. If desired, only or selected within the limits specified by the nature of the cohesive end. Selective repair can be performed by supplying dNTPs. Kleno After treatment with -, the mixture was extracted with phenol/chloroform and ethanol precipitate and then pass through a Sephadex G-50 spin column. under appropriate conditions Treatment with 31 nuclease at 31 results in hydrolysis of the single-stranded portion.

Synthetic oligonucleotides are described by Matteucci et al., J. Am. Chem.

Soc, (1981) 103: 3185 by the triester method, or Prepared using a commercially available automated oligonucleotide synthesizer. prior to annealing , or single chain kinase treatment for labeling in 50mM Tris (pl!7 ．． 6), 10mM Mg (11!□, 5mM dithiothreitol, 1-2 m MAT　P, 1.7　pmole32P　-AT　P(2.9m　Ci　/　m mole), 0.1 mM spermidine, and 0.1 mM EDTA. About 10 units of polynucleotide for a remainder of, for example, Q, ln mole of substrate. This is accomplished using leotide kinase.

The ligation is carried out in a volume of 15-30μβ at the following standard conditions and temperature: 20mM TrisilC! ! (pH 7,5), 10mM MgCjiz, 1, Om MDTT, 33 pg/m RBSA, 10mM ~ 50mMNa1 ! ; and 40 μM ATP, 0.01-0.02 (W eiss) unit of T, DNA ligase, 0°C, or “for blunt end ligation. is 1mM ATP, 0.3 to 0.6 units of T, and DNA ligase. , 14℃. Intermolecular “adhesive end” linkage is usually 33 to 100 μg/m! l of total DNA concentration (5-1100n total end concentration). Intermolecular blunt end ligation (usually using a 10- to 30-fold molar excess of linker) is performed at a 1 uM total end concentration.

“In the construction of vectors using vector fragments, the vector fragments are generally bacterial Treat with alkaline phosphatase (BAP) to remove the 5' phosphate, and to prevent vector religation. BAP digestion is approximately 150mMT at pH 8. Approximately 1 unit of B per 1 μg of vector in the presence of Na'' and Mg'' in RIS. This is carried out using AP at 60°C for about 1 hour. To recover nucleic acid fragments, fertilize the preparation. Extraction with alcohol/chloroform and ethanol precipitation followed by Sephadec Desalt by applying to a G-50 spin gel. Alternatively, the unwanted fragments Additional restriction enzyme digestions can avoid religation in double-digested vectors. I can do it.

For portions of vectors derived from CDNA or genomic DNA that require sequence changes , using site-directed primer-directed mutagenesis. This represents the mutation of interest In addition to eliminating limited mismatches, we also use a primer complementary to the single-stranded phage DNA to be mutated. Performed using imer-synthesized oligonucleotides. In summary, complementary to phage Synthetic oligonucleotides are used as primers to direct strand synthesis; The resulting double-stranded DNA is transformed into a phage-supporting host bacterium. shape Cultures of transformed bacteria are plated in top agar and a single phage-bearing Causes the formation of plaques from cells.

Theoretically, 50% of new plaques are composed of phages that have a mutated form as a single strand. 50% will have the original sequence. Accurately measure the resulting plaque Allows hybridization that matches but does not match with the original strand. at a temperature that is sufficient to avoid hybridization. hybridize with a synthetic primer treated with enzyme. Next, probe and Hybridizing plaques are plated, cultured, and DNA collected. Part special Details of the constant mutation method will be described later in specific examples.

C95, Approval of the Emperor of the Bottom Blood In the following constructs, to confirm correct ligation of the plasmid constructs, first, E. coli MM294 strain obtained from E. coli Genete Inc. Stock Center (CGSC #6135) with the ligation mixture. Successful transformation the body due to ampicillin, tetracytalline or other antibiotic resistance, or Other markers are used to select depending on the mode of plasmid construction. Next, transformation Plasmids from the transectants are optionally subjected to chloramphenicol amplification (Clew ell, D, B, + J, Bacte-riol, (1972) 110:6 67), followed by Clewell, D. B., et al., Proc., Natl.

Acad, Sci, (USA) (1969) 62: 1159 method Prepare accordingly.

The isolated DNA was analyzed by restriction enzyme treatment and/or as described by Messing et al. , Nucleic Ac1ds Res (1981) 9:309 Further described in Sanger, F., et al., Proc, Natl., Acad, S. ci, (USA) (1977) 7↓: 5463 by the dideoxy method, and Maxam et al., Methods in Enz molo (1980) 6 5: Determine the sequence by the method of 499.

C, 6,! ! l jade beans i and soil The host strains used for cloning and expression in this invention are as follows: It is.

Cloning and sequencing and under the control of most bacterial promoters Due to the manifestation of the construct of E. coli? 1M294 strain (listed above), Talmadge , et al., Gene (1980) Uni 235; Mesel-son+M, et al. , Nature (1968) 217:1110 was used as a host.

PLN! I11! For expression under the control of the promoter, E, Co'J K1 2 MC100O lambda lysogen strain N7Nsic18575u-Pso, ATCC 39531 (hereinafter sometimes referred to as MC1000-39531) .

Because of the M13 phage recombinant, E. coli strains susceptible to phage infection, e.g. 12 strains D098 are used for coli.

Strain DC98 was deposited with the ATCC on July 13, 1984, and has accession number 19. It has 65.

D. Cloning of human TNF and Next, we obtained the coding sequence for human-TNF 1 and created an expression vector for this sequence. A method for obtaining expression of a protein of interest is exemplified.

D.1. Human TNF words j, +■ and "shallow," 1.a, Centrifugation of stationary phase HL-60 cells at a high concentration (approximately 2 x 106 cells/ml) washed with RPM11640 medium in the absence of serum, and then plated with 1 x 107 cells. cells/mR. Cells were then incubated at 37°C with constant agitation for 3 1100 n/ml phorbol ester, 12-0 in suspension culture for 0 min. - treated with tetradecanoylphorbol-13-acetate (TPA). culture Centrifuge the material, decant the supernatant, and transfer the cells to 10 μg/ml of condensed ribopolysate. Contains Ca ionophore (LPS) and 10 μM Ca ionophore (A23817). 1 x 107 cells/m in RP medium with II at 37°C under constant agitation. resuspended for an hour. Cells were spun down at 1200 rpm for 10 minutes and then The supernatant was recentrifuged at 800 rpm for 20 minutes. Pour the obtained supernatant, 1. Section b Natural TNF was obtained using the purification method of

D.1. b. Made of bamboo blinds ri, 1. a, About 4-8β supernatant prepared from HL-60 induced in section Amicon Hollow Fiber (1 square foot cartridge/10.OOMW cut) Approximately 300 mj due to off)! Concentrated into Centrifuge the concentrated culture medium to remove cell debris was removed and the supernatant was added to 30mM ammonium bicarbonate buffer (pH 8.2). The conductivity was adjusted to 6.2 mS. Apply this solution to a PMIO (Amicon) membrane. It was further concentrated by ultrafiltration, and the concentrated liquid was centrifuged (20,0 OOX g for 10 minutes).

