CS275838B6 - Method for production of hybridoms used for monoclonal antigen against hiv virus producing - Google Patents

Description

The invention relates to a method for producing monoclonal antibodies which can be used in the diagnosis, treatment and prevention of viral infections. In particular, the monoclonal antibodies and peptides obtained by the method of the invention can be used in the diagnosis and vaccination against human viral infections associated with immunological system (HIV) deficiency.

The infectious organism responsible for acquired immunodeficiency syndrome (AIDS) and the early stages of the disease, as well as for complex disease, AIDS-related (ARC) and lymphadenopathy syndrome (LAS) is a new lymphotropic retrovirus. This virus has been called variously to date, for example, LAV, HTLV-III, ARV, and more recently HIV.

As HIV prevalence is currently pandemic, attention is being paid to the treatment of infected individuals and to the prevention of transmission to uninfected individuals. Various measures have been proposed that target different stages of the virus life cycle and are reported in Mytsuya and Broder, 1987, Nature 325, 773. One approach is to use antibodies that bind to the virus and inhibit the replication of the virus, either by preventing the virus from entering host cells or other mechanisms. Once a virus component accessible to the antibody has been found, it is hoped that an amount of antibody sufficient to neutralize the infectious capacity of the virus by vaccination or at least passively administering an immunoglobulin or monoclonal antibody with the desired specificity could be delivered.

The envelope glycoproteins of most retroviruses appear to react with receptor molecules on the cell surface of a possible host, thus determining the infectivity of the virus for a particular host. Antibodies that bind to these glycoproteins can block the interaction of virus and cellular receptors and thus neutralize virus infectivity, as generally described in The Molecular Biology of Tumor Viruses, 534, (published by J. Tooze, 1973) and RNA Tumor Viruses. , 226, (R. Weiss et al., 1982) and references cited therein, as well as Gonzales-Sacrano et al., 1982, Virology 120, 42 (La Crosse Virus); Matsuno and Inouye, 1983, Infect. Immun. 39, 155 (Neonatal Claf Diarrhea Virus); and Mathews et al., 1982, J. Immunol., 129, 2763 (Bcephalomyelitis Virus).

In the general structure of HIV, the ribonucleoprotein nucleus is surrounded by a lipid-containing envelope that the virus retains during penetration into the host cell. Encoded viral glycoproteins are bound in the packaging. HIV envelope glycoproteins are usually synthesized only in-vivo. 150-160,000 units, which is then processed in the cell into an N-terminal fragment of 110,000-120,000 units to form an external glycoprotein and a C-terminal fragment of 41,000-46,000 units which represents a transmembrane envelope glycoprotein.

For the above reasons, the N-terminal glycoprotein weighing 110,000 units (gp 110) has HIV properties for which it has long been pursued as a possible target at which the life cycle of the virus could be interrupted. Blood serum from HIV infected individuals has been shown to neutralize this virus in vitro, and it has been shown that antibodies that bind to purification of gp110 are present in this serum. These results have been reported, for example, in Robert-Guroff et al., 1985, Nature 316, 72; Weiss et al., 1985, Nature 316, 69; and Methews et al., 1986, Proc. Nati. Acad. Sci. U. S.A., 83, 9709. Purified and recombinant gp110 stimulated the production of neutralizing antibodies in blood serum when used to immunize animals as described in Robey et al., 1986, Proc. Nati. Acad. Sci. USA, 83, 7023; Love et al., 1986, Science 233, 209 and Human, Zagury et al., 1986, Nature 326, 249. Binding of the gp110 molecule to the CD4 receptor (T4) has also been demonstrated, and monoclonal antibodies that recognize certain epitopes of the CD4 receptor and they block HIV binding, syncytia formation and infectivity. This has been reported, for example, in Mc. Dougal et al., 1986, Science 231, 382, Putney et al., 1986, Science 234, 1392, which also describes neutralization of antibodies in serum of animals after immunization with a recombinant composite protein containing the C-terminal half of gp 110 molecules and publications have shown that glycosylation of the envelope protein is not necessary to neutralize the antibody.

CS 275 838 B6

Thus, it is apparent that at least a partial AIDS vaccine could be generated using the HIV gp110 molecule or portions thereof. These vaccines are another option in addition to vaccines prepared from inactivated or attenuated virus. Inactivated vaccines are often associated with difficulties due to the possibility that all virus particles are not killed, in the case of attenuated virus, the possibility of mutation remains with the re-ability to cause disease. For partial vaccines, only those parts of the virus that contain antigens or epitopes that are capable of eliciting an immunological response, i.e., the generation of neutralizing antibodies, ADCC and the cytotoxic T-cell response, are used to immunize a possible host. The main advantage of these vaccines is that they exclude portions of viral material that are insignificant for antibody production.

Viral subunits for use in a vaccine can be obtained in various ways. For example, envelope glycoproteins can be expressed in a bacterial host with subsequent purification, although most modifications that occur after translation, such as glycosylation, will be lacking in this molecule. Such modifications can be obtained using a eukaryotic expression system, for example in yeast or mammalian cell cultures. Virus genes have been introduced into mammalian cells using chickenpox virus as a vector, as described by Mackett M. et al., 1982, Proc. Nat. Acad. Sci. USA 79, 7415; Panicali D. and Paoletti E., 1982, Proc. Nat. Acad. Sci. USA, 79, 4927 Recombinant chickenpox virus can be constructed according to Hu et al., Nature 320, 537 (1986) or Chakrabarti et al., Nature 320, 535 (1986). In these systems, the virus glycoprotein produced by cells infected with recombinant chickenpox virus is appropriately glycosylated and can be transferred from the cell surface to the culture medium and then isolated.

An important step in the production of a partial vaccine is sufficient purification of the desired glycoprotein from the complex mixture obtained from the expression system. There are several methods for cleaning. These may be, for example, preparative polyacrylamide gel electrophoresis, gel chromatography, and various other types of chromatographic purification, for example, using ion exchange, reverse phase, immunoaffinity, hydrophobic interactions, and the like. Most of these techniques are used in various combinations to yield a substantially pure substance. as described in Kleid DG et al., 1981, Science 214, 1125; Cabradilla C. D. et al., 1986, Biotechnology 4, 128, Dowbenko D. J., 1985, Proc. Nat. Acad. Sci. USA, 82, 7748.

Obviously, there would be a need for procedures that would allow these vaccines to be obtained by reducing the number of purification steps required from the mixture in which a particular viral antigen was expressed. Effective separation of the antigen from other raw materials could be performed by immunoaffinity chromatography. This procedure, also known as immunoadsorption, consists essentially in selective adsorption of the antigen to a solid support to which a specific antibody is covalently bound. The selectively adsorbed antigen can then be eluted from the adsorbent by, for example, changing the pH and / or ionic strength of the buffer used.

Polyclonal antibodies obtained from animals immunized with a particular antigen or naturally infected individuals, as described in Lasky et al., Have often been used by immunoadsorption, but these often have significant drawbacks, for example in that

i) not all of the antibodies bound to the insoluble support are specific for the molecule under consideration, requiring further purification, ii) antigen yields are low, and iii) affinity of antibodies often varies from formulation to formulation, requiring modification in elution procedures.

The use of monoclonal antibodies specific for the desired viral antigen would eliminate all the above disadvantages.

Mouse monoclonal antibodies capable of binding HIV antigens have been described. A number of monoclonal antibodies specific for the p-25 core protein have been described, for example, in di Marzo Veronese et al., 1985, Proc. Nat. Acad. Sci. USA 82, 5199 and Chassagne J.

CS 275 338 B6 et al., 1986, J. Immunol. 136, 1442. Monoclonal antibodies specific for the membrane giycoprotein gp41 have also been described, for example, in di Marzo Veronese et al., 1985, Science 229, H02.

Obviously, it would be desirable to obtain a monoclonal antibody specific for epitopes of a well-defined region of the gp110 major envelope glycoprotein. Monoclonal antibodies that would bind to these regions and cause reduction of replication and HIV transmission capabilities or would prevent these phenomena would be of great use in therapy and prophylaxis. Furthermore, monoclonal antibodies could also be used to purify the desired region of gp110 from virus fragments or a recombinant expression system for use in, for example, vaccines. In addition, it would be possible to chemically synthesize the epitope-containing region recognized by monoclonal antibodies, thus avoiding the difficulties of purifying and administering larger fragments of the gp110 molecule.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a solution to overcome the aforementioned drawbacks of the known processes.

SUMMARY OF THE INVENTION The present invention provides a method for the production of hybridomas for the production of a monoclonal antibody against HIV, wherein the lysed virus extract binds to a carrier, such as a lens lectin, and is used to immunize mice whose spleen B cells are then subjected to fusion with ITS-1 myelomatic cells to form hybridomas, the culture medium in which the hybridoma cells are maintained is screened for the presence of antibodies and monoclonal antibody producing cells capable of reacting with the HIV gpHO envelope glycoprotein antigen, contained in the blocking peptide T, or they are further cultivated in its derivative or analogue.

The invention therefore relates to a process for the production of novel substances which neutralize the HIV virus and prevent or substantially prevent the virus from penetrating the host cells. Peptides that mimic HIV neutralizing regions and monoclonal antibodies that react with them can be used to diagnose, treat, and vaccinate against HIV infections. The term neutralizing region refers to those portions of HIV, particularly proteins, that contain stretches of amino acid sequences defining one or more epitopes capable of reacting with antibodies that the cell, individually or in combination with other antibodies obtained by the method of the invention, can neutralize HIV infections . Suitable neutralization assays are known, for example, reduction of HIV infection in T-cell lines, reduction of plaque formation units in VSV (HIV) pseudotypes carrying HIV envelope glycoproteins, assays for syncytial inhibition and virus and receptor binding. If desired, the neutralizing potency can be compared to the reactivity of the antibody in immunochemical assays such as immunofluorescence, immunoblot and radioimmunoprecipitation.

The novel peptides usually contain less than 50 amino acids, and in each case five or more related amino acids which form epitopes substantially similar to epitopes in the HIV neutralizing regions of gpHO or p25, for which they encode the env and gag regions of the HIV genome. Of particular interest are regions from amino acid residue 301 to residue 336 in gp110 and residue 278 to residue 319 and residue 315 to residue 363 in p25, all residues refer to the LAV _BRU- labeled HIV strain. Data Bank (AIDS virus sequence database, Los Alamos National Laboratory, Theoretical Division, Los Alamos,

NM 87545).

