JPH03504729A - A fusion CD4 polypeptide produced by the fusion of a substance with affinity for maltose (MalE) and a CD4 protein fragment with neutralizing properties against the HIV virus. - 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] A substance with affinity for maltose (MalE) and ) against ITV virus. The fused CD4 polypeptide generated by fusion with a τM-gment that has neutralizing properties Petit de The invention relates to at least a portion of the CD4 (also referred to as T4) molecule, more particularly The region consisting of the region near the N-terminus that contains the HrV virus fixation site and the region that is compatible with maltose. resulting from fusion with a bacterial periplasmic protein (MalE) that has affinity for The present invention relates more particularly to hybrid polypeptides of this type. CD4 min isolated from polypeptides and also from these hybrid polypeptides It concerns the said part of the child.

More particularly, they provide that the various said hybrid polypeptides are Origination of the corresponding DNA sequence in Escherichia coli (E, Co11) under the control of the element These expression products are transported to the bacterial periplasm and extracted. and preferably purified in a single step by i, t affinity column, If so, the sequence of the CD4 protein or the above part of the CD4 protein is the basic sequence. so that the desired polypeptide is cleaved and recovered from the hybrid polypeptide. It was treated with a protease selected as follows.

In this specification, the former abbreviation V refers to AIDS (AIDS) in the body of a host, especially a human. Refers to all retroviruses that can be induced. Therefore, HIV is IIIVI, l ! IV2 or all pathogenic variants of these viruses; numbers in parentheses is the number of the reference list attached to this specification.

Protein CD4 is a T lymphocyte (helper/inducer) integral membrane glycoprotein. It is of good quality. The outer part (located on the N-terminal side) is the domain of the immunoglobulin long chain. It consists of four domains similar to In. This outer part has a transmembrane followed by a cytoplasmic portion and a cytoplasmic portion (1), this protein is a viral protein. Receptor of IIIV virus (AIDS-inducing virus) through interaction with gp120. (2).

The first two domains (which do not contain glycosylation sites) contain gp120 binding sites. In addition, there are 10 anti-CD4 monoclonal antibodies that have the ability to neutralize viral infection. It was also found to contain binding sites for T4 and 0KT4^ (3), (4), (5), The cleavage protein secreted by eukaryotic cells containing these two domains is 1 nvitro tests (3), (4), (6), (7), (8), (9) and It showed the ability to inhibit viral infection in Gezaru (10).

A liliplasmic bacterial protein with an affinity for maltose and maltodextrin. Method for expressing C[14 functional receptor fused to protein MalE in E. coli For example, European Patent Application No. 299,810 published on January 18, 1989 has been disclosed. This method uses the MalE gene and other bacterial genes that have already been realized. This is to fuse genes (11) and (12). hive obtained in this way The lid protein retains the functions of the two components. these proteins Again. Can be transported into the periplasm.

The hybrid protein contains the fused MilE gene and CD4 gene. Transcription signals specific to nuclear cells (e.g. SV40 virus origin of replication and polyadenyls) If transfected with a structure combined with C1 102 strain (expressed in eukaryotic cells such as hamster embryonic fibroblasts J12) You can also For this operation, methods known to those skilled in the art are used.

The method described in the prior patent application specifies the type of product that can be easily purified in a short time. to produce a hybrid protein. Indeed, the hybrid protein Like the protein MalE, it can be produced in large quantities and is It can be purified in a single step eluting with maltose at neutral pH.

The present invention shows that this environment is present in Durham-negative bacteria, particularly in E. coli, when CD4, more specifically C. Promotes the establishment of disulfide bridges important for the biological activity of the N-terminal portion of D4 The fact that this appears to be particularly important is that it is derived from CD4 and It is said that it is likely to be a leap sequence if the sequence present in the sequence does not have the C-terminal region of CD4. Use facts.

As demonstrated in the present invention, this method involves the use of a hybrid hybrid combination of CD4 proteins. It allows for the evaluation of the therapeutic potential of a virus and its properties (stability, interaction with viruses, etc.). This may also provide clues for research such as the selection of CD4 mutants with altered effects. Ru. In addition, the absence of the C-terminal portion of the CD4 molecule results in hybridization in host cells, especially in E. coli. The production rate of lid protein increases significantly.