Next, the supernatant was dissolved in 30mM ammonium bicarbonate/1mM NaCl (pH 8,2). applied to a DEAE ion exchange column equilibrated in the same buffer. Washed with. Both fractions were collected and protein was monitored at 280 nm. these The unbound fraction was assayed using the L-929 cytotoxicity assay and TNF activity was determined using the L-929 cytotoxicity assay. Both fractions were pooled and concentrated again by ultrafiltration.

This concentrate was equilibrated in 30mM ammonium bicarbonate buffer (p) 17.4). The method was applied to Sephadex G75 Super Fine (Pharmacia). same The unbound fraction obtained by washing with the same buffer was measured at 280 nm. monitored and assayed for TNF. Contains peak TNF bioactivity Both portions were lyophilized.

Resuspend the lyophilized protein in Laemmli SDS sample buffer and and electrophoresed on a 5DS-polyacrylamide gel. Cut the gel into two crane sections. cut the protein from each section into 1 mβ of 30 mM ammonium bicarbonate buffer. Elute by diluting into a solution (pH 7,4) and shaking overnight at room temperature. Ta.

Equilibrate sections with TNF bioactivity in 0.1% trifluoroacetic acid (TFA). applied onto a modified Vydac C4 reverse phase HPLC column and using a linear gradient of 0% to 60% acetonitrile in 0.1% TFA. The characteristics were eluted.

Protein was monitored at 280 nm and 214 nm and both fractions were frozen. Bioassay was performed after drying and 30mM ammonium bicarbonate buffer (pH 7,4). Both aliquots containing TNF activity were lyophilized again.

The resulting protein was of sufficient purity to be used for sequencing analysis.

Sequences were determined using a gas phase sequencer (Applied Biosystems) . The sequence obtained from the first 22 amino acids is shown below.

Val -8rg -Ser -8rg -Thr -Pro -Ser -A sp -Lys -Pro -Val -8la -Vat -Ser -Va t - 8 la - Asn - Pro - 1'9 20 21 22 (Gin)-(Ala)-Glu-Gly Furthermore, the purified protein (G -75 gel) from other human tumor and normal cell lines as a substrate. Tested using a modified L-929 cytotoxicity assay used as e). L-9 The G-75 fraction that was toxic in this assay on Hs93 cells was 9T (melanoma), BT-20 (breast cancer), A427 (lung cancer), HT-1 It was also toxic to 080 (colon cancer) and HT-29 (colon cancer). these Both parts are Hs939sk (skin fibroblast), HeLa cell (headache), Hs27 F (foreskin fibroblasts) or C0S7 (monkey cells transformed with SV-40) It was not toxic to the cells.

D.2. 1-F! Ready-made to D The intron-free DNA sequence encoding human TNF is described in this specification. It was prepared by the method. Human promyelocytic leukocytes that produce large amounts of TNF when induced Blood disease cell line HL-60 series (obtained from ^TCC as NoCCL240), c Used as an mRNA source to obtain a DNA library. these cells An oligonucleotide constructed based on the protein sequence determined from TNF purified from Search this cDNA library using a leotide probe to obtain a complete copy of the protein. The code sequence was recovered.

D, 2. a, - U of mRNA Total Metsenger RNA was extracted and purified from HL-60 cells as follows. Ta. Harvesting HL-60 cells for TNF production, 1. Attract as described in a. and a 4-hour cell suspension was obtained by centrifugation. Holocytoplasmic ribonuclei Acid (RNA) was isolated as follows. All steps are performed at 4°C. cells to P Wash twice with SB (phosphate buffered saline solution) and add 10mM vanadyl adenosylate. C Berger, S. L., et al., Biochem, (1979) ±8:5 143) containing IHB (140mM NaCA, 10mM Tris, To 1.5mM Mg (J!z, pH 8)? ! , become cloudy.

Add ethylene oxide polymer type nonionic detergent to 0.3%. lysed the cell membrane rather than the nuclear membrane. 1. Centrifuge at 0OOXg for 10 minutes The nucleus was removed by this. The postnuclear supernatant was treated with TE (10mM Tris, 1mM phenol saturated with M ethylenediaminetetraacetic acid (EDTA, pl+7.5) alcohol/chloroform (+, :1) (0.5% sodium dodecyl sulfate (S DS) and 10 mM EDTA]. Re-extract the supernatant four times. The mixture was removed and centrifuged at 2000×g for 10 minutes to separate the phases.

Prepare the sample to 0.25M NaCβ and add 2 volumes of 100% ethanol. RNA was precipitated by heating and storing at -20°C. RNA 5, Pellet for 30 minutes at 000Xg and wash with 70% and 100% ethanol. and dried. Polyadenylation (polyAM metsenger RNA (mR NA> from total cytoplasmic RNA to oligo dT cellulose. (Av+v+J, et al., Proc, Natl.

Acad, Sci, (1972) 69: 14084412), Suna Specifically, RNA was subjected to ETS (10rnM Tris, 1mM [EDTA, It was dissolved in 0.5% 5DSXp) 17.5) at a concentration of 2 mg/me.

The solution was heated to 65°C for 5 minutes and then rapidly cooled to 4°C. RNA solution After reaching room temperature, it was adjusted to 0.1 M NaCβ and added with binding buffer (50 0mM NaC12, 10mM Tris% 1mM EDTA, p The mixture was passed through a dT cellulose column pre-equilibrated with H7,5). effluent was passed through the column two more times and the column was washed with 10 volumes of binding buffer.

PolyA″ mRNA was eluted with an aliquot of ETS and TE-saturated phenol. Extract once with chloroform and add 2 volumes of NaCβ to 0.2M. of 100% ethanol. RNA was reprecipitated twice, Wash once in 70% ethanol and then in 100% ethanol before drying. It was dry.

Poly A' mRNA was mixed with 10 mM Tris-H (1 (pH 7,4), 1 m M EDTA, 10 mM NaC# and 0.1% SDS fractionation using a gradient. Beckman SW40 rotor medium 38. OO After centrifugation at Orpm for 17 hours, mRNA was ethanol precipitated from the gradient. Collected by. Injecting mRNA into oocytes and making oocyte extracts cytotoxic Both fractions containing TNFmRNA are assayed for activity. was identified. Both fractions containing peak activity are pooled to construct a cDNA library. It was used for the purpose of

D.2. b, cDNA library〇′ oligo dT primin of polyA tail Okayama, tl., et al., Mo1. Ce1l Biol, (1983) 3: 280 (incorporated herein by reference). cDNA was prepared from the 16S mRNA fraction concentrated using this The method yields a high proportion of sufficiently long codons, and there The two vectors described in and readily available from the authors, That is, the pcDV1 and pLl portions are used. The resulting vector is proximal B Contains an insert between vector fragments containing amHI and Xho1 restriction sites. child The vector contains the pBR322 origin of replication and the Amp resistance gene.

Other methods for preparing cDNA libraries are of course within the skill of the art. well known in the world. One now classic method is to use oligo dT printers. Tail formation of two-terminal cDNA by primer, reverse transcriptase, and Bo1JdG, and open at the desired restriction site and tilled by poly dC. using annealing to a suitable vector such as pBR322 or its derivatives. Ru. A detailed description of this alternative method may be inherited by the same successor as the present successor, for example. No. 1,564,224, incorporated herein by reference. (incorporate it into a book).