Those skilled in the art will recognize that analogous homologous regions from other HIV isolates can be identified depending on their location. These homologues can be identified by reference to DAV _BR data as follows:

(a) The amino acid sequence of the HIV isolate and the DAV _BRU strain can be compared to obtain maximum homologues between the two sequences;

b) it is possible to identify peptides which contain amino acid isolates corresponding to the placement of the peptides in DAV _BRU , which also immunologically mimic the proteins of said strain. Peptides containing the amino acids of the HIV isolate so identified immunologically mimic the properties of the HIV isolate proteins.

CS 275 838 B6

This procedure is applicable to HIV strains that have not yet been discovered. For example, in detecting new strains of HIV, their amino acid envelope sequences and core sequences can be compared to the LAV _BRU sequence for maximum homology. These procedures are known in the art. In particular, it is necessary to maintain the greatest homology between the cysteine residues. The amino acid sequence of a novel HIV strain or group of strains can be synthesized and used in accordance with the method of the invention.

Another method for determining the sequence of a homologous region in other HIV strains is the method of Scharf et al., Science (1986), 233, 1076. This method uses two oligonucleotide primers that bind to conserved sequences outside the region of interest. wherein a different restriction enzyme site is located in each primer. The DNA of the HIV strain is then amplified in vitro, the resulting oligonucleotides can be cloned in appropriate vectors to obtain sequence analysis, and then incorporated into a vaccine in the form of a cassette which represents a particular epitope of the HIV strain.

It is not necessary that the epitopes contained in these sequences exhibit cross-reactivity with antibodies against all strains or types of HIV. Peptides containing immunological epitopes that distinguish one type or serological group from another are useful for identifying certain types or serological groups and can be used to identify individuals infected with one or more types or serological groups of HIV. They may also be combined with other peptides, either from a homologous region or from another neutralizing region for therapeutic purposes.

These peptides are preferably derived from the gp110 region of the virus. Of particular importance in this region are peptides that are encoded in the env region from pairs of bases 666? 6774 LAV _BKU isolate _pairs> Different homologous regions of other HIV isolates thus include homologous sequences that can be obtained from the Los Alamos Data Bank (except LAV2) as shown in Table 1 below.

Table 1

HXB2 TGTACAAGACCCAACAACAATACAAGAAAAAGA

Cys ThrArgProA snA snA snThrArgLy sArg

ATCCGTATC

IleArglle

BH102 ----------------------------- Ser BH8 ------------------ ----------- Lys HXB3 ------------------------- Lys H9M ---------- ------------------- Ser BRU ------------------------- Ser MAL - --------- Gly ---------- ArgGly ELI - Ala - TyrGln ——Gin —— ARV2 ------------------ ----------- Ser WMJ2 ————— Tyr —- Val- ArgSer

KPENV

Ser

Z6 ----------- TyrLys ------- GlnSer --------- Z3 ----------- GlySerAspLysLysIle ------- ----- NY5 ----------------------- Lys-Gly --------- CDC42 -------- --------- His ----------- ValThrLeu

LAV ----------- Gly-Lys-Val-Gin ---------— HisPhe ThrPro ~ - Tyr - LeuSer - - ThrLys ThrPro - GlnSer - - Ala -

MetLeu

309

309

309

309

309 314 314

(+420) 310 312 306 322

311 306 304 320 302

HXB2 CAGAGA .......................... GGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATG

GlnArg .......................... GlyProGlyArgAlaPheValThrlleGlyLysIleGlyAsnMet

GH102 ------------------------------------------------- ----------------------------- BH8 -------------------- -------------------------------------------------- -------- HXB3 ----------------------------------------- ------------------------------------- H9M ------------ -------------------------------------------------- ---------------- BRU --------------------------------- --------------------------------------------- MAL ---- - ----------------------------------- Gin-LeuTyr-Thr-IleVal-Asplle

ELI GlyLeu ——----------------------- ... GlnSerLeuTyrThr - Arg - IleValSerArgSer

326

326

326

326

326

331

329

323

CS 275 838 B6

Table 1 - continued

ARV2 · «···· WMJ2 ·· ♦ ··· REENV ·· * ··· Z6 GlyLeu Z3 Arglle NY5 GlyPro CDC42 LAV2 MetSer

-------------------------------------------- His - Thr - Arg - IleGlyAsp 327

------------------------------------- Arg - ArgGlu ... - IleGlylle 320

-------------------------------------- ValIleTyrAlaThr-Gin-IleGlyAsp 337

------------------------------. ,, GlnAlaLeuTyrThr-Arg-ArgThrLysIlelle 327

----------------------------------- LysVal - TyrAlaLys - Gly .......... 319 —---—-----------------—---...------ ThrLeuTyrAlaArgGlu -----— Asplle 320

------------------------------------- ValTrpTyr-Thr-Glu-LeuGlyAsp 335

----------------------------------- HisVal - HisSerHisTyrGlnProIle - Lys 323

XHB2 ... AGA ... CAAGCACATTGT ... Arg ... GlnAlaHisCys

BH1 02 ------------------------ BH8 ----------------------- HXB3 ----------------------- H9M ----------------------- BRU- ---------------------- MAL ------—— Arg - Tyr -

ELI IlelleGly ------------- ARV2 Ile - Lys ... --------- WMJ2 Ile ----------------- --REENV Ile - Lys ... ----------- Z6 Gly ... --------------- Z3 IleThrGly ---------- --- ΝΪ5 ----------------------- CDC42 Ile ------------------- LAV2 ArgProArg ------- Met

331

331

331

331

336

334

330

333 326 343

(+420) 334 326 325 341 330

Other peptides that are suitable for the use or screening of monoclonal antibodies are those encoded by the env region from the 7246 to 73'7 pair of LAV _BHU . These antibodies and reactive peptides are particularly useful in immunoassays.

In the gag region of the LAV _H g _U isolate, the amino acid sequences of p25 are from 278 to 319 and from 315 to 363 are additional HIV neutralizing regions. It will be apparent to those skilled in the art that other neutralizing regions can be identified based on the above, in particular, combinations of monoclonal antibodies with different HIV epitopes that may have neutralizing activity.

Peptide I, also referred to as peptide 29, is encoded in the env region from about amino acid residue 308 to residue 328 and has the following amino acid sequence, wherein the polypeptides included in this sequence will form linear epitopes in the region of said sequence:

I (29)

Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y '

Y1 ', if present, means sequences of up to twenty amino acids.

Where Y and Y * are present, they may consist, for example, of one or more amino acids, sequences that delimit amino acids 308 to 328 in the HIV sequence, or may be any part of these delimiting sequences. For example, Y may be part or all of the LAVggy envelope amino acid sequence from residue 301 to residue 307 and Y 'may comprise part or all of the LAV-ggy sequence from residue 329 to residue 336 as follows:

II (28a)

Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg.Ile-Gln-Arg-Gly-Pro-Gly-Arg-A3a Phe-Val-Thr-Ile- Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys.

It is also possible to prepare portions of the peptide sequence obtained by the method of the invention. Of particular value in this regard may be the following sequence derived from peptide 29:

III (28b)

CS 275 838 B6

Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Y 'where

Y and i ', if present, are sequences of up to about 20 amino acids, and

IV (28c)

Y-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y '

Y and Y1, if present, represent sequences of up to about 20 amino acids.

In another embodiment, the homologous regions of the ARV-2 isolate of particular interest and encoded by the env region from amino acid residue 306 to residue 323 typically have the following amino acid sequence, wherein the oligopeptides included in the sequence also include linear epitopes within the sequence. sequence:

V (177)

Y-Thr-Arg-lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ie-Y where

Y and Y ', if present, represent a sequence of up to twenty or more amino acids.

When Y and Y 'are present, they may contain one or more amino acid residues that flank the amino acid sequence within residues 306 to 323 of the ARV-2 envelope portion or any portion of these sequences. In particular, Y may comprise all or part of the HIV envelope amino acid sequence sequence from amino acids 299 to 306 and Y 'may comprise all or part of the HIV amino acid envelope sequence within the amino acid residues 324 to 333.

It is also possible to prepare a sequence comprising portions of sequence V. In this sense, the following sequences are very useful:

VI (177b)

Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Y 'and VII

Y-Asp-Cys-Lys-Thr-Ile-Leu-Lys-Ala-Leu-Gly-Pro-Ala-Ala-Thr-Leu-Glu-Glu-Norleu-Norleu-Thr-Ala-Cys-Y 'where

Y and Y, when present, are sequences of up to twenty or more amino acids.

Another example may be any of the homologous regions of the LAV-2 isolate as encoded in the env region from amino acid residue 311 to residue 330 with the following typical sequence:

VIII (1-0-2-2)

Y-Lys-Th-Val-Lya-Ile-Nor-Leu-Nor-Ser-Gly-His-Val-Fhe-His-Ser-His-Tyr-Gl.n-Pro-Y 'where

Y and / or Y ', if present, means sequences of up to twenty or more amino acids.

These sequences have been described in Nature, 326: 662 (1987).

In carrying out the method of the invention, new cell lines capable of producing monoclonal antibodies capable of recognizing at a high titer (from 10 ² to 10? 7) can be obtained.

10 'or more) neutralizing regions comprised in a predetermined sequence of the gpliO or g25 coat glycoprotein, their protein precursors, recombinant proteins, obtained by expression, and synthetic peptides containing one or more epitopes in the predetermined region of gp110 or p25. These hybrid cells contain an identifiable chromosome in which the original DNA has been redesigned to contain a code for an antibody with a binding site. 7

GS 275 838 Βδ epitope in gp110 or p25, common to some or all of the clinical HIV isolates. These monoclonal antibodies can be used in a variety of ways, including diagnostic and therapeutic procedures, as well as to identify other cross-reactive and blocking antibodies. The epitope-containing peptides or polypeptides with which they interact may find particular use as immunogens for vaccine production or as therapeutic agents.