The invention more particularly relates to fusion with a first sequence upstream of the N-terminus and optionally In addition, the peptide derived from the CD4 protein fused to a second sequence is also downstream of the C-terminus. It relates to a hybrid polypeptide containing a tide chain. said first arrangement and an optional The second sequences are each derived from the MalE protein. Hybrid polype of the present invention More specifically, peptide is a peptide chain derived from -CD4 protein. Park.

containing at least a part of the protein, that is, the N-terminal region containing the former V virus anchoring site. Mi, and - the length of said first and optionally second sequence derived from MalE; may be used when producing hybrid proteins using genetic engineering methods. Transfected microorganisms, especially E. coli, are transported outside the cytoplasm. MalE protein involved in transport and translocation (excretion) of the microorganism into the periplasm Quality characteristics and specific affinity of MJIIE for maltose or amylose and long enough that they are simultaneously held by the hybrid polypeptide. Features.

A preferred hybrid polypeptide of the present invention is the second compound derived from Ma□IE. In fact, the cultured bacteria producing these hybrid polypeptides are , a sequence derived from CD4 is inserted between the two sequences of MalE. hybrid polypeptides than to hybrid polypeptides of said type. shows greater resistance to Another noteworthy point is that the CD4 protein The MalE-derived chain and fusion occur at the N-terminal level near the fixation site with V-type viruses. Even when the CD4 protein is combined, the specific activity of the CD4 protein is retained.

In particular, the hybrid polypeptide of the invention has at least a Vl of CD4 protein. The hybrid polypeptide of the present invention is more Specifically, it does not contain the v3 region and v4 region of CD4.

More particularly, the present invention relates to hybrid polypeptides of the above type, or sequences derived from MalH. It also relates to polypeptides derived from separated chains.

The polypeptides of the invention are particularly those described in European Patent Application No. 299.810, supra. It can be produced by These methods were used in the Examples below. Other features of the invention Features will become clearer in the description of these embodiments with reference to the accompanying drawings.

Of the attached drawings, - Figure 1 shows the essential parts of the CD4 protein and the proteins that commonly carry it. FIG. 3 is an explanatory diagram that briefly shows the relationship of the portion to the membrane of a lymphocyte.

- Figure 2 shows the polypeptides of the invention and the ability to produce these polypeptides in E. coli. FIG. 2 is an explanatory diagram briefly showing the structure of a corresponding plasmid.

For the peptide sequence of the CD4 protein and the numbering of these amino acids, see P. roc, Natl, Aeacl, Sci, USA, vol, 84. pp.

9155-9159. December 1987, P, J described in fmunology , Maddon et al. and the Lit described in Ce1l 55, 541.1988. See the correction by t+nan et al. In this specification, this numbering is used as is. use.

In particular, the polypeptide of the invention comprises amino acids 1 and 1 of the N-terminal portion of the CD4 protein. 77 including the following areas extending between: pne Leu thr lys gl y pro ser lys l@u asn asp arg ala as pglu val glu asp gin lys glu glu vaL gln lau lau val phaser lau thr lau thr lau glu sar pro pro gly sar sar pr.

Although the present invention includes only a portion of the sequence of amines w11-177, This type of sea urchin is suitable for exporting the hybrid polypeptide out of the cytoplasm. It concerns all hybrid polypeptides.

The hybrid protein of the present invention reduces CD4 levels in the absence of v1 protein. The amino acid sequence extending from the 15th amino acid to the 85th amino acid (V 1 region). The hybrid protein of the present invention also has the above arrangement. Sequences m and v2 extending between the 15th amino acid and the 16060th amino acid of the column region).

Of course, Ma transports the expressed hybrid protein from the corresponding nucleic acid sequence. Perform local deletions or substitutions in the amino acid sequence as long as they do not affect the ability of lH. You can also do that.