In the method used in this invention, enriched mRNA (5II g) was denatured by treatment with 10mM methylmercury for 5 minutes at 22°C. , and detoxified by adding 100mM 2-mercaptoethanol ( Payvar.

Fo et al., J. Biol, Chem, (1979) 254: 7636-764 2), cleave plasmid pcDV 1 with Kpml and til it with dTTP. formed and annealed to denatured mRNA. This oligo dT primed m The RNA is treated with reverse transcriptase and the newly synthesized DNA strand is treated with dCTP. A tail was formed. Finally, remove the undesired portion of the pcDV1 vector with HindI [Removed by cleavage with I.

Separately, pLl was cleaved with PstI and a tail was formed with dGTP. cleaved with tlindlll and then cleaved into the pcDV1 vector fragment. Into a more extended polyT-til formed mRNA/cDNA complex, E9 coli ligase is used to ligate and this mixture is ligated using DNA polymerase ■ (Klenou), E. coli ligase, and RNase H. occured The vector was transformed into E. coli 12MM294 to create 8mpR.

D, 2. c, Bu0-7"O"1 provisional An oligomer complementary to the coding sequence for amino acids 8-12 of the purified TNF sequence was Prepared. Due to codon redundancy, a total of 64 different 14-mers were used to code this part. is a candidate for complementarity to Mensen. A total of 64 types of 14-mers were prepared and divided into 4 pools of 16 types each.

Each pool was filled with a concentrated sucrose gradient fraction prepared as described above. mixed with the reduced mRNA preparation and injected this mixture into the oocyte translation system. Ta. Control trials were performed using untreated Metsenger RNA. Live in oocyte system The produced protein was subjected to L-929 cytotoxicity assay (:ls release) and Derived from oocytes injected with a mixture of light and mRNA and three oligomer pools. of the proteins showed activity. In this one hybrid capture” assay, the following sequence :Only oocytes injected with Metsenger treated in a pool with inert Met. The specificity of this oligomer pool was tested for induced HL-60 cells and non-induced HL-60 cells. Concentrated mRNA prepared as above from both induced HL-60 cells, and the corresponding m obtained from cells known to produce lymphotoxin. Further determination using “dot plot” hybridization with RNA fractions did. This pool hybridized well to the induced mRNA, but only The corresponding components from uninduced cells or lymphotoxin-producing cells are hybridized. It didn't work. However, using the kinased pool as a probe This is the 188 (ribosomal) RNA fraction and p It was shown that it contains a sequence that cross-hybridizes with BR322DNA.

Therefore, we synthesized the members of this successful pool as eight pairs of 14-mers, By performing a “bibrid capture” assay using each pair as described above, It was further fractionated. The following array: Only the pair with there were. Fractionated induced HL-60 mRNA fraction, induced total HL-6 0 polyA'' RNA, uninduced HL-60 polyA'' RNA, and pBR322D Dot plot experiments using N The above 14-ma pair is specific and the other pairs are incapable of It was confirmed.

D.2. d. Danyu Jotobi Sword Thief 11゜ D, 2. cDNA prepared above. Using the 14-ma pair identified in c. searched. Pick up 28 colonies that hybridize with the probe, culture them, and Plasmid DNA was isolated. Insert long enough to encode the entire sequence and B. as described above in Section 5. Correct using hybrid translation combined with 358 release format for cytotoxicity assays Several plasmids were assayed for sequence. The hybrid translation assay is This method takes advantage of the fact that the test sequence recovers correct mRNA from unfractionated preparations. is a protein produced by the oocyte translation system injected with the recovered mesosenger. Confirmed by assaying.

The plasmid cDNA to be tested was bound to the filter and the induced H The filter is treated with polyA'' RNA isolated from L-60 cells.

The filter is then eluted and the eluate is injected into the oocyte translation system. The oocytes are White matter was extracted and then subjected to the 353 format of the L-929 toxicity assay. test. Some hybridizing clones designated E2-E4, E6, and E8 The results for pBR3229 B and 24 (A and B were obtained by sucrose gradient? NA is used as a control, El and pBR322 are negative controls. ) Restriction analysis and partial sequencing of the insert revealed two candidate plasmids, pE4 and pBl. 1 appears to have a complete TNF coding sequence. About pE4 The results of this analysis are shown in Figure 2.

Sequenced pE4 and translated all three possible reading frames. The deduced amino acid sequence was determined by N-terminal sequencing of the purified protein. By matching the known N-terminal sequence of natural mature TNF, We identified the correct reading frame for (see Figure 1). mature Amino acid sequences in proteins are numbered starting from valine at position 1. The above In particular, the homology was not complete. However, due to the high degree of homology, the correct c It was shown that the DNA was selected. Experimentally determined limits shown in Figure 2 Proof of the cleavage site is also provided. 1.1, the 3'Pst1 site in the bPstl fragment The old ndlII upstream of After transformation of , it is possible to extract the code sequence as the old ndln cassette.

D.3. The detailed diagram of human TNF derived from the DNA arrangement is shown in Figure 1. The mature TNF protein has a length of 157 amino acids, as deduced from the cDNA sequence. residue and has a molecular weight of approximately 17,354 without glycosylation. Ru. The leader sequence starts from the first available Met start codon and begins at approximately 7 It appears to contain 6 amino acids. Two cysteines remain in 69th and 101st place group is present, leading to the possibility that the active structure contains a disulfide bond.

D.4. -　solid　-8゜-1 D, 4. a, N-〇“codon・ノ In order to express the mature protein, the N-terminal valine (marked as 1 in Figure 1) of the mature protein was added. An ATC initiation codon is introduced immediately preceding the GTC sequence encoding the , and the former ndI just upstream of ATG for ligation to a suitable host expression vector. It was convenient to provide site II. This is a site-directed mutation described in Section C.4. Achieved by induction.

More specifically, a DNA fragment containing the upstream portion of the coding sequence is plucked by the Ps inlet. Excise from E4, isolate by agarose gel electrophoresis, and collect by electroelution. and ligated into the PstI site of bacteriophage M13mp18.

The ligated phage was frozen into competent E and coli strains D G 98 (ATCC #39768) and from Sigma (St. Louis). The obtained isopropylthiogalactoside (IPTG) 5×10-’M and Culture by plating on medium containing 40 μg/ml of X-gal. did. Non-α-complemented white plaques were picked into fresh medium. expected (i, For recombinant single-stranded phage DNA containing inserts of 1 kb) size, We screened ruchi, 1a.

The structure of the desired recombinant phage, designated clone 4.1, was determined using restriction enzyme analysis. Teli! Approved.

The following array: 5’-GAAGATGATCTGACCATAAGCTTTGCCTGGG Chemically synthesized and purified 33-mer oligodeoxyribonuc having CC-3' G encoding the first amino acid (valine) of the mature TNF protein using leotide. A former ndIII restriction enzyme site and an ATG-initiation coton were introduced before the TC codon. .