Blocking peptides

Primarily for use in conjunction with the above peptides or neutralizing monoclonal antibodies, other peptides or antibodies that interfere with binding of HIV to receptors or otherwise reduce HIV infectivity are intended. Preferably, so-called blocking peptides capable of inhibiting viral proliferation can be used, as well as monoclonal antibodies specific for epitopes contained in these blocking peptides to increase the effectiveness of treatment against HIV infection. HIV-blocking peptides typically correspond to the sequence of HIV amino acids that are likely to be essential for linking the virus to the host cell, for example, amino acid residues in the range 190-197 LAV1 and 185-192 ABV2 and HTLV-III (BH-10), code that is env. These peptides include octapeptide T, i.e. Ala-Ser-Thr-Thr-Thr-As-Tyr-Thr and various derivatives thereof, for example the peptide IX below and analogs, for example the peptide XI below, as described in Pert and next, why. Nati. Acad. Sci. USA 83: 9254-9258 (1983), the peptides are deposited in the gp110 or 120 envelope glycoprotein region.

Of particular interest are blocking peptides having the following sequences, preferably with NH3-terminal acetylation and amidation to terminate COOH:

IX (173B). Y-jAla-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Y ',

X (186)

Y-Thr-Thr-Asn-Tyr-Thr-Y ',

XI (187)

Y-Thr-Thr-Ser-Tyr-Thr-Y ',

XII (188)

Y-Thr-Asp-Asn-Tyr-Thr-Y ',

XIII (188)

Y-Asn-Thr-Ser-Tyr-Gly-Y '

XIV (190)

Y-Asp-Thr-Asn-Tyr-Ser-Y & XV (191)

Y-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Y '

Y and Y ', if present, represent an amino acid sequence of up to 20 amino acids.

The epitopes or antigenic determinants within these peptides are typically defined by a sequence of at least five amino acids, and can be used, for example, to mimic naturally occurring antigenic regions of HIV to obtain HIV-responsive antibodies and vaccines. Obtaining monoclonal antibodies

Monoclonal antibodies can be obtained by immortalizing the expression of nucleic acid sequences coding for HIV-specific antibodies by incorporating these sequences, typically cBNA, coding for said antibodies into a host grown in a tissue culture. culture. The immortalized cell line may be a mammalian cell line transformed by oncogenesis, transfection, mutation, and the like. For example, myeloma cells,

CS 275 838 B6 or other cell lines capable of expressing and secreting the antibody in vitro. The antibody may be a naturally occurring immunoglobulin of a mammal, obtained by transforming a lymphocyte, particularly a splenocyte, by a virus or by fusing a lymphocyte with a neoplastic cell, for example. a myelomatic cell to form a hybrid cell line. Typically, soulenocytes can be obtained from animals immunized against HIV or an epitope-containing fragment thereof.

Immunization procedures may vary quite a lot and still remain effective. These procedures have been summarized in Goding, Monoclonal Antibodies: Principle and Practice, Academic Press, Second Edition (1986). Disrupted virus, synthetic peptides, and bacterial composite proteins containing gp110 or p25 antigenic fragments can be used as immune-inducing agents. Preferably, the immunogen from the disrupted virus, peptide or recombinant protein may be enriched for such epitope-containing proteins or fragments for which it is desired to grow antibody-producing B cells or splenocytes. In particular, disrupted virus-containing solutions, lysates or extracts or supernatants containing recombinant protein after expression or disrupted expression vectors can be enriched for glycoproteins using various purification procedures, for example by polyacrylamide gel electrophoresis. Affinity purification using lectin is a convenient and convenient procedure for purifying the glycoproteins gp110 and others, for example using lens lectin. The extent of purification of glycoproteins from solutions for use as immunogens may vary within a wide range, for example from less than 50%, but at least 75 to 95 purity is usually desired, 95 to 99% purity and most preferably absolute homogeneity.

After purification of the proteins to the desired degree, the proteins may be suspended or diluted with an appropriate physiological carrier for immunization or coupled to an adjuvant. A preferred procedure is, for example, adsorption of proteins and fragments thereof to the lectin from the lens or to agarose containing the lectin from the lens or to other macromolecules. carrier for injection. HIV-enriched antigen compositions, including the glycoproteins gp110 and p25 n < 3 &gt; portions thereof, are usually used for immunization in an amount of 1 µg to 20 mg / kg host. The material is administered by injection, for example, intramuscularly, intraperitoneally, subcutaneously, intravenously, and the like. The agent may be administered once or repeatedly, usually at an interval of one to four weeks. The immunized animals are screened for antibody production to the desired antigen, then the spleens are removed and B-lymphocytes are isolated, then fused to or transformed with the myeloma cell line. Transformation or fusion may be carried out in a conventional manner. Fusion procedures have been described in a number of patents, for example 4,172,124, 4,350,683, 4,363,799, 4,381,192 and 4,423,147. The method has also been described by Kennett et al., Monoclonal. Antibodies (1980) and Goding, supra.

The immortalized cell line can be cloned and subjected to conventional selection, antibodies that will be present in the supernatant bind to gp110 and p25 viral proteins, recombinant composite proteins, or synthetic peptides containing the desired region of the epitope. Appropriate immortalized cell lines can then be grown in vitro or injected into the peritoneal cavity of the host to produce ascites. Since there are already a number of antibodies known to be specific for certain epitopes, for example in the region for which they encode the hAV-gy genome region from base pair (bp) 6688 to bp6750 including peptide 29 or from bp7246 to bp7317, including peptide 36, the supernatants can be monitored in competition with the appropriate monoclonal antibody in a competitive assay. Base pairs are referred to in Wain-Hobson et al., Cell 44: 9 (1985). Thus, other immortalized hybridoma cell lines with the desired binding properties can be readily obtained based on the antibodies obtained by the method of the invention specific for a particular antigen. These cell lines can then be fused to other neoplastic B cells, which B cells can serve as a recipient for the genome DNA encoding the antibody.

Preferred cells are rodent cells, particularly mice, but other mammalian cells, such as cattle, horses, sheep, swine, birds and the like, may be used. Immunization of these

The animals are easy to carry out and their lymphocytes, especially the thumb splenocytes, can be fused.

The monoclonal antibody secreted by the transformed or hybrid cell lines may be from any immunoglobulin class or subgroup, e.g., IgM, IgD, IgA,

IgG _{U (1} or IgB) The most preferred of the IgG family is the common isotype used in diagnostic procedures. Monoclonal antibodies can be used as such or in the form of fragments thereof, for example Pv, Fab, F (ab) ₂ ! as such.

In order to overcome the possible antigenicity of non-human antibodies to humans, it is possible to construct chimeric antibodies in which a binding fragment of an immunoglobulin molecule for an antigen (variable region) is linked by peptide binding to at least a portion of another protein not perceived by the human organism. for example, a stretch of a human immunoglobulin molecule. This goal can be achieved by combining the variable exon of another animal with the constant exons of the human kappa or gamma region. These procedures are known in the art and have been described, for example, in PCT 86/01533, EP 17H96 and EP I73494.

Pharmaceutical compositions and their use

Monoclonal antibodies obtained by the method of the invention that have a neutralizing effect, such as those that react with epitopes in gp110 or p25 or that react with a blocking peptide, can also be incorporated as components of pharmaceutical compositions to alleviate HIV infections. A composition of this type should contain a therapeutic or prophylactic amount of at least one monoclonal antibody obtained by a method of the invention on a pharmaceutical carrier. The pharmaceutical carrier in this case can be any non-toxic agent with which the monoclonal antibody can be administered to a patient. These may be sterile water, alcohols, fats, waxes and inert solids. Conventional excipients such as buffers, dispersants and the like can also be used. The composition may contain a single monoclonal antibody, for example if it is to be specific for HIV strains with envelope glycoproteins that contain epitopes in the pb region of 6688 to 6750. However, the composition may also contain one or more additional monoclonal antibodies to form a cocktail. For example, monoclonal antibodies against various strains of HIV can be mixed into a cocktail to provide a versatile monoclonal antibody composition with therapeutic and preventive action against the vast majority of clinical HIV isolates. The composition may comprise monoclonal antibodies that bind to HIV proteins or glycoproteins other than gp110 or p25, for example, gp41 glycoprotein or p34 nuclease. The molar ratios of the various monoclonal antibodies will usually not vary by a factor of more than 10, usually no more than a factor of 5, preferably the molar ratio will be in the range of 1: 1 to 2.

The monoclonal antibodies obtained by the method of the invention may also be used in conjunction with other anti-retroviral agents including blocking peptides. Common current knowledge of anti-retrovirus and anti-HIV agents is reviewed in Mitsuya et al., Nature 325: 773-778, 1987.

Monoclonal antibodies, peptides and pharmaceutical compositions containing them are particularly useful for oral and parenteral administration. Preferably, it is parenteral, i.e. subcutaneous, intramuscular, or intravenous. For parenteral administration, the composition comprises a solution of the monoclonal antibody, peptide or mixture thereof in a suitable, preferably aqueous, medium. Various aqueous media can be used, such as water, buffered water, 0.4% sodium chloride solution in water, 0.3% glycine in water, and the like. These solutions are sterile and usually do not contain a solid. They can be sterilized by conventional means. These formulations may contain other excipients customary in the pharmaceutical industry as required to meet physiological requirements, for example to adjust pH, eliminate toxicity, and the like, such as sodium acetate, sodium chloride, potassium chloride, chloride The concentration of antibodies in these compositions can vary within wide limits, for example from less than 0.5%, usually from 1% to 15 to 20% by weight, the concentration being selected depending on the volume of the liquid, viscosity and the like, and depending on the route of administration chosen.

CS 275 838 B6

Thus, for example, a typical pharmaceutical composition for intramuscular administration may comprise 50 mg of monoclonal antibody and sterile water with buffer up to 1 ml. A typical composition for intravenous infusion will contain, for example, 150 mg of monoclonal antibody and 250 ml of Ringer's solution. Conventional procedures for the manufacture of parenteral compositions are known and are described in more detail, for example, in Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pennsylvania, <1980).