Therefore, in the present invention, an amino acid sequence selected within the N-terminal region of CD4 and MalE It is formed between proteins and produced in mammalian (CHO) cells. Obtaining a hybrid polypeptide with the same activity level as a soluble CD4 molecule be able to. The similarity of activity levels as described above can be evaluated by the following items. Runi - Binding to the CD4 molecule or the ability to form monoclonal antibodies.

- Fixed inhibitory properties of the HIV protein gp160 to CD4.

- Ability of strain Ca2 to inhibit immobilization of IIIVI virus particles to lymphoid cells.

Particularly noteworthy regarding the hybrid protein CIMER described below, In the three tests above, a significant correlation between soluble CD4 molecules produced in CHO cells and No significant difference in activity was observed.

The overall structure of the CD4 protein is briefly shown in FIG. In this diagram, - The part indicated by the thick line (arrow pointing from )IIV is the fixation site. handle. The description herein will be made with more particular reference to this site.

- The code written on the right side of the simple structural illustration of this protein various parts of the quality (Vl, v2, v3 and V4) and the monochrome shown in this figure. represents the position of the site of the CD4 protein recognized by the null antibody.

- The letter G indicates the normally glycosylated site of the CD4 protein.

- Letters E, M and C are the extracellular region of CD4 protein (E), crotch region < M) and the cytoplasmic region (C).

Bi1・Number A dual expression vector carrying the chimeric gene (section 1) was constructed. first vector The 177 amino acids in the N-terminal part of CD4 are converted into MalE (amino acids 369 defines a hybrid protein (CIMER) fused to the C-terminus of . The second vector contains the same CD4 protein fused to the N-terminus of the MalE protein. fragment encoding a hybrid protein (CISREM>). In the case of individuals, the N-terminal portion of CD4 is modified with Ma to facilitate its transfer to the periplasm. It was fused with the signal peptide of lH. Therefore, the soluble portion of CD4 is the molecule of MalE. It is present in the gap between the gnal peptide and the mature protein (see Figure 1).

These structures are briefly shown in FIG. Below are these configurations (based on Figure 2): This is an explanation of the main characteristics of the structure.

MalE-CD4 = codons -4 to +177 of CIMERCD4 gene The containing DN^ fragment (Fnu4H-Nclel) was treated with ampicillin and tet The gene resistant to lacycline, the 1ael gene, and the tac promoter ( Plus carrying the MalE gene under the control of IPT (inducible by IPT) and inserted into Mid (pBR322 derivative). Adapter on the distal end of MalE , and the CD4 insertion is in phase at the level of the final codon of MalH. I did it like that. The structure of this protein is shown above the plasmid structure diagram.

CD4-Ma l E Δ = CTSREM First, use the same CD4 fragment as above. downstream of the MalE signal sequence (after the 27th codon of the mature protein) inserted into.

Next, the entire MalE (codons -2 to +370) was inserted into the 177th codon of CD4. Later, they were inserted in the same phase. Remains of plasmid (ambicillin and tetracycline) resistant gene, fact gene, and tac promoter) in the case of IjMER. It's the same. The structure of this protein is shown above the plasmid structure diagram.

In both cases, transcription of the chimeric gene is under the control of the tac promoter (13 ), this transcription is unaffected by catabolite repression and is (propyl β-D-thiogalactoside). plus expression level (determining which repressor binds to the tac promoter) on mid is obtained.

cytoplasmic proteases and periplasts, respectively, to promote stable protein production. Carrying mutant Ion- and degP that inactivates rasp proteases Cerebrum strain was used (14), (15). Expression vector-bearing cells are incubated at 30℃ for 3 generations. If induced during one or four generations, these cells Proteins that are mostly exported (CIMER) or partially exported to Protein (CISREM) is produced.

This protein has a size of 60 KD) and its antigenic determinants MalE and It can be identified by antibodies specific for CD4.

has a size intermediate between the complete hybrid protein and the MalE protein The synthetic molecular fraction produced is probably the product of altered or abortive expression; Only a small portion of the hybrid protein remained.