10 pmole of oligonucleotide was added to l Q m M NaCl-%20 mM Tris-HCA’ (pH 7,9), 20 mM MgC6z and at 67°C in a mixture 15μβ containing 20mM β-mercaptoethanol. 2.6 μg of ss clone by heating for 5 minutes at 42°C and 25 minutes at 42°C 4.1 DNA. Cool this annealed mixture on ice. and then 0.5mM of each dNTP, 17mM Tris-H (J ! (pH 7,9), 17mM Mg1z, 83mM NaCf, ], 77m DN A polymerase I Klen with 5 units of Mβ-mercaptoethanol The reaction mixture containing the ow fragment was prepared to an initial volume of 25 μp and incubated for 1 hour at 37°C. Incubated for a while. The reaction was stopped by heating to 80°C and this The reaction mixture was used to transform Combiten) D098 cells and plated on agar plates. Phage plaques were obtained by plating and incubating overnight.

Plates containing mutated clone 4.1 plaques and non-mutated clone 4.1 plaques. 4. Cool the two plates containing the Farziptak to 4°C and Place the first dry filter on the agar plate for 5 minutes, then place the second filter on the agar plate. Remove phage plaques from each plate by incubating two nitric plates for 25 minutes. Transferred to cellulose. Next, this filter was mixed with 0.2NNaOH, 1.5M 5 minutes on a thick filter soaked in NaCl and 0.2% Triton X-100. Interval, and Q, 5 MTris-11C (1 (pif 7.5) and By placing the filter on the filter soaked in 1.5 M NaCe for an additional 5 minutes. Neutralized. This filter was washed in the same way on the 2XSSC soaked filter. Cleaned, dried and heated in a vacuum oven at 80°C for 2 hours. 2 fi 10rrl of DNA hybridization per filter for 4 hours at 42°C. tion buffer [5x SSC, pi (7,0,4x Denhardt's solution (polyvinyl Lupyrrolidone, Ficoll and bovine serum albumin, 1x - 0.02% each), 0 ．． 1% SDS, 50mM sodium phosphate buffer, pH 7.0, and 100μg /mE denatured salmon sperm DNA) was pre-hybridized. Primer 32p-labeled proteins are generated by kinase treatment of 32p-labeled protein with labeled ATP. Lobes were prepared. Add 1 to 5 ml of DNA hybrid per filter to the filter. 32p-labeled platinum of 5×1106 cp/mβ in diization buffer Hybridization was performed on the imer for 8 hours at 64°C.

Filters were incubated at room temperature for 10 minutes in 0.1% SDS, 20mM I. (buffer) and once in eXSSC; 20 min at 37°C, buffer and 2XS once in SC; once in buffer and 2X SSC for 20 min at 50°C; and This was followed by washing at 60° C. for 20 minutes in buffer and IX5SC.

Filters were air dried and autoradiographed for 4 hours at -70″C. did. A new HindI [[option to create a restriction site] is inserted into the mutated clone. Now that you have selected a oligonucleotide primer, hybridize with this primer. RF-DNA from a number of clones was digested with this restriction enzyme. new Pick one of the mutated clone 4.1 plaques with the old ndI[I restriction site, and inoculated into a culture of DG98, 5sDNA was prepared from the culture supernatant, and dsRF-DNA was prepared from seven cell pellets. Correct sequence to dideoxy sequence Confirmed by decision method.

Isolate the correctly synthesized strand and transform it by Pstl and old ndn [(partial) , or old ndlI[ and ligated into the expression vector.

D.4. b. Vector height For prokaryotic expression, the coding sequence (along with some 3' untranslated nucleotides) is It was cut out from sM13-AW701 using two methods.

In the first method, dsM13-AW701 is digested with Pstl and then Partially digested with old ndlI[ and old ndlll-Pst I TN F code arrangement. Got a line. (Because there are several old nd111 sites in M13-AW701, ndlI+ partial digestion is required. ) Partial digestion of DNA fragments results in complete digestion of DNA. This can be done by using 1710 of the amount of restriction enzyme required for total digestion. Ru. The mixture is incubated at a temperature appropriate for the enzyme and quenched. Aliquots of the reaction mixture were removed at 10 minute intervals for up to 1 hour. Then these An aliquot of was loaded onto a gel and the DNA fragments were analyzed. DN required The time point that gave the highest yield of A fragment was used for preparative digestion with restriction enzymes. , and the appropriate fragments were purified from the gel by electroelution.

The PstI/BamHI fragment containing the 3'-noncoding sequence of the TNF gene was , purified from pE4 after digestion of the DNA with the enzymes PstI and BamHI. .

HindlI[/PstT fragment and PstI/Bam)I fragment are the same This constitutes the coding sequence + 600 bp 3' untranslated portion of the DNA.

The two fragments were transformed into the old ndm/BamHI digested host vector pT as follows. Connected to RP3.

pTRP3 (see below) is an E. coli trp promoter and ribosomal Contains a protein binding site. pTRP3 was digested with old ndI [[ and BamHI; The vector fragment was purified on an agarose gel. Next, add the isolated fragments described above. old ndI[[/Pstl segment and PstI/BamHI segment E. coli MM29 using a three-way linkage and using this mixture. 4 was transformed into All1pH to obtain pAW701.

In the second method, dsM13-AW701 was digested with old ndI[I; The fragment containing the gene was then isolated on an agarose gel. This isolated fragment The fragment was ligated into pTRP3 cleaved with HindI[I and treated with BAP. and transformed into E. coli MM294 to obtain pAW702.

Contains PL promoter and Bacillus positive retroregulatory sequences pFC54,t (ATCC39789) or pPLOP (see below) ) can also be used as a host vector. These vectors are Ill and BamHi and the large plasmid fragment containing the control sequences was extracted. Purify the pieces on agarose. Old ndI of TNF gene prepared as above The II/PstI part and the PstI/Bamt part are connected by three-way connection. 7 into the old ndIII and BamHI sites of the vector to create plasmids, respectively. pAW711 and pAW712 are obtained.

Alternatively, the purified 1lindIII fragment from pH 4 can be ] pFC54 opened with T and treated with BAP, or by ligating it to pPLOP. pAW713 and pAW714 are obtained, respectively.

D.4. c, TNF in the primordia - pAW701 and pAW702, E, Co. 294 and cultured under conditions that repress the trp promoter. Proliferate. After induction by tryptophan depletion, TNF production begins. It was. Similarly, pAW711 was constructed and E. coli MC1000-39 531 and cells were induced by high temperature. several hours under induction conditions After culturing, the cells were sonicated and the sonicated product was determined by L-929 cytotoxicity assay. was confirmed to contain TNF. The results are as follows.

Plasmid TJ/m! 701 1, 3 x 10' 702 1, 3 X 10' 711　2　XIO’ Units of INF activity are as defined in B, Section 51.