The monoclonal antibodies and peptides obtained by the method of the invention may be lyophilized and reconstituted in a suitable environment prior to use for extended storage. This technique is effective in conventional immunoglobulins and conventional lyophilization and reconstitution methods can be used. Obviously, efficacy losses may occur to varying degrees, for example, in the case of conventional immunoglobulins, IgM usually has greater efficacy losses than IgG antibodies, but these losses can be compensated in advance.

The composition comprising the monoclonal antibodies obtained by the method of the invention, peptides or mixtures can be administered for prophylactic or therapeutic purposes against HIV infections. In therapeutic use, the compositions are administered to patients who have already been infected with HIV in an amount sufficient to cure or at least stop the infection and its complications. The amount required to achieve this goal is called a therapeutically effective dose. This dose will depend on the severity of the infection and the general condition of the patient's own defense system, but is usually in the range of 1 to 200 mg of antibody per kg of patient weight, with doses of 5 to 25 mg / kg being commonly used. It will be appreciated that the materials obtained by the process of the invention will generally be administered in serious, life-threatening or potentially life-threatening conditions. In such cases, at the discretion of the attending physician, the above dosages may be exceeded.

For prophylactic administration, compositions comprising peptides, antibodies, or mixtures thereof may be administered to patients who are not yet infected with HIV but are currently exposed or likely to have been exposed to a virus to support the patient's resistance to a potential infection or vaccinate. against the virus. The dose used in this case is a prophylactically effective dose. In this use, the dose depends on the condition to be treated, generally from 0 to 25 mg / kg, in particular from 0.5 mg to 2.5 mg / kg, depending on the general state of health and immunity.

Single or repeated administration of the compositions and the dosage employed are selected by the attending physician.

In any case, the compositions containing these agents should contain sufficient amounts to treat the patient.

In addition, the monoclonal antibodies obtained by the method of the invention may be used as carrier molecules that are a specific target. Any antibody can bind to a toxin to form an immunotoxin or a radioactive material or drug. Methods of making immunotoxins and radiopharmaceuticals are known and are disclosed, for example, in Cancer Treatment Report 68: 317 (1984).

Is it also possible that the hetero-aggregates of monoclonal antibodies obtained by the method of the invention with human T-cell activators, for example monoclonal antibodies against the C3-antigen or against the βθ-gamma receptor on T-cells, can enable human T-cells or cells carrying? _c- gamma (such as K-cells or neutrophils) destroy cells infected with HIV cell-mediated antibody-dependent cytolysis. (ADCC). Such heteroaggregates may be prepared, for example, by covalently cross-linking an anti-HIV antibody and an anti-CD3 antibody, using N-succinimidyl-3- (2-pyridyldithiol) propionate as a bifunctional reagent as described by Karpowski et al. J. Exp.

Copper. 160: 1686 (1984).

Peptide compositions may also be used for therapeutic purposes, since administration thereof results in a reduction in the amount of HIV in the infected host or in its removal. These compositions, such as peptide 29, blocking peptides, and peptide 126 (described in U.S. Patent Application No. 930,785), can be administered intravenously in a suitable physiological carrier, e.g.

The various vehicles may be sodium chloride solution with phosphate buffer, sodium chloride solution, water, potassium chloride, sodium lactate and the like. The peptide concentration will vary over a wide range depending on its use. , its efficacy and route of administration. Preferably, peptides with an amide group at the COOH end group are used, which are formulated at the amino terminus or other pharmaceutically acceptable derivatives thereof may be used. Přidá-. By blocking peptides to mimic and neutralize regions of HIV and / or specific reactive antibodies, the therapeutic effect is greatly enhanced. It is also possible to incorporate other anti-HIV agents in these compositions, for example, non-monoclonal antibodies such as 3'-azido-3'-deoxythymidine, 2 ', 3'-dideoxycytidine, 2', 3'-dideoxy-2 ', 3'. didehydrocytidine and the like. Use of monoclonal antibodies in immunoaffinity purification

Monoclonal antibodies specific for polypeptides containing gp110 or other antigenic determinants, particularly those obtained by the biological expression of recombinant composite proteins or lysates or HIV extracts grown in culture, are particularly suitable for use in purification procedures. These antibodies will usually have an affinity constant of the order of 10 to 10 M. These antibodies can be used to purify recombinant composite proteins from the culture medium of the recombinant expression system when the protein formed in this way is secreted or from components of the disrupted biological expression system. if the expression material is not secreted from the cell. Typically, monoclonal antibodies capable of reacting with gpH0 or another antigenic determinant bind to a carrier or support on which they are immobilized. The solution containing the HIV antigenic determinants is then contacted with the immobilized antibody under conditions suitable for forming an immunological complex between the antibody and polypeptides containing the gp110 antigenic determinants. Unbound material is separated from the bound immunological complex and the gpHO complex or antigen fragments are then separated from the carrier.

The monoclonal antibodies are usually at least partially purified upon recovery from ascites or cell culture supernatant prior to binding to the carrier. Such techniques are well known in the art and may include high salt neutral salt fractionation. Other techniques, such as DEAE-cellulose chromatography, gel filtration, gel electrophoresis, or affinity chromatography using protein A may be used to achieve at least partial purification of the monoclonal antibody prior to binding to the immunoadsorbent material.

The carrier to which the monoclonal antibody is bound and immobilized should have the following general properties:

a) poor protein interaction to minimize non-specific binding,

(b) good flow to allow high molecular weight substances to flow,

(c) the content of chemical moieties which may be activated or modified to allow chemical binding of monoclonal antibodies;

(d) be physically and chemically stable under the conditions used to bind monoclonal antibodies; and

(e) be stable to the conditions and components of the buffers required for antigen uptake and elution.

Some carriers that are commonly used are agarose, polystyrene derivatives, polysaccharides, polyacrylamides, activated cellulose, glass and the like. There are various chemical methods for binding the antibody to the substrate, as described, for example, in Cuatrecasas P., Advances in Enzymology 36: 29 (1972). Antibodies obtained by the method of the invention can be coupled directly to the support used or through a binding chain or the like. The general conditions required to immobilize monoclonal antibodies to chromatographic materials are well known in the art. They have been described, for example, in Tijssen P., 1985, Practice and Theory of Enzyme immunoassay. The specific binding procedures will depend somewhat on the type of antibody to be bound. Monoclonal antibodies have the property that they do not usually vary from batch to batch, so that their binding conditions can be optimized. The binding of obCS 275 838 B6 results in covalent attachment to the support.

The HIV extract extract or lysate suspension or supernatant for culturing the biological system for expression or the disrupted cell suspension is then added to the separation matrix. The mixture is incubated under conditions and for a time sufficient to form an immunological complex, usually at least 30 minutes, typically in the range of 2 to 24 hours. The immunological complexes containing the polypeptides with the antigenic portions of gp110 are then separated from the mixture. The mixture is then removed, for example, by elution, and the bound immunological complex is washed extensively with adsorption buffer.

Then, the complexes can be eluted from the separation matrix using a solvent compatible with the carrier, and can be selected by any person skilled in the art. It is also possible to selectively remove polypeptides containing gp110 or other antigenic portions. For example, peptides containing epitopes recognized by antibodies can be used to compete for binding sites in the antibody, a possible elution technique that can be performed under gentle elution conditions, selectively adsorbed gpl10 antigen containing polypeptide can be eluted with affinity adsorption material, by changing the pH and / or ionic strength of the buffer.

Chaotropic agents can also be used to remove bound antigen. The choice of the chaotropic agent, its concentration and other elution conditions are dependent on the interaction between the antigen and the antibody, but once determined, there is usually no need to change them, as is usually the case in polyclonal separation systems.

The material so obtained will usually need to be adjusted to physiological pH when high ionic strength buffers or too low or high pH buffers have been used to separate the bound gp110 antigen from the matrix. Sometimes, dialysis or gel filtration is also required to remove excess salts in the elution material to reconstitute gp10 or polypeptides containing gp'10 fragments with antigenic properties.

Thus, by the method of the invention, substantially purified gp110 or polypeptides containing fragments thereof having an antigenic effect can be obtained, either from a natural source from infected cell cultures or after expression in bacteria, yeast or cultured insect or mammalian cells. The gpHO polypeptide, or fragments thereof, or other purified proteins are generally pure to more than 50%, more often to more than 75%, and typically to more than 95 to 99%. These molecules can then be used for a variety of purposes.

HIV gp110 proteins, polypeptides containing fragments of these materials with antigenic properties, or other substantially purified proteins can be used for a variety of purposes, including the production of an AIDS vaccine. The vaccine comprises an effective dose of determining antisera, for example gp10 or a neutralizing region thereof. As an additional component, antigenically active portions of HIV proteins or glycoproteins that stimulate the production of antibodies (preferably neutralizing antibodies) in the immunized host can be used, which antibodies are then capable of protecting against future HIV infection.

Diagnostic use of monoclonal antibodies

The monoclonal antibodies obtained by the method of the invention can also be used for diagnostic purposes. They can be marked for this purpose. The diagnostic procedure typically involves detecting complex formation by binding the monoclonal antibody to the HIV antigen. In the absence of labeling, the antibodies can be used, for example, for agglutination. In addition, unlabeled antibodies can be used, for example, in combination with other labeled antibodies (second antibody) that react with a monoclonal antibody, such as antibodies specific for immunoglobulin. In addition, monoclonal antibodies can be directly labeled. A wide variety of labeling means can be used, for example, radionuclides, fluorescent substances, enzymes, enzymatic substrates, enzyme cofactors, enzyme inhibitors, binding chains (especially haptenes, etc.). Various immunological procedures are available, for example at least those which have been disclosed in U.S. Patent Nos. 3,817,827 »3,850,752,

901 654, 3 935 074, 3 984 533, 3 996 345, 4 034 074 and 4 098 876. Usually, the monoclonal antibodies and peptides obtained by the method of the invention may be used in enzymatic immunoassays, for example, with the antibody or other antibodies from different species bind to the enzyme. In the case of a biological sample containing HIV antigens, for example η

CS 275 838 Β6 human blood serum, saliva, semen, vaginal secretion or suspension of virus-infected cells contacted with antibodies, binds between the antigen and those molecules that contain the epitope in question. These proteins or virus particles can then be separated from unbound components and the second enzyme labeled antibody added. The presence of the antibody-enzyme conjugate specifically bound to the antigen is then determined. Other conventional methods known in the art may be used.