Under low level expression (non-inducing level) conditions, the fusion protein is deficient for MalH. can complement the loss (normal growth on synthetic media containing maltose as the only carbon source) do). Under fully inducing conditions, overproduction of CCl5RE becomes toxic to bacteria However, overproduction of CIMEH is not toxic. This protein CIMER is manufactured by N. and investigated its characteristics.

As a result, the purified hybrid protein, more specifically the amylose column The two ellipsoidal components of tanno-chrystalline CIMER purified by It has been found.

l -MalE is maltose and maltostrin: η: Umeichi Shibicho 3 1 澾－ Passing the contents of the periplasm through an amylose column (osmotic shock) A portion of the protein is specifically immobilized on amylose. This part (frac tion) in a single step by eluting the column with maltose. Can be purified. The yield is approximately 500 μg of hybrid protein per gram of bacteria. be.

This group of molecules exhibits an affinity for amylose and maltose. Appearance molecular weight of 60 kD suitable for hybrid protein on Mido-5DS gel and anti-MalE polyclonal antibody and anti-CD4 monoclonal antibody. Therefore, it is recognized. The molecule Ji 60kD is ^mershan RAINB The hybrid was prepared according to the conditions described for the use of the O-High Molecular Weight Kit. Compare the migration distance on a gel of a protein with a known high molecular weight as a reference protein. It was evaluated by The following proteins were used as reference proteins. Tani - Phosphorylase of 92,500 Daltons, Oba of 46,000 Daltons Rubumin, -69, QOO Dalton's bovine serum albumin, -14,300 Dalton Tonorizozyme.

2- CD4o1 of the “separate hybrid protein” is an envelope protein 120-　　　　　　　　　　　　　　old ■ i. In experiments using “neutralizing” monoclonal antibodies, these monoclonal antibodies The clonal antibodies (10T4 and 0KT4^ above) were tested in ELIS^ test or in mammals. The species was isolated by immunoprecipitation with soluble CD4 purified from mammalian cells at the same concentration. It was demonstrated that it reacts with hybrid proteins.

ii, the hybrid protein and HIVI purified protein 8p120 or Direct interaction between gp160 and co-immunoprecipitation and immunotransfer - verified by.

iii. When the hybrid protein is present together with virus particles, virus particles ``neutralized'', which means that the cells of the lymphoid cell line CEH) are ``neutralized''. This was clearly demonstrated in a test that measured the dyeing power of R. be.

Pre-incubating the virus with CIMER at a concentration of 1 μg/ml Didothymidine (i.e. ΔZT, one of the anti-old V substances also widely used 1) The same neutralizing ability is obtained when used at a concentration of 10 nM. CIMER concentration to 101 When the concentration is higher than 1 g/ml, the virus inhibition rate exceeds 90%. These results are Consistent with results for soluble CD4 produced by other methods (3), (7), (8 ), (9).

All of these consistent data indicate that the CD4 component of the hybrid protein is This indicates that it retained a structure capable of binding to complete virus particles. especially , it appears that the disulfide bridge of CD4 was formed correctly.

Using the MalE-74 structure described in European Patent Application No. 299,810, from sonicated bacteria or osmotic shock due to its affinity for amylose columns. The expressed hybrid protein MalE-74, which can be purified in a single step, is It was also found to have the properties: 1) This protein is a polyclonal antibody against MalE and CD4 (also known as T4). ) with a monoclonal antibody (IOT4.0KT4.0KT4^) against Recognized.

2) This protein interacts with viral proteins Bp120 and gp160. use This was demonstrated in three ways:

a) "Dot blot" (natural protein) experiment.

b) MalE-mediated by polyclonal antibody against MalE or gp160 Co-immunoprecipitation of T4:gp160 (native protein) complex.

C) "denatured") g by hybrid protein immobilized on nitrocellulose Retention of p160 (incubation with gp160 and anti-gp160 ``Western plot'' that was realized in the body).