D.4. d, Makiritachi, Chichuoki 11 Doyoki LΔ, Satomori, 2. d-term cDNA The vector pB11 isolated from the library is capable of acting on the TNF coding sequence. Contains the SV40 promoter linked to the SV40 promoter. Hybridized to 28 positives All of the colonies containing this linkage (including especially pH4 and pBll) contain this linkage. is expected to be, and therefore can be expressed in a suitable mammalian host. subordinate Therefore, pBll was used to transform CO3-7 monkey kidney cells, and the cells were transformed into CO3-7 monkey kidney cells. Cells were cultured under conditions that induce induction of the SV40 promoter. Signal sequence is pB TNF is still present in the culture medium and functions in mammalian cell systems. secreted into the earth.

TNF in the supernatant on the CO3-7 cells that formed a monolayer was The following results were obtained.

Plasmid 3SS (transferred by λ modified ratio) B 11 22,763 E9 (negative control) 2.739 -DNA 2,565 D, 51 Sarasayu Σri and D.5.a-Fukiji E. coli D.G.95 transformed by p.711 (MC100O-59 A lambda lysogenic strain similar to 531) was grown at 37°C in standard growth medium at approximately 0.5 O D b. . and then induced by increasing the temperature to 42°C. guided. After 2 hours, cells were sonicated and L-929 cytotoxicity assay ( (above) was used to confirm that the sonicated product contained TNF activity. next, Apply the sonicate to a DEAE Sepharose column (Pharmacia) and gently Wash with buffer solution (10 m MTris, pH 8, 2, 1 m NaCj2) Purified. 10m MTris (pt+8.2) 0.02M, 0.04 TNF activity was inhibited by stepwise extraction with M, 0.1 M, and 0.8 M NaCA. Both fractions were obtained.

Most of the TNF1 activity was produced in 0.04M NaCβ).

Both of these fractions were concentrated by ultrafiltration and then passed through a phenyl TSK-5PW column. Further purification was performed by IPLc (using LKB). TNF is 0.1 In the presence of 1.8M ammonium sulfate in M sodium phosphate (pl+7.0> The ammonium sulfate concentration decreases to When developed, it eluted at about 0.4M ammonium chloride. Including T　N　F Both fractions were concentrated by ultrafiltration and loaded onto a GH75 size fractionation column (Amico pure TNF was obtained.

D.5. b. TNF mutin\ Isoelectric focusing showed that TNF prepared as described in the previous section was 5.8 to 6. It was shown to consist of several species with different pi values between 5 and 5. All major species are It was shown that it was the expected mature TNF (mTNF), but the contaminated mutin form 4TNF was also present. Isoelectric focusing gel results indicate the existence of numerous variants of TNF. It was shown that

To separate mTNF from 4TNF on a preparative scale, use the method described in Section D, Section 51. The EDTA Sepharose column was eluted using high fractionation, and the main mTNF Before the elution of the peak, both 4TNF-enriched cells in about 0.03N NaCl were added. I got my share. Enriched 4TNF4TNF was contracted and under the above conditions 11 It was applied to a phenyl TSK-5PW column for PLC. enriched ryobun ) IPLc is determined by mTNF-containing peaks at about 0.4 ammonium sulfate as described above. and reverse from 0.1M phosphate (pH 7) to deionized water. Apply the purified TNF eluting in the gradient to the column with deionized water. used. The 4 TNF peak fractions were concentrated and run on a GH75 as described above. homogeneous 4 with a pr of 5.8 in isoelectric focusing on a gel. I got TNF. .

obtained from the above GH75 purification step, identified by absorption at 280 nm. Zero-order results were obtained by testing the specific activity of both components, including the TNF peak.

fraction protein concentration biological activity specific activity tlh (mg/ml) (U/m j! ) (U/mg) 16 0.043 4.7X10' 1. lX10”1 7 0.091 1.9 x 10' 2. IX 10”18 0, 102 1.4 X 10" 1.4 X 10" 19 0.063 7. Ox 106 1,1 x 10'20. 0.035 2゜4 x 10' 6.9 x 1 The average specific activity that is consistent across the expected peaks for 0' pure protein is 1. 3 XIO' U/m g. This is about 10 times higher than that of mTNF. .

D. The compositions were compared with the following results.

Amino acid predicted composition TNF, TNF, difference (cDNA) p15. L6.5 pH5,8 TNF+-TNFzAsx 12 12.7], 2.4 0.3 Thr 6 6.2 6.1 0. l 5er 13 12.6 10.9 1.7 Glx 20 20.5 20.2 0.3Pro 10 10.1 10.3 -0.2cry 11 11.1 11.1 0.08 Ia 13 13.4 13.1 0.3 Val 13 10.9 10.2 0.7Met OO,200,2 1ie 8 6.5 6.6 -0. ILeu 17 18.7 18.5 0 ．． 2Tyr 7 6.9 7.0 0. I Pike 4 4.0 4.0 0.0Lys 6 6.1 6.2 -0. I His 3 3.0 3.0 0. O Arg 9 9.0 8.0 1. O n = 152 151.9 147.4 4.5 Both proteins were compared in terms of composition. However, if the composition of 4 TNF lacks two serine, one valine, and one arginine residues It seemed like that. Removal of these residues from the N-terminal sequence is performed using an automated protein sequencer. - Confirmed by sequencing of 4TNF above, the sequence of the first 10 amino acids is It was given as follows.

A comparison of this sequence with the deduced amino acid sequence in Figure 1 shows that this sequence corresponds to the first four amino acid sequences. Lacks the mino-terminal residue Val-Arg-Arg-3er but matches the position following it. It is shown that it has an N-terminal sequence. The following array: 5’-CACTCGGGGTTCGAGACATAAGCTTTGCCTG Chemically synthesized purified 15-mer oligodeoxyl with GGCC-3' nucleotides and thereby the methionine initiator The 12 nucleotides encoding the four N-terminal amino acids downstream from the don are removed. Ta.

l　Q　pmole oligonucleotide was added to 2.6 μg of SS clone 7M13- AW701DNA, 100mM Na1J, 20mM Tris -HCj2 (pH 7,9), 20mM MgC1z and 20mM β-mercap at 67°C for 5 minutes and at 42°C in 15μβ of a mixture containing ethanol. Hybridization was performed by heating for 25 minutes. annealed mixture Cool on ice and then add 0.5 mM each dNTP, 17mM Tri s-H(J (pH 7,9), 17mM hgcI22.83mM NaC , g, 1.7mM β-mercaptoethanol, and 5 units of DNA polymer. Prepare the reaction mixture containing the Klenow fragment to a final volume of 25 μβ. , and incubated for 1 hour at 37°C. The reaction is started by heating to 80°C. stopping and using the reaction mixture to transform Combiten 1-DG9B cells; Plate on agar plates and incubate overnight to remove phage plaques. I got it.