It is also possible to arrange kits for use of said antibodies in the detection of HIV infections or the presence of HIV antigen. These materials comprise a monoclonal antibody, obtained by the method of the invention, usually in lyophilized form, either singly or in conjunction with other antibodies specific for other HIV epitopes. Antibodies that may be associated with the labeling moiety are used together with buffers such as tris-buffer, phosphate buffer, carbonate and the like together with stabilizers, biocides, inert proteins such as bovine serum albumin and the like. However, 5% by weight, based on the amount of active antibody, are present in an amount of at least 0.001% by weight, based on the antibody, it may often be desirable to incorporate inert material to dilute the active ingredient in an amount of 1 to 99%. If a second antibody capable of binding the monoclonal antibody is used, it will usually be present in a separate vial. The second antibody is typically bound to the labeling material and processed to the adjuvant in a conventional manner.

The detection of the gp110 and g25 antigens may find its use in the diagnosis of HIV infection or whole virus in various biological samples. Such biological samples may be, for example, blood serum, saliva, semen, biopsy tissue samples (brain, skin, lymph nodes, spleen, etc.), cell culture supernatants, disrupted eukaryotic and bacterial expression systems, and the like. The presence of the virus is determined by incubating the monoclonal antibodies with the biological sample under conditions in which the immunological complex may be formed with subsequent detection of this formation. In one embodiment, complex formation is detected using a second antibody that is capable of binding a monoclonal antibody, the second antibody typically being bound to a labeling material. In another possible embodiment, the monoclonal antibody is bound to a solid support, which is then contacted with the biological sample.

Preparation and use of synthetic peptides

According to the method of the invention, novel peptides can be obtained which, inter alia, immunologically mimic protein epitopes encoded by HIV retrovirus, in particular epitopes encoded in the env or gag region of the virus genome which encode gp110 or p25. In order to capture inter-strain variations between different isolates, various adjustments can be made, balancing conservative substitutions and selecting between alternatives in which non-conservative substitutions occur. The peptides can be used as immunogenic agents to inhibit or eliminate HIV antigen production in vivo or in vitro in the detection of virus or anti-virus antibodies in a physiological sample. Depending on the method selected, the peptides may be coupled to a carrier or other compounds, labeled or unlabeled, to a solid carrier, and the like.

In one embodiment, the desired peptides can be derived from the gp110 region of the virus. Of particular importance is the env region from bp6688 to bp6750 and from bp7246 to bp? 317.

Said peptides, including blocking peptides, comprise at least five, sometimes 6, 8, 12, 21, usually less than 50, more often less than 25 amino acids in the sequence encoded by the retrovirus HIV. It is desirable that the peptide be as small as possible while retaining substantially all of the immunological reactivity or antiviral activity of the large peptide. In some cases, it may be desirable to couple two or more oligopeptides that do not overlap to form a single peptide or use them as individual peptides simultaneously so that the same sensitivity can be obtained simultaneously or separately.

The peptide can be modified by introducing conservative or non-conservative substitutions into the peptide structure, typically less than 20% more often less than 10%

CS 275 838 B6

Η be changed. In those situations in which the regions are polymorphic, it may be desirable to exchange one or more specific amino acids to more efficiently mimic differing epitopes of different retrovirus strains. In many cases, methionine, norleucine (Nor), can be substituted to increase chemical stability.

It will be appreciated that the peptide used in the method of the invention need not be identical to any of the specific HIV sequences if the compound used is in competition with the proteins of at least one HIV retrovirus strain. Thus, peptides can be altered in various ways, for example, by waving, deleting, and substituting certain amino acids, conservative or non-conservative, and these changes may entail certain advantages in use. Conservative substitution is a substitution within gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; phe, tyr; and nor, met; the sequence will typically not differ by more than 20% from the sequence of at least one HIV retrovirus strain except that the amino acids are added at the ends to obtain a linker (arm) that can be used to immobilize the peptide. These linkers consist of at least one amino acid, but may also contain 50 or more amino acids, but are usually 1 to 10 amino acids.

A peptide in which the amino acid chain is modified by substitution, addition or deletion of amino acid residues is intended to retain substantially all immunological reactivity or antiviral activity of the unmodified peptide, which can be measured in a variety of ways. The d-isomer of one or more amino acids can be used, if desired, to modify biological properties such as potency, disintegration rate, and the like.

In addition, one, two or more amino acids may be added to each end of the peptide to facilitate peptide binding, binding to a carrier consisting of a larger peptide, modification of the physical, chemical or other properties of the peptide or oligopeptide and the like.

For example, the amino acids tyrosine, cysteine, lysine, glutamic or aspartic acid and the like can be attached to the N-terminus or C-terminus to improve the next bond. Cysteine is a particularly useful amino acid to facilitate covalent bonding or to form polymers by oxidation.

In addition, sequences of peptides or oligopeptides may differ from natural sequences in that the terminal -NH ₂ group is acylated, for example acetylated or amidated with thioglycolic acid, or converted to an amide by, for example, ammonia or methylamine with another molecule or carrier or for polymerization.

For example, when Y and Y 'are present in peptides I to VIII and IX to XV, there is a preferred embodiment wherein Y or Y * is one or more cysteine residues or a combination of cysteine residues with other amino acids that delay the cysteine residue from ending and lengthening the string. A preferred amino acid in this regard is glycine. A preferred peptide for use in oxidative polymerization is one that contains at least two cysteine residues in Y and Y '. When two cysteine residues are present at the same end of the peptide, it is preferable to separate these residues from one to three amino acid residues, preferably they are glycine residues. The presence of cysteine residues may allow formation of peptide dimers and / or increase the hydrophobicity of the resulting peptide, which facilitates immobilization of the peptide to a solid phase or immobilization system.

Of particular interest is the use of the mercaptan group of cysteine or thioglycolic acids used to acylate terminal amino groups and the like for coupling two peptides or oligopeptides or linking them with a disulfide group or a longer chain to form a polymer that contains a number of epitopes. These polymers have the advantage of increased immunological reactivity. If different peptides are used to form the polymer, there is another possibility of inducing the production of antibodies that react immunologically with several antigenic determinants or with different HIV isolates.

In order to achieve the formation of antigenically effective polymers (synthetic multimers). compounds containing bis-haloacetyl groups, nitroaryl halides may be used

CS 275 838 δδ and the like, wherein the reagent is to be specific for thio groups. Thus, the linkage between two mercapto groups may be a single bond or a chain containing at least 2, usually at least 4, but not more than 16, and usually no more than 14 carbon atoms, when using different peptides or oligopeptides.

The peptides may be used on a macromolecular carrier (i.e., a carrier with a molecular weight of at least 5 units). The carrier may be, for example, a polyamino group (polyamino acid), either naturally occurring or synthetic. Examples of such carriers include, for example, poly-L-lysine, hemocyanin, thyroglobulin, albumins, such as serum bovine albumin, tetanus toxin, and the like. The choice of carrier depends primarily on the target for which the antigen is to be used and its availability.

Thus, in the case of these conjugates, at least one molecule of at least one peptide per macromolecule and not more than 1 to 0.5 units, usually no more than 1 to 2 units of the macromolecule. One or more different peptides may be linked to the same macromolecule.

A conventional coupling method, reagents such as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic anhydride, succinic anhydride, glutaraldehyde and the like are used. Binding may occur at the N-terminus, C-terminus, or at a site located in the central portion of the molecule. The peptide obtained can be derivatized by binding to a carrier and the like.

Various methods known in the art can be used to detect the presence of antibodies to the retrovirus proteins or the presence of the retrovirus own proteins. Of particular interest is the use of labeled reagents, where the label causes a detectable signal or peptide binding directly or indirectly to a surface on which proteins may bind to antibodies. The presence of human antibody bound to the peptide can then be demonstrated using exo-. gene antibodies specific for human immunoglobulin, usually for IgM and IgG, or by using a labeled protein specific for an immunological complex, for example S. aureus protein factor A.

A typical embodiment of these assays is a method using, for example, microtiter plates with a certain number of wells in which the polypeptides or conjugates are adsorbed to the bottom and / or walls of the wells by covalent or other binding. The sample, usually human blood or serum at the appropriate dilution in buffer, is added to the wells of the plate and left there for a sufficient time for. forming a complex between the polypeptide and any antibody in the sample. The superaatant is then removed and the wells are washed to remove non-specifically bound proteins. A labeled specific protein that specifically binds to a complex, such as an anti-human immunoglobulin antiserum, is used for detection.

The peptide can be prepared in a number of ways. Because it is a relatively short-chain substance, it can be synthesized in solution or on a solid support in a conventional manner. Various automated synthesis devices are also available today and can be used. These devices and their use have been described, for example, in Stewart and Young. Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co., 1984 and Tam et al., J. Am. Chem. Soc. 105: 6442 (1984).

It is also possible to use hybrid DNA technology and prepare a synthetic gene using single strands that encode said polypeptides or strands that are substantially complementary, the single strands overlapping and can be joined and hybridized. The hybridized chains can then be ligated to form the full-length gene, and by selecting the appropriate termini, the gene can be inserted into a suitable vector as currently available. These procedures are described, for example, in Maniatis et al., Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982. It is also possible to clone a region of the viral genome that encodes a peptide in a conventional manner using recombinant DNA and then expressing it as also described in Maniatis et al., supra.

CS 275 838 B6

The DNA coding sequences of LAV-γ and ARV, two HIV retrovirus isolates that can be used to express peptides, can be shown as follows:

LAV.

BRU

ARV-2

TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC CGT ' ATC CAG AGG GGA CCA GGG AGA TTT GTT ACA ATA GGA AAA ATA GGA AAT ATG AGA CAA GCA CAT TGT TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC MELT ATA GGA CCA GGG AGA GCA TTT CAT ACA ACA GGA AGA ATA ATA GGA GAT ATA AGA AAA GCA CAT TGT.

Sequence fragments can be used to express peptide fragments, conservative base sequences can be made in which the modified codons encode the same amino acid or non-conservative changes in the code sequence, and the resulting amino acid can cause a conservative or non-conservative change in the amino acid sequence, above.