3) This protein was able to inactivate the old ■1 virus in in vitro tests. I can do it. While increasing the concentration of this fusion protein (0, 2, 20 and 50u g/...1) Pre-incubation with the virus causes replication in CEM cell lines. The rate of inhibition of the ability of the virus to be produced increases (0.55, 90 and 100 %, these values indicate the inhibition rate of reverse transcriptase activity after 15 days).

The protein MalE purified and used under the same conditions completely inhibited virus replication. I didn't.

produced by a fusion between the bacterial MalE gene and a fraction of the human CD4 gene. The hybrid protein expressed in E. coli is therefore composed of the two components mentioned above. The characteristics of CD4 are particularly strong against HIV viruses, especially the old v1 virus. Retains neutralizing ability.

The present invention therefore provides a method of administration, particularly a pharmaceutical vehicle suitable for parenteral administration. In a combination of conditions, retroviruses belonging to the HIV family are neutralized in vivo. an amount of a MalE-CD4 hybrid polypeptide as described above; is derived from the hybrid polypeptide by cleavage and separation of the MalE sequence. Compositions for use in in vivo therapy comprising the soluble peptide CD4 provide. Although non-limiting, the daily dose of this composition is 1001.1 g to 10 g, for example about 1 g, is suitable.

The hybrid proteins of the invention can be produced in large quantities. CD4 expression Since E. coli is used as a protein, it is also possible to introduce modifications at the CD4 gene level. It is also possible to develop selection methods to isolate more effective mutants. It will be done. The hybrid protein of the present invention can further improve the structure of CD4 by, for example, X-rays. It also makes research possible. These hybrid proteins also exhibit affinity forces. culture, especially polymerized maltose or amylose-based columns. It is also used to purify the old V virus contained in the culture medium, and selectively removes the virus upon contact. Based on retention.

The present invention investigates whether the HIV virus or the antigen of the virus is present in a biological sample. It also relates to an in vivo detection method for determining whether or not the present invention occurs. This method uses HrV or HI If V antigens are present, these HIV or biological samples provided that the lid or non-hybrid polypeptide is immobilized. contact with the polypeptide and detecting whether a complex is produced as a result. It consists of

ward"L"2 [1] Middon, P.J., Litmun, D.R., Godfre y, M, , Mmdon, D, E, , Chess, k et R, Ax el.

Ceu 42 (1985) 93-104° [2] Mc Dougal, J, S, , Kennedy, M, S, , Sligh, J, M,, C art, S, P, lawle, A, et J, A.

N1cholson, 5science 231 (1986) 382-38 5゜[3] Triunecker, A, L (lcke, W, etc. K, K xual, 1nen, Namr: 331 (198g) 84-8U゜ [4] Berger, E, A, Fuerst, T, R, et B , Mo5s, Proc, Nzyama Acad, Sci, @tJsA 85 (198g) 2357-2361° [5] R4chardson, N, E,, Brown, N, R,,) Iussey, R, E, , Vaid, A, , Mat 狽■■Next sword A T, J.

Bolognesi, D, P, et E, L, Rheinhertz, Proe, Natl, person czd, Sei, USA 85 i198g) 6102-6106゜ [6] Sm1th, D.H., Byrn, R.A., Marster s, S, A,, Gregory, T,, Group Ko≠ Mr. A, J, E, et D.

Capon, 5science 23g (1987) 1704-1707゜ [Sword Fisher, R, A, Bertonis, J, M, Mei er, W,, Johnson, V, A,, Cos+Shield Tape Shield Floating Shield Sword { D.S., Liu.

T., Tizara, R.Walker, B.D., Tari, M.S. , 5chooley, FL Ding, et L Flavck Nature 331 (1988) 76-78° [8] Hussev, R,E,, R4ehardson, N,E, , Kowilski, M., Erown, N-R., AChang, H. ,-C,.

5iciliano, R.F., Dorfman, T., Walker, B. 5odroski, J, et　El　Re1nhenz　N≠狽 Floating Nitrogen■R31 (198g) 7g-81゜ (9) Deen, LC, Me Dougdl, J, S, In@c ker,' R,, Fole+za-Wasserman, @G,, Arth os, J.