Plates containing mutated clone M13-AW701 plaques and non-mutated Two plates containing clone M13-AW701 phage plaques were incubated at 4°C. and place the first dry filter on the agar plate for 5 minutes. Remove phage pullers from each plate by placing a second filter for 25 minutes. The sample was transferred to two nitrocellulose filters. Then add these filters to , 0.2 M Na, 1.5 M NaCl and 0.2% Triton X-100. Place on soaked thick filter and 0.5 M Tris-HCj! ( Place on the filter soaked in pH 7.5) and 1.5 M NaCj2 for 5 minutes. It was neutralized by this. Filter immersed in 2xSSC in the same way Washed twice on top, dried and then heated in a vacuum oven at 80°C for 2 hours. did. Two filters were heated at 42°C for 4 hours, 10 ml of DNA per filter. Hybridization buffer C3XSSC (pH 7,0) 4x Denhardt's solution (Polyvinylpyrrolidone, Ficoll and bovine serum albumin, 1x - each 0.02%), 0.1% SDS, 50mM sodium phosphate buffer (pH 7.0 ) and 10 (bJg/m7! of denatured salmon sperm DNA). Ta. The filters were subjected to DNA hybridization of 1 to 5 mj2 per filter. 32p-labeled primer at 5 x 10' cpm/mβ in buffer at 64°C. The mixture was hybridized for 8 hours.

Filters were soaked in 0.1% SDS, 20mM sodium phosphate for 10 minutes at room temperature. (buffer) and once in 5XSSC; 20 min at 37°C, buffer and ZXSS once in buffer and 2×5SC for 20 min at 50°C; and finally Washed in buffer and IX5SC for 20 minutes at 60°C.

Filters were air dried and autoradiographed for 4 hours at -70°C. Ta.

RF-DNA from positive clones was treated with 1lindIII and mutated The fragment containing the TNF coding sequence was isolated by gel electrophoresis. The collected distribution The column was ligated into old ndIII-cleaved and BAP-treated pAW711 to create pA M736 was obtained. The presence of a 12 nucleotide removal in the former ndlI [and pvu Confirmed by restriction analysis by II. 146b produced by pAW711 pAW736 is 134 bp compared to the pH4nd III/Pvu II fragment. Contains the l1indnl/Pvu II fragment.

pAW736 was deposited with the ATCC on April 10, 1985, and is designated No. 53. It has an accession number of 092.

E. coli DC95 transformed with pAW736 was grown as described above. And guided. The sonicate was prepared as described above and aliquots were divided into 1 Analysis was performed using 2.5% 5OS-PAGE. 4TNF exactly as described above. isoelectrically purified from the sonicate and found to be identical to the previously prepared 4TNF Shown by spot electrophoresis. Isolated 4TNF was subjected to L-929 cytotoxicity assay. and was shown to have a specific activity of approximately 2X10''U/mg. .

D. By exactly the same method as described above in Section 6, compared to the arrangement shown in FIG. TNF-depleted mutins lacking the first 3 to 11 amino acid residues were prepared.

Figure 3 shows the name of the resulting vector and the site characteristics used to cause the deletion. Oligomers used in asymmetric mutagenesis are shown. This figure also shows 156-115 0-1 and 1.4O-TNF, and des-ser3des-ser4 and v Names of al+5ser+6 myetein vectors and used for their construction. The oligomers used are shown below.

5er5 is similar to Ser + o + mutin, and the oligonucleotide command is changed. Using mutagenesis, CySbq, which has TNF activity, was changed to other amino acids. and/or cys. , is replaced with Obtaining a mutant TNF gene encoding a mutin that is present or deleted . Preferred oligonucleos for exemplary conversion of CyS69 to 5erbq Tidobrimer is 5'-CATGGGTGCTCGGGCTGCCTT- It is 3'. This oligonucleotide pairs with codon 69 of the TNF gene. Has a T-A change in the libretto. Similarly, it pairs with the codon at position 101. CyS, using primers containing changes corresponding to the triplet. , Se r　l　can be converted to OI.

D, 4. Clones prepared by Pst treatment of pE4 as described in section a. 4.1, D, 4. Substantially the same as described in paragraph a, but with the following Rymer: 5’-CATGGGTGCTCGGGCTGCCTT-3’ This is 69th place but the change from TGC to AGC site-directed mutagenesis using I put it on. Mutated plaques were identified as described above and confirmed by sequencing. did. One plaque, MB-AW731, containing the mutation of interest was incubated with Ava I and pjV digested with PstI and this fragment was digested with Pstl/Aval. Connected to 711. Transform the ligation mixture into E. coli MC1000/39531. Amp” and use primer-probes to identify the transformants in the correct configuration. Screened for columns. One colony, designated pAW731, was transformed. was used for the expression of the sequence. pAW731 was transferred to ATC on January 25, 1985. It has been deposited with C and has accession number No. 53007.

Similarly, ser. , pAW74, an expression vector for TNF Made around the world.

TNF muti with cysteine-69 and/or cysteine-101 removed mute in which the N-terminus or C-terminus has been removed using a DNA sequence encoding a transform the pin to obtain the corresponding “double” mute shape. These muti expression vectors for DNA coding regions containing these variants. Fragment the H part using an appropriate restriction enzyme and insert it into the vector prepared above. Created by

5erbQ, 513r+O+s and 5Gr69Ser+oIT N F type. obtained from plasmids pAW731, pAW732, or pAen735, respectively. Special Plasmid pAW731 has been extensively described previously, and pAW732 and pAW 735 is Ser + o T N F and 5erbqserlo + T N F and manufactured in the same manner. The expression vector thus obtained is E.

coli, and the cells were cultured and induced as described above to achieve the desired of protein. The resulting TNF mutin is comparable in activity to mTNF. be.

D.7. b, Mu-in no Dan E. coli MC100O-3951 carrying p.731 was grown and D. ,5. a. Induced at elevated temperature as described in section a.

Sonicates from induced cells were assayed and pAW711 transformants was found to have approximately the same TNF activity per ml as . however, SDS analysis showed that the amount of 17kD TNF protein in these extracts was about 5 times lower; The specific activity of 5eriqTNF is higher than that of the unchanged protein. It has been shown.

The purified (95%) intact protein was treated with DTT and Reduced and alkylated by treatment with iodoacetate. untreated The protein had an activity of 2.6X10'U/ml, but the reduced protein protein, or reduced and alkylated protein is 4.4 to 4.8 XIO' U/m It had β activity.

Processing activity No DTT 2.6X10' 0.1n+M DTT 3.3X10'1mM DTT 4.8X10' 2mM DTT 3.9X10' 10mM DTT 1.2X10' 20mM DTT 1.7XXO' Buffer + 2.4mM IAA 1.5X10’1mM DTT + 2.4mM IAA 4.4X10'pAW711 in exactly the same way as described above 3, which encodes an N-terminal deletion mutin of TNF. transform E. coli, and the transformed cells are cultured and induced. and obtained the production of the target protein. Same as above for mTNF and 4TNF. The recombinant protein thus obtained was purified and assayed in a similar manner. L-92 In the 9-cytotoxicity assay, the specific activity of these mutins was higher than that of 6-8 TNF. A bell-shaped curve with the maximum profit is shown. The specific activity of these mutins is similar to that of mTNF. It is calculated to be 5 to 10 times higher than that. These results are shown in FIG.

Furthermore, these mutins have a more homogeneous form than mTNF under conditions of m-conversion production. It seems to be produced in This is illustrated in Figures 5 and 6, which are for SDS polyacrylamide gel electrophoresis and isoelectric focusing gel, respectively. Figure 3 shows purified preparations of mTNF and various mutins when isolated. each The figure shows pAW711 (mTNF) and 4, 6, 7, 8, 9. Reach and lacking the 10 N-terminal amino acids (p736, pAne739, pAW737, Results are shown for the expression products of pAAl2O2p741 and p742.