The coding sequence can be extended to the 3-terminus, the 5'-terminus, or both, while maintaining its epitope. The elongation can be carried out with a binding chain, for example, for a labeling reagent such as an enzyme, for coupling two or more peptides, for obtaining greater antigenic activity, for introducing restriction enzyme sites for cloning and the like.

The DNA sequence itself, fragments or longer regions thereof, usually containing at least 15 bases, preferably at least 18 bases, can be used as a nucleotide probe for detecting RNA retroviruses or DNA provirus or for identifying homologous regions or for detecting a sequence. A variety of methods can be used for this purpose, such as the Grunstei-Hogness process, the Southern technique, the dot blot, and various improvements to these processes, as described, for example, in U.S. Patent No. 4,358,535.

Said peptides including blocking peptides and analogs thereof may be used as such or in combination in vaccines. Anti-idiotypic antibodies, i.e., antibodies reactive with antibody idiotypes obtained by the method of the invention and containing epitopes mimicking HIV neutralizing regions, may also be used in vaccines. The peptides or anti-idiotypic antibodies can be processed in a conventional manner, usually at a concentration of 1 µg to 20 mg / kg. Physiologically acceptable media are used as carriers, for example sterile water, physiological sodium chloride solution, the same phosphate buffer solution and the like. The vaccine may also contain adjuvants such as aluminum hydroxide gel, wetting agents such as isolecithin, polyols, polyanions, peptides, proteins such as diphtheria or cholera toxins and oil emulsions. The peptides may also be encapsulated in liposomes or conjugated to polysaccharides, polypeptides or polymers to form a vaccine. The vaccine is administered by injection, for example, intramuscularly, peritoneally, subcutaneously, intravenously, and the like. The immune-inducing dose is administered once or repeatedly, usually at an interval of one to four weeks. An immune-inducing dose is the amount of vaccine that is capable of eliciting an immunological response in the host.

Further details of the process of the invention will be explained in the following examples.

Example 1

Formation and characterization of monoclonal antibodies

The example describes the generation of hybrid cell lines that produce monoclonal antibodies specific for HIV envelope glycoproteins. This procedure involves the use of purified LAV-γ lectin extracts bound to the agarose lens lectin as an immunogen. Monoclonal antibodies that are sequentially generated by hybrid cell lines are characterized by their ability to immunoblot and radioimmune the precipitation of gp110 from purified LAV and as a recombinant composite protein whose expression has been achieved. Monoclonal antibodies that bind to epitopes of gp110 are also reactive using an excited whole virus BLISA system, composed proteins and synthetic peptides, and react with the whole virus under indirect fluorescence.

Hybrid cell lines capable of producing monoclonal antibodies were obtained subsequently

CS 275 838 B6

LAV virus purified from infected CEM cells (ATCC No. CRL8904) was disrupted in 50 mM Tris pH 7.4, 0.15 M sodium chloride, 1.0% Aprotinin, 2.0% Nonidet P-40® (NP -40) (octylphenoxypolyethoxyethanol). The extracts were clarified by centrifugation twice and were adjusted to 0.5% NP-40 by adding virus disruption buffer (3 volumes) without NP-40. Sepharose with lens lectin (Pharmacia, Pis cataway, NJ) was prewashed with virus disruption buffer (50 mM Tris, pH 7.4, 0.15 M NaCl, 1.0% Apritinin, 0.5% NP.4P) . The clarified virus extracts were adsorbed to the lectin from the lens for 42 hours at 4 ° C. Unadsorbed material was removed by washing with excess buffer. The adsorbed material was then eluted with 0.2 M alpha-methylmannoside in adsorption buffer. The eluent was dialyzed against PBS to remove sugar, and the material was then re-adsorbed onto Sepharose with lens lectin.

The Sepharose glycoprotein complex with lens lectin was used to immunize Balb / c mice by triple administration intraperitoneally without adjuvant at 2 to 3 weeks interval. The spleens were then removed and the presence of antibodies in circulation was detected by immunoblot analysis. they were antibodies to the glycoprotein HIV. ELISA and RIP provided the same result.

To obtain cell lines, the procedures of Kohler and Milstein, Nature, 256: 495 (1985) were modified with the modification of Goldstein LC et al., Infect. Immun. 38: 273 (1982). Spleen B cells were fused to NS-1 myelomatic cells using 40% polyethylene glycol. After fusion, the cell mixture resuspended in HAT medium (RPMI-1640, supplemented with 15% fetal calf serum, 1 x 10 "^ M HY-7 -5 poxanthinu, 4χ10 M aminopterin and 1 x 10 6 ^J M thymidine) For selection for hybrid cell growth, the cells were distributed into 96 well microtiter plates at a concentration of 1-3 x 10 6 ml cells and then incubated at 37 ° C in a humidified atmosphere containing 6% carbon dioxide. The cultures were fed by replacing one half of the supernatant with a new HAT medium. The wells were then monitored using an inverted microscope for signs of cell proliferation, and once sufficient cell density was reached, the supernatants were examined for the presence of antibodies against the LAV isolate.

Wells that contained hybrid cells producing anti-LAV antibodies were identified by ELISA by measuring binding to purified complete disrupted virus or complex proteins after biological expression. Tests using an excited virus ELISA system were performed. £ LAV EIA plates (Genetic Systems, Seattle, Washington). The plates were incubated with cell culture supernatant at 37 ° C for 45 minutes and then washed three times with 0.05 $ Tween 20 in phosphate buffered saline (PBS-Tween).

Peroxidate-bound goat anti-mouse IgG antibody (i: 2000 dilution in PBS-Tween (Zymed Laboratories, Inc., South San Francisco, California)) was added at 100 µl per well for incubation for 45 minutes at temperature 37 ° C and then washed as above. Substrate (0.025 M citric acid, 0.05 M sodium hydrogen phosphate pH 5.0 containing 14 mg o-phenylenediamine and 10 µl 30% hydrogen peroxide per 50 ml) was added. and the plates were incubated for 30 minutes at room temperature in the dark, the reaction was stopped by the addition of 3 N sulfuric acid, and the colorimetric reactions were measured quantitatively by an automated reader, the wells shown to be positive were further cloned by dilution, retested for specificity and grown .

The monoclonal antibodies that were produced and secreted by the resulting hybrid cell lines were further characterized for their specificity and reactivity by immunoblot reaction, immunoprecipitation and ELISA system using disrupted LAV, recombinant composite LAV proteins, and synthetic LAV peptides. All antibodies were of the IgG isotype. The HIV-gp110-1, HIV-gp110-2 and HIV-gp110-3 cell lines were deposited with the American Type Culture Collection and designated ATGC No. HB 9175, HB 9176, and HB 9177.

Recombinant composite proteins tested for reactivirus were designated ENV2, ENV3, ENV4 and ENV5: The ENV2 protein was expressed by expression of the plasmid pENV2 (ATCC 5307 '),

CS 275 838 B6 is a BAV region ranging from bp6598 up to bp7178 (numbering according to Wain-Hobson et al., Cell 44: 9 (1985), BHV3 was expressed by plasmid pENV3 (ATCC)

53072), which contains the BAV region from bp7178 to bp7698, ENV4 was obtained by expression using plasmid pBNV4 (ATCC 53073) and contains bp7698 to bp8572, and BNV5 was obtained by expression using plasmid pBNV5 (ATCC 53074), and contains a BAV region from bp5889 to bp 7698. Production of recombinant composite proteins has been described in US Patent No. 721237.

Synthetic peptides

Peptides I (29) and VIII (100-2-2) were assembled on a benzhydrylamine resin (Applied Biosystems Inc., Poster City, Califomia). The benzhydrylamine is supported on a polystyrene / divinylbenzene support. Peptide V (177) was assembled on a target. butoxycarbonyl (Boc) -ethylbenzylcysteine-phenylacetamidomethyl (PAM) -polystyrenedivinylkenzene resin. Symmetrical anhydride bonds were performed in an Applied Biosystems 430 A apparatus. Cysteine was added as the first residue for both peptides.

Dicyclohexylcarbodiraid linkages in the presence of hydroxybenzyl triazole were used for asparagine and glutamine. Side chain protection with benzyl groups and Boc-alphaamine was used. Additional side chain protection commonly used was Boc (formyl) tryptophan, Bocmethionine sulfoxide, Boc (tosyl) arginine, Boc (methylbenzyl) cysteine, Boc (tosyl) histidine, Boc (chlorobenzyloxycarbonyl) lysine and Boc (bromobenzyloxycarbonyl).

When the peptides were radiolabelled, this was done by acetylation of the amino-terminated end with H-acetic acid and an excess of dicyclohexylcarbodiimide.

Removal of the protecting groups and cleavage of the peptide from the resin were carried out according to Tama et al. Extraction from the resin was performed with 5% acetic acid and the extract was subjected to gel filtration in 5% acetic acid.

Synthetic peptides tested for reactivity against monoclonal antibodies were peptides 29, 36 and 39. For peptide 20 the BAV? Y genomic region is encoded from bp6688 to bp6750, for peptide 36 the code is from bp7246 to bp7317 and for peptide 39 it is encoded region from bp75-6 to bp7593. Peptides 36 and 39 are described in detail in U.S. Patent No. 4,629,783.

Blocking peptides IX-XV were also assembled essentially as described above on methylbenzhydrylamine-polystyrene-divinylbenzene resin (Applied Biosystems Inc., Poster City, California). Symmetrical anhydride bonds were performed on an Applied Biosystems -430A synthesizer. For asparagine, a dicyclohexylcarbodiimide bond was used in the presence of hydroxybenzotriazole. In the case of the need for protecting groups, groups based on benzyl and Boc-alpha-amino were used, whereas in the case of tyrosine-containing side chains, Boc (bromobenzyloxycarbonyl) was used. Acetylation was performed using acetic anhydride or glacial acetic anhydride and dicyclohexylcarbodiimide, if necessary. Deprotection of peptide and peptide from resin was performed according to Stewart et al., Supra. Extraction from the resin was performed with 50% acetic acid and the extract was then gel filtered in 20% acetic acid. High pressure liquid chromatography on a Vydac C18 column (Rainin Instrument Co., Bmeryville, Calif.) Was performed as necessary using 0.1% trifluoroacetic acid and acetonitrile gradient.