Rosenberg, J., Maddon, P.J., R.e. t R, S, SweeLNature 331 (1'1W) g2-gs- [10] Waunabe, M,, Re1r+unn, K, ^,, D e Long, P, A,, uu, T,, Fishe Chichi A LA, at N, Lervin.

[11] B6douelle, H,, Duplay P, et M, Hofnung, CI'L, Aca Self ScL Par Kanto@305 (1 987) 623-626゜ [12] B6doue11e, H, etP, Duplay, EurJ, Bi ochem, 171 (198g) 541-549 [13] Amm, E,, B rosius, J, etM, Ptashne, Gene24 (19g3) 167 -171[14] Howard-Flanders, P,, 51m5o n, E, at L, Tbcx’rot Genetics 49@(196 4) 237-246° [15] 5trauch KL et J, Beckwith, F’ roc, Natl-Acad, SCL USA N5 i198g) 15 76-1580° F electric, 1 1 N I HrV international search report international search report FR9000182 S^　　　35778

Claims (10)

[Claims] 1. It is fused to the first sequence upstream of the N-terminus and optionally also the second sequence downstream of the C-terminus. A hybrid comprising a peptide chain derived from the CD4 protein fused to the sequence of a polypeptide, wherein the first sequence and the optional second sequence are each MalE. More specifically, this hybrid polypeptide is derived from a protein. - A peptide chain derived from the CD4 protein has a small amount in the N-terminal region of the protein. at least a part, that is, a part containing an HIV virus fixation site, and - the length of said first and optionally second sequence derived from MalE; is used when producing hybrid proteins using genetic engineering manufacturing techniques. Transduced microorganisms, especially E. coli, are transported out of the cytoplasm. Characteristics of the MalE protein regarding transport and translocation into the periplasm of the microorganism At the same time, it increases the specific affinity of MaIE for maltose or amylose. A hybrid characterized by being long enough that the hybrid polypeptide retains polypeptide. 2. The hybrid according to claim 1, characterized in that it does not contain the second sequence. polypeptide. 3. Contains the soluble portion of protein CD4, especially the N-terminal region of protein CD4 The hybrid polypeptide according to claim 1 or 2, characterized in that: 4. A claim characterized in that it does not contain the V3 region and V4 region of protein CD4 The hybrid polypeptide according to item 3. 5. It is characterized by containing the V1 region of protein CD4 but not the V2 region. The hybrid polypeptide according to claim 4. 6. V1 region and V2 region of protein CD4, especially the following amino acid sequences 1-1 77: [There is an array] The hive according to claim 3, characterized in that the hive contains lid polypeptide. 7. from expression products in hosts such as eukaryotic cells or Gram-negative bacteria such as E. coli. a polypeptide whose expression product comprises a nucleic acid sequence encoding the polypeptide. and a regulatory element bound to the nucleic acid sequence that allows said polypeptide to be present when the bacteria are cultured. Transformation with a recombinant nucleic acid containing regulatory elements that express the peptide in the host This expression product transforms the polypeptide previously extracted from E. coli into α( 1-4) Contact with an insoluble polymer of glucose or amylose and Purification is achieved by separating and recovering the immobilized polypeptide from non-immobilized substances. The polype according to any one of claims 1 to 6, characterized in that Petite. 8. Contains only a peptide chain derived from the protein CD4, and this peptide chain is cleaved. and a MalE-derived sequence obtained after separation. A polypeptide derived from the described polypeptide. 9. A composition suitable for neutralizing the HIV virus in vivo, the composition comprising: in combination with a vehicle that is physiologically acceptable to the host to which the composition is administered. and adding the polypeptide according to any one of claims 1 to 8 to said neutralizing composition. A composition containing only an effective amount to confer fitness. 10. HIV or HIV antigens, particularly envelope glycoproteins, are present in biological samples. A method for detecting whether or not HIV is present in vitro, the method comprising: Or, if an HIV antigen is present, under conditions such that the polypeptide is immobilized thereon. contacting a biological sample with a polypeptide according to any one of claims 1 to 8; , and optionally detecting the formed complex. How to get out.