In Figure 5, all proteins appear to be homogeneous and the preparations are isolated proteins. It shows that quality is pure in terms of size. However, the results in Figure 6 is a mixture of proteins whose pAW711 (mTNF) expression products have different pl values. , thus indicating variations in the side chains of the primary sequence.

The product of pAW73G appears to contain a small amount of protein with modified side chains. However, the expression products of other plasmids appear to be clean.

D.8 In vivo assay '7- The recombinantly produced mTNF (rTNF) is B, 5. b. It was active in an in vivo assay. Figures 7a and 7b are mouse 0.5, 1.0 and 2 of rTNF on fibrosarcoma and proliferation and host survival. ．． The effect of 0 μg injection is shown. In these assays, each indicated amount of TNF Dosage level indicates the amount per individual injection. Injections begin 9 days after tumor implantation and continued every other day for a total of 6 injections. Figure 7a shows the TNF injection. Figure 3 shows the effect of TNF on tumor growth starting from time point 0 indicating initiation. Along the bottom line The arrow indicates the time of administration. These results demonstrate that 6 x 0.5 μg r We show that even TNF prevents a significant increase in llff1 tumor volume compared to controls. are doing. Figure 7b shows time 0 as the last day of TNF injection, i.e. Figure 7a. The survival rate starting from 10 days) is shown. This result shows that the survival rate (%) is at 3 dose levels. ・MCF-7 tumor implanted Intravenous TNF injections were started 7 days after tumor implantation in the mice. first injection (2 μg) was toxic (5 out of 10 mice died) and a total of 5 follow-up Subsequent injections, given every other day during treatment, were at 1ag/mouse. to the mouse In addition, 1 g of estradiol was administered intramuscularly on days 0, 2, 4, 7, and 9. and injected for 11 days. Tumors in mice injected with TNF for 14 days The volume increased slightly from 300' to 50mmff, whereas that of the control was 30f1 3 to 130mm'.

D.9. -Remains of death pPL OP was deposited with the ATCC on December 18, 1984, and It has the number No. 39947. Its creation is described below.

D.9. a. Replicative type juxtaposition pCS3 replicates at high temperatures leading to high copy numbers of pPI, OP host vectors Provide a starting point. Its creation is based on a US patent filed on October 14, 1983. 1b541.948 (which is incorporated herein by reference). (incorporate). pC33 was deposited on June 3, 1982, and has accession number It has the number ATCC 11h39142.

pC33 is derived from pEW27 and pOP9. pEW27 is E, M, Won g.

Proc, Natl, Sci, (USA) (1982) 79: 35 70.

This substance contains mutations that bring about temperature control of copy number near its replication origin. Ru. These mutations result in high copy number replication at high temperatures; However, at low temperatures, low copy number replication occurs.

pOP9 is a high copy number plasmid at all temperatures, and this one El type plasmid pOP6 (Gelfand, D. et al., Proc. Natl, Acad, Sci, (USA) (1978) 75: 58 Insert the EcoRI/Pvu II start point-containing fragment from 69) into pBR322. It was created by Prior to insertion, this fragment was transformed as follows. 5 0 μg of pOP6 was completely digested with 20 units each of BamHI and 5stl. did. Remove the 5stT3' overhang and fill in the Bam 5' end. In order to convert the digested pOP6 DNA into E. coli DNA polymerase I ( enow) at 20°C to first remove the 3'5stl overhang. , and then treated in a two-step reaction at 9°C to repair the 5′ end. . Digest the blunt-ended fragment and use 0.02 μmole to create competent DG. 75 (0' Farrell, P, etc.) acteriolo (19 78) 134:645-654] was transformed. 50μ of transformants g/mβ ampicillin on a plate containing 3.3 kb Regarding the loss of the Sst 1 site and the presence of the newly formed old Bam site Screened.

One candidate, designated pOP7, was selected and 25 μg of pOP7 was injected into 20 units. E. coli DNA polymerase I fragment (Klenow ) and religated with T4 DNA ligase. One site was removed. Competent DG75 was treated with 0.1ag of DNA and Transfer the transformants onto plates containing 50 μg/m +2 ampicillin. I selected it. Candidates were screened for loss of the Bam1 day restriction site. p I chose OP8. To obtain pOP9, AvaI from pBR322 (repaired )/EcoRI Tet” fragment was prepared and isolated and extracted from pOP8. ligated to the isolated PvulI (repaired)/EcoRI 3560 bp fragment.

1.42k bEcoRI/Ava I (repair) Ter” (fragment A) and 3 ．． The connection of 56k b EcoRI/Pvo II Amp” (fragment B) is In a two-step reaction, 0.5 μg of Fragment B and 4.5 μg of fragment A were used.

Conbiten) DG75 was transformed with 5 μl of the ligation mixture and the transformants Selection was made on plates containing ampicillin (50 μg/m6). AmpRTet pOP9 isolated from R transformants is high copy number, colicin resistant, EcoR I, BamHI.

1 restriction site for pvo II and old ndlII, 2 for old ncII Restriction sites were shown, as well as the appropriate size and Haem digestion pattern.

To obtain pC33, 50 μg of pEW27DNA was added to Pvu II and E It was completely digested with coRI. Similarly, 50 μg of pOP9 was added to Pvu n and E Complete digestion was performed with col'I T, and the 3.3kb fragment was isolated.

0.36 μg (0,327 pmole) of pEW27 fragment and 0.35 μg (0,16 μmole) of pop9 fragments and form E. coli 294. It was used for quality conversion. AmpRTetR transformants were selected. good result koro The needles were first incubated on β-lactamase assay plates at 30°C and 41°C. After cleaning and then growing at 30°C and 41°C 6,' plus Screened for mid DNA levels. A successful candidate called pC33 was confirmed by sequencing.

Ll, 9. b. PLNR phage promoter and N-remains The DNA sequence containing the ribosome binding site for the gene (NRII5) was added to pFC. 5 and finally ShimaLake and Rosenberg, Nat. Derivatives of pKc30 published by ure (1981) 292: 128 Obtained from. pKc30 is l1indnl/BamHI from pBR322 A 2.34kb fragment from camelid phage cloned into a vector fragment contains. PL promoter and NR11, Bgl II and H in pKC30 It occupies between the paI sites. pKc30 is BglT converted to HcoII 1 site It has an I site.

BglII immediately preceding the PL promoter was inserted into the EcoR1 site as follows. Converted. pKc30 was digested with BglIl and digested with Klenow and dNTPs. repair and add New England bismodules to the EcoRI linker with T4 ligase. (obtained from Orapus) and converted to E. coli with 12 strains and 294 lambda 7. did. Plasmids are isolated from AmpRTet' transformants and the desired sequences was confirmed by restriction analysis and sequencing. Pvu the resulting plasmid pFC3 Double digestion with I and IIpaT yielded an isolated fragment of approximately 54 obp, and treated with Klenow and dATP, then treated with Sl nuclease to remove the 3' end. Plain with the end sequence -AGGAGAA (-AGGAGA part is NRII3) A slip-end fragment was generated. This fragment was restricted with HcoII I and 5' -EcoRI (sticky end) and H4nfl (partial repair, S1 blunt) -3' end A 347 base pair DNA fragment is obtained.