Immunoblot reactions

These assays were performed using clone supernatant or ascites effusion and purified BAV and recombinant composite proteins as antigen. The antigens were first separated by acrylamide gradient gel electrophoresis (7.0-15.0%) and then transferred to a nitrocellulose membrane (NCM) of electrophoresis for 4 hours at 25 V in 25 mM sodium phosphate pH 7.0. After transfer, CM was blocked to prevent non-specific reactions by incubation in PBS-Tween or Blotto (5% non-fat milk powder in PBS) for one hour at room temperature. Then the NCM was incubated with the cell culture supernatant or

CS 275 838 B6 with ascites liquid diluted in PBS-Tween for one hour at room temperature and then washed three times with fresh PBS-Tween each time. In the second step, NCM was incubated with goat anti-mouse IgG antibody bound to horseradish peroxidase, diluted in PBS-Tween for one hour at room temperature. This incubation was followed by washing with PBS-Tween and then immersion in peroxidase development solution (Bio-Rad Laboratories, Ricbmond, California) for 20 minutes. The reaction was then stopped by immersion in deionized water. The reactivity of the monoclonal antibody was compared to a positive control serum that was reactive to the purified disrupted virus or the composite protein obtained by expression. The results show that all antibodies bound to gp110 and its precursor, gp150, using the disrupted virus. Antibodies 110-1 and 110-2 also recognized the composite ENV3 protein, while antibodies 110-3, 110-4, 110-5 and 110-6 formed a complex with the ENV2 protein.

Immunoprecipitation

Viral extracts for radioimmunological precipitation were prepared from OEM cells infected with LAVggy HIV isolate, adapted by continuous passage for lytic growth. Once the first signs of cytopathic effect were apparent, the cells were transferred to a labeling medium containing ^ 5 S-methionine (0.05 mCi / ml) or HH-glucosamine (0.025 mCi / ml) and then incubated for 24 hours. By this time, most cells had dissolved and the virus was released into the culture supernatant. Then the virus was pelleted by centrifugation for 1 hour at 100,000 g of cell-free supernatant and extracts were prepared in P-RIPA buffer (sodium chloride phosphate buffer solution containing 1.0% Triton X-LOO, 1.0% deoxycholate, 0.1% SDS) and 1% Aprotinin). Similar extracts were prepared from supernatant of uninfected CEM cells.

Immunoprecipitation assays were performed with 100 µl virus extract incubated with 100 µl hybrid cell line supernatant for 1 hour on ice. Then, 4 µl of rabbit anti-mouse IgG serum (Zymed Laboratories, San Prancisco, California) was added to each sample and incubated for a further 30 minutes. Immunoprecipitin (100 µl, Bethesda Research Laboratory, Bethesda, Maryland) was then added to each sample suspended in P-RIPA buffer containing 1.0% egg albumin and incubated for an additional 30 minutes. The bound complex was then washed and separated by SDS-polyacrylamide gel electrophoresis (15.0% acrylamide, DATD gel). After electrophoresis, the gels were fixed, dipped in Enhance (New England Nuclear, Boston, MA), dried, and exposed to Kodak XR-5 film. Reference positive serum that immunologically precipitated with all HIV proteins was reacted with CEM cells infected with the virus and other virus as positive and negative controls.

The results showed that all six monoclonal antibodies were specific for gp110 and gp150 and precipitated both of these materials.

Enzyme-linked immunoadsorption tests

In order to detect the gpl10 epitopes recognized by the monoclonal antibodies obtained by the method of the invention from hybrid cells or from ascites fluid, these were further characterized by their reactivity using an ELISA system and compound proteins obtained by biological expression and using synthetic peptides. The same procedures as above were used except that the composite proteins or synthetic peptides were used instead of the purified virus as antigens adsorbed to the microtiter plates.

When peptides were used as antigens, the lyophilized peptide was dissolved in a 6M solution of guanidine in hydrochloric acid. Prior to plating, the guanidine solution was diluted to a final peptide concentration of 100 / µg / ml with 0.05 M carbonate and bicarbonate buffer, pH 9.6, and then 50 µl of the peptide solution was placed in one well of the microtiter plate and the plates were then incubated overnight at 4 ° C. Excess peptide solution was removed, the plates were blocked with Blotto, and the whole procedure was terminated as usual using an ELISA system. Similarly, the recombinant protein was diluted to a final concentration of 2 / µg / ml in 0.05 buffer with carbonate and bicarbonate pH 9.6 prior to the actual procedure.

The results are shown in Table II. Monoclonal antibodies produced by cellular antibodies

20 lines of HIV gp110-1 and HIV gp1Q-2 were reacted with ENV3, ENV5, peptide 36, and disrupted virus. Antibodies from the HIV-gp110-3, HIV-gp110-4, HIV-gp4110-5 and HIV-gp110-6 cell lines were reacted with ENV2 and with peptide 29 as well as with the disrupted virus.

Table II

Using an ELISA system to determine the reactivity of monoclonal antibodies with recombinant proteins and synthetic peptides phiocombine composite protein Synthetic peptide BAV CEM

ENV2 ENV3 ENV4 ENV5 29 36 39 English Translation: 1 10-1 0,077 3,000 0,113 3,000 ND 2,421 0,054 0,908 0.125 110-2 0.003 3,000 0,000 3,000 ND 2,305 " 0.005 1,214 0.009 110-3 3,000 0.011 ND ND 3,000 ND 0.017 0.363 0,046 110-4 3,000 0.020 ND .ND 3,000 ND. 0,01 6 0.383 0,067 110-5 3,000 0.014 ND ND 3,000 ND 0.016 0.368 0,025 110-6 3,000 0,033 ND ND 1,937 ND 0.017 0.468 0,032 Results from Tables II show that monoclonal antibodies HO-2 and ί 10-1: recognize

an antigenic determinant encoded by a DNA sequence in the pENV3 region, in particular the HIV genome region, which is defined by the amino acid sequence that corresponds to peptide 36. That is, the monoclonal antibodies gpl.10-1 and gpl'0-2 bind to a region of the gp110 peptide encoded by a region in the range of bp7246 to bp7317 as evidenced by the formation of an immunological complex with peptide 36 and with ENV3. This region of the HIV genome was previously considered to be relatively conservative, ie only a small number of changes in the DNA sequence coding for peptide 36 in various virus isolates from different geographical regions were expected, as described in Starce et al., Cell 46 637 (1986). In contrast, the monoclonal antibody gp110-3, -4, -5, and -6 binds to the HIV peptides identified by regions coding for peptide 29, i.e., from bp6688 to bp6750. The gp110 region, as defined by peptide 29, appears to contain several nucleotide substitutions in various virus isolates. Monoclonal antibodies that specifically bind to gp-polypeptides that contain conserved epitopes, for example, antibodies 110-1 and 110-2 may have increased utility in a variety of circumstances, such as affinity chromatography and the like. Also in ELISA assays, peptide 110-2-2 reacted with the individual serum from which the LAV-2 isolate was isolated.

Indirect immunofluorescence test

Indirect immunofluorescence using monoclonal antibodies against HIV gp110 antigen was performed on acetone fixed and viable cells. Acetone-fixed cells on a slide prepared from CEM cells infected with LAV isolate were incubated with diluted culture supernatant or ascites fluid for one hour at 37 ° C, whereas live cells were incubated with culture supernatant or ascites fluid for one hour at 4 ° C before placing the cells on a glass slide and fixing them with acetone. Both methods consisted of using a fluorescein isothiocyanate labeled anti-mouse IgG antibody to detect cells carrying the reactive gp110 antigen. The monoclonal antibody HIV-gp110-1 provides positive results using cells infected with LAV isolate, both live and fixed with acetone.

Example 2

Neutralization of virus infectivity. HIV monoclonal antibodies anti-gpHO

This example describes and characterizes the neutralization of HIV infectivity using monoclonal antibodies that bind to gp110 and peptides in the gp110 region. The results show that the monoclonal antibodies gp-110-3, -4, -5 and -6 have a neutralizing effect and that in particular gp-110-3 and gp-110-4 have a high level of this effect.

Neutralization tests

A sensitive neutralization test was developed with the possibility to quantify the effect

Monoclonal antibodies to HIV infectivirus. OEM cells were selected as target cells and then compared. The ascites fluid was prepared as described in Example 1, or an IgG fraction purified by ammonium sulfate precipitation was used, followed by inactivation at 56 ° C for 30 minutes and then diluted as necessary with RPMI containing 10% fetal calf serum. A suspension of the HIV strain, LAV _BBU isolate, was obtained from a four day old OEM culture in the growth log-phase after filtration through a 0.2-0.45 µm filter, after aliquoting and freezing to -70 ° C. One aliquot was heated by thawing and titrated to determine TCID4, the following tests were performed with freshly thawed material diluted to 1: 500 in culture medium to achieve a concentration of approximately ten times the amount required to infect 50% of the OEM cells in culture ( 10 x ΤΟΙϋ ^ θ). The virus suspension was mixed with an equal volume (250 µl) of the monoclonal antibody solution at a five-fold sequential dilution of 1: 5.1: 9.1: 765, 625. The virus-antibody mixture was incubated for 45 minutes at 37 ° C and then the dilutions were again increased and the mixture was re-incubated for 45 minutes at 37 ° C, after which the samples, re-diluted in half, were used to inoculate wells of microtiter plates containing approximately 2 x 10 6 OEM cells per well of 1.0 ml. The cultures were then incubated at 37 ° C in 50% carbon dioxide humid atmosphere for a total of 14 days before being separated, pelleted and disrupted with 1% Triton-X-100 in PBS for 10 minutes. The amount of virus or viral antigen present in the disrupted cells was quantitated using an enzyme-sensitive HIV antigen. The neutralization potency titer, if detectable, was determined as the reciprocal of the dilution of the monoclonal antibody, resulting in inhibition of antigen production by more than 50% in cultures incubated without antibody or monoclonal antibody of the same isotype that had no neutralization prior to this measure. effect.