To complete pFC5, pβLZ15 was used to inject tlin into the 3′ of N, l, . A dII1 site was formed. pβI-Z 1-5 was AT on January 13, 1984. CClTh39578 and L7 were deposited. This is fused to ATG + 1acZ. The sequence containing 140 bp of combined β-IFN was fused to pBR322. It was prepared by In pβl-215, EcoR of p[1R322 one site is maintained, and the insertion is immediately adjacent to the ATG start codon of β-IFN. Contains the preceding old ndl11 site.

pβI-Z15 was restricted with old ndlll and repaired with Klenow and dNTPs. and then digested with EcoRI. The resulting EcoRI/BindlI The I (repair) vector fragment was added to the above EcoRI/1linf I (repair) fragment. ligate the fragments and use the ligation mixture to transform MC100O139531. Ta. Transformants containing successful constructs were grown on lactose minimal medium at 34°C. It was identified by its ability to grow at 30°C but not at 30°C. (3 transformants X-gal-Ampmp-rates at 0°C and 34°C and 30°C and and plated on minimal lactose plates at 34°C. appropriate structures Transformants with X-gal-Ampmp-rate are superior at both temperatures. However, it grows only at 34°C on minimal lactose medium. ) good results constructs was designated pFc5.

D, 9. c, l1 Next, pC33 was modified to provide PL and NRH5 restriction sequences. pC33 was digested with tlindII and then with EcoRI.

Isolated EcoRI vector fragment from pFC5 containing PLNRB3 /1lindm and transformed into E. coli 294. isolated The correct composition of the assembled plasmid DNA is confirmed by restriction analysis and sequencing, and The plasmid was named pPLOP.

D, 10. Yuho pTrlP3 was deposited with the ATCC on December 18, 1984, and has accession no. It has Th39946. The construction is as follows.

Construct a host vector containing the trp control sequence after the tlindIIT site Therefore, it lacks the attenuator region (trp promoter/operator/ribosome). pVI binding site sequence obtained from Stanford University C, Yanofsky. Obtained from I153. The trp sequence can be found on various plasmids well known in the art. obtained from. Hhal pVI+153 (this leaves the sticky ends exposed). (to cut just 5' of the trp promoter) and Kleno The ends were blunt-ended with w and partially digested with TagI. trp reader The restriction at the Tag 1 site, which is 6 nucleotides preceding the ATG start codon of A 99 bp fragment corresponding to the pTRP3 was obtained by ligation to I-digested pBR322.

The plasmids listed below are from the American Type Culture Collection, Le, MD, USA (deposited with the ATCC. These deposits are The provisions of the Budapest Treaty and its Regulations on the International Recognition of Deposits (Budapest Treaty) The same was done. This would allow maintenance of viable cultures for 30 years from the date of deposit. Guaranteed retention. These organisms are classified under the Budapest Treaty and under applicable U.S. special regulations. Pursuant to an agreement between the applicant and the ATCC that guarantees unrestricted availability after the license becomes effective. will be made available by ATCC. Availability of deposited strains , infringing the rights conferred under the authority of any government under its patent laws. It shall not be construed as a license to practice any invention.

Plasmid CMCCNo ATCCNo deposited pE4/E, coli MM294 2318 39894 October 15, 1984 pAW711/ E. coli DG95 2162 39918 November 8, 1984 p AW736/IE, coli DG95 2317 53092 1985 April 1st pAW742/E, colt DG95 2345 53161 June 21, 1985 pAW741/E, colt DG95 2344 53162 June 21, 1985 pAW739/E, coli DG9 5 2342 53163 June 21, 1985 plV737/E, co li DG95 2340 53164 June 21, 19B5 pAW740 /E, coli DG95 2343 53165 June 21, 1985 p AW731/E, coli DG95 53007 January 25, 1985 FIG. 2 Procedural amendment (formality) January 1986 Commissioner of the Patent Office Kuro 1) Akio 1.Display of the incident PCT/US 85101921 , name of invention human tumor necrosis factor 3. Person who makes corrections Relationship to the incident: Patent applicant Name: Sitas Corporation 4. Agent Address: Shizuko Toranomon Building, No. 8-1o, Toranomon-chome, Minato-ku, Tokyo 105 Phone: 504 -07215, date of correction order December 23, 1985 (Shipping date) 6. Subject of correction (1) Translation of the description and claims (2) Power of attorney 7. Contents of correction (1) Translation of the description and claims (no change in content) (2) As per attached sheet 8. List of attached documents (1) Translations of the specification and scope of claims (1 copy each) (2) Power of attorney and its translations (1 copy each) 1 letter international search report lmfialnMI A*1mmM N, PCT/US 85101921AN NEX To T)! E INTERNATIONAL 5EARCHREPO RT ON

Claims (19)

[Claims] 1. Recombinant human tumor necrosis factor (TNF). 2. Recombinant TNF comprising the amino acid sequence shown at positions 17-157 in Figure 1 . 3. TNF (mTNF) according to claim 1 having the amino acid sequence shown in FIG. ). 4. TN according to claim 1, which is a mutein from which cysteine has been removed. F. 5. ▽1-, ▽3-, ▽4-, ▽5-, ▽6-, ▽8-, ▽9-, and ▽10- TNF according to claim 1 selected from the group consisting of TNF. 6. Claims having the following N-terminal sequence: TNF according to paragraph 1. 7. Claim 1 having the following N-terminal amino acid sequence: described in T.N.F. 8. Recombinant DNA encoding human TNF. 9. The amino acid sequence of the encoded TNF is shown in positions 17-157 of Figure 1. The DNA according to claim 8, comprising: 10. Claims that the encoded TNF has the amino acid sequence shown in Figure 1 (mTNF) DNA according to Range Item 8. 11. Claims that the encoded TNF is a cysteine-removed mutein DNA according to Range Item 8. 12. The coded TNF is ▽1-, ▽3-, ▽4-, ▽5-, ▽6-, ▽8- , ▽9-, and ▽10- TNF. DNA as described. 13. The encoded TNF has the following N-terminal sequence: Ser-Asp-Lys-Pr The DNA according to claim 8, which has o. 14. The encoded TNF has the following N-terminal amino acid sequence: [Sequence available] of a claim with DNA according to Range Item 8. 15. Expressing the DNA of claims 8 to 14 in compatible host cells. A recombinant DNA comprising control and coding sequences effective for. 16. Recombinant host cells transformed with the DNA of claims 8 to 14 . 17. Recombinant host cells transformed with the DNA of claims 8 to 14 A method for producing human recombinant TNF, which comprises culturing. 18. In a mammal, the active ingredient is the TNF of claims 1 to 7. Compositions useful for performing tumor necrosis. 19. Antitumor efficacy of recombinant human tumor necrosis factor defined in claims 1 to 7 A method of treating a tumor by administering an amount to a host.