The above HIV antigen assays were performed with two monoclonal antibodies specific for p25. The respective hybridoma cell lines were obtained as described above with minor modifications including purification of the gag composite protein as an immunogen and characterization of the resulting monoclonal antibody with respect to its specificity and reactivity using recombinant composite protein, pre-labeled GAG-1, GAG-2 and GAG-3 and synthetic peptide 141. GAG-1 protein is obtained by expression of plasmid pGAG-1 (ATCC 53379), GAG-2 protein is obtained by expression of plasmid pGAG- 2 (ATCC 53111) and GAG-3 was obtained by expression from pGAG-3 (ATCC 53112). The production of recombinant composite protein is described in US Patent Nos. 764,460 and 828,828. The code for synthetic peptide 141 is the genomic region of LAVggjj, which corresponds to amino acid residues 198-242. The monoclonal antibodies produced by the hybridoma cell line p25-2 and p25-3 are reactive with respect to the recombinant composite proteins GAG-1, GAG-2 and GAG-3, and the monoclonal antibodies p25-3 are also reactive with respect to the synthetic peptide 141.

In order to perform the antigen binding assay, the individual reagents were first bound to a solid support. The ascites fluid obtained by using hybridoma cells of p25-2 and p25-3 lines was diluted 1: 5000 in 25 mM Iris buffer and pH 8.5 and 200 µl of the material so obtained were placed in individual wells of microtiter plates.

The wells were then sealed and incubated at 4 ° C for 16 hours. The solution was then removed from the wells and a blocking solution of Blotto (0.3%) in PBS was added. The blocking was continued for 5 minutes at room temperature. The blocking solution was then removed by suction and a sample was added. To each well was added 200 µl of the disrupted cell suspension and 5.0 µl of the conjugate prepared as described above. The plates were incubated at 37 ° C for two hours and then the suspension was aspirated and the wells washed 4 times with buffer (0.05% Tween 20 in PBS). The conjugate was prepared as follows: The monoclonal antibodies p25-6 and β 5-7 were conjugated to horseradish peroxidase (HRP) at a 3: 1 molar ratio (Ab: HRP) for a total of three hours using periodate oxidation as described by Kakane et al.

J. Histochem. Cytochem. 22: 1084 (1974). The conjugates were diluted 2: 1 in 1500 with non-fat skim milk at a concentration of 2.5%, 0.01% thimerosal and 0.005% Antifoam A in 20 mM sodium citrate. The rest of the procedure using the ELISA system was performed in the same way

CS 275 838 B6 as in Example 1.

The results of the neutralization experiments with gp-110-3, -4, -5 and -6 antibodies are as follows. Tittry maintaining the highest neutralizing activity were: 110-3 ^GP-1 = 5625, GP-9,765,652 = 110-4, 110-5 = gp-125 and gp-110-6 625Vzhledem = the estimated genetic variability region defined by peptide 29 the ability of the monoclonal antibody gp-110-4 to recognize other HIV isolates was investigated. Immunofluorescence assays were performed as described above. The gp-110-4 antibody was able to recognize antigen in cultures of at least three of the 16 HIV isolates that were tested.

The gp-110-4 antibody was able to neutralize the viruses isolated within 15 weeks from chimpanzees inoculated with isolate 1A-1g as control animals in an AIDS vaccine experiment. The monoclonal antibody was able to neutralize isolates despite the fact that the animal had serum antibodies that neutralized HIV in vitro and despite the animal developing a measurable cell-mediated immunological response against HIV infection. This demonstrates the absence of in vivo antigen shift for the epitope recognized by the gp-110-4 antibody. Example 3

Neutralization of HIV infectivity with a mixture of monoclonal antibodies against gp-110 and p-25

This example describes the neutralization of HIV infectivity using monoclonal antibodies that bind to gp110 and to peptides in the gp110 region in combination with monoclonal antibodies that bind to p25 and to peptides in this region that as such have little or no neutralizing efficiency. The results show that gp_110-2 monoclonal antibodies in combination with p25-6 or p25-7 have a particularly high level of neutralizing activity.

The p25-6 and p25-7 hybridoma cell lines were obtained as described above with modifications that include the use of inactivated disrupted virus or purified Gag recombinant composite protein, obtained by expression in E. coli as an immunogen, followed by characterization of the resulting monoclonal antibodies for specificity and reactivirus in using recombinant composite proteins, designated GAG-1, GAG-2 and GAG-3, and synthetic peptides 15, 88, 150, 147, and 148. The synthetic peptides are encoded in the LAV 1 region, corresponding to the following amino acid residues:

peptide 15 - amino acid residues 329 to 350, peptide 88 - amino acid residues 3'5 to 350, peptide 150 - amino acid residues 318 to 363, peptide 147 - amino acid residues 278 to 319 and peptide I48 - amino acid residues 290 to 319.

The monoclonal antibodies produced by cells of the p25-6 and p25-7 hybridoma cell lines are reactive to GAG-3, the p25-6 cell line antibodies react to synthetic peptides M7 and 148, and p25-7 is reactive to synthetic peptides 15, 88 and 150.

Neutralization experiments were carried out as described above except that, when mixtures were used, the individual monoclonal antibodies were first diluted 1: 5 with nutrient medium and then mixed in equal proportions to a final 1: 10 dilution. as described above.

The monoclonal antibodies gp110-2, p25-6 and p25-7 neutralize the antigen to less than 50% when used as such. When a 1: 10 dilution of gpT'0-2 monoclonal antibody with p25-6 or p25-7 is used, complete neutralization occurs. When a mixture containing monoclonal antibodies p25-6 and p25-7 is used, the neutralizing effect is 60 to 90 (S.

Example 4

Immunological activity of peptide 29 and its homologues

CS 275 838 B6

The ability of peptides 29 and homologous peptides, including peptides 177, to stimulate an immunological response against HIV was examined in two mouse strains. The procedure for producing immunogenic peptides, immunizing and evaluating the immunological response will be described below.

Peptide 29 was prepared for immunization by conjugation with purified thyroglobulin. Thyroglobulin can be used for conjugation as a derivative with N-succinimidyl 4- (N-maleimidoraethyl) cyclohexane-1-carboxylate as described in U.S. Patent 4,629,783, column 10, lines 28-51. the immunogen was thyroglobulin used as a glutaraldehyde derivative as follows. 27 mg of swine thyroglobulin was dissolved in 1 ml,

0.1 M sodium bicarbonate, to which was added dropwise 8.3 µl of a 25% glutaralaldehyde solution, and the mixture was stirred overnight at room temperature. Then 1 ml of sodium carbonate and bicarbonate buffer pH 9.3 was added and the solution was dialyzed against two liters of the same buffer at 4 ° C for 8 hours with complete dialysate exchange after 4 hours. Then peptide 29 in the thyroglobulin derivative was added. of an excess of approximately 100M and the mixture was stirred at room temperature overnight. Unreacted glutaraldehyde was blocked with 200 µL of 0.2 M lysine solution by stirring the mixture for several hours or overnight at room temperature. The peptide-thyroglobulin conjugate was then thoroughly dialyzed against PBS at 4 ° C.

Two strains of mice, C57 (black) and BALB / c, were vaccinated with peptide prepared by conjugation.

At the start of vaccination, animals aged 2 to 4 weeks were used. The animals were vaccinated intranasally, intraperitoneally, to the removed skin of the tail, subcutaneously or to the paw. The inoculum material consists of 25 µg of conjugated peptides in suspension in 0.5 ml of complete Preund's adjuvant, followed by inoculation at 2, 3 and 5 weeks using the same material except that incomplete Preund's adjuvant was used. Serum samples from individual mice were collected before immunization, 4 days after immunization after 3 weeks and 4 days after final immunization after 5 weeks. Serum samples were analyzed for antibodies against homologous peptides or whole virus by BLISA. Sera containing active anti-LAV antibodies were further monitored for neutralizing potency and immunoblot reaction with LAV antigen disrupted and radioimmunoprecipitated with radiolabeled gpHO.

Immunization results show that mice immunized with peptide 29 produced antibodies that reacted with peptide 29 and with disrupted LAV-1 using an ELISA system. By conjugating peptides 29 to glutaraldehyde, a higher titer of anti-peptide 29 antibodies could be achieved in Balb / c mice, although some of these mice could also induce the production of these antibodies by conjugation with maleic acid imide. Mice immunized with peptide 177 produced antibodies against the peptide, but conjugation of peptide 177 to glutaraldehyde induces improved immunological responses. C57 mice were more sensitive to immuno-stimulation after injection of peptides 177 than Balb / c mice. Mice immunized with LAV-2 110-2-2 isolate peptide produced antibodies against the 110-2-2 peptide and LAV-2 virus as determined by ELISA. Glutaraldehyde-conjugated peptide 110-2-2 was immunogenic in C57 and Balb / c mice, although antibody titers against peptide 110-2-2 were usually higher in Balb / c mice than in C57 mice, whereas titers for LAV-2 virus were usually higher in C57 mice than in Balb / c mice. ,

Serum samples from immunized mice that

(i) contain antibodies against whole virus; (ii) are capable of neutralizing HIV, for example in the tests set forth in Example 2; and (iii) react with peptide 29 in the BLISA assay, demonstrating the efficacy of using peptide 29 as a vaccine.

Example 5

Immunoaffinity division of gp110 using monoclonal antibodies

Monoclonal antibodies against the HIV gp110 antigen can be used in immunoaffinity separation to yield substantially pure recombinant composite proteins resulting from bacterial expression. If the protein as a product of expression is secreted by the bacterium, CS 275 838 B6 24 can be isolated from the culture supernatant. If it is not secreted, it may be necessary to disrupt bacterial cells.

The construction of plasmid pENV-5 (ATCC No. 53074) is described in U.S. Patent Application Ser.

721 237. This plasmid contains the code for the major portion of the carboxyl terminus of gp'10 and for the portion of the gp41 terminus, containing the amino group of the LAV isolate, which codes are incorporated into the trp vector for expression. E. coli C600 is transformed with this vector to express but not secrete the gp110 composite protein.

E. coli C600 containing pBNV-5 was grown in a medium containing 20 µg / ml tryptophan and 100 µg / ml ampicillin overnight at 37 ° C with aeration. The culture obtained is then seeded at a ratio of 1: 100 into fresh minimal nutrient medium containing 100 µg / ml ampicillin but not tryptophan. These cultures are then grown under aeration for 2 to 3 hours, i.e. until the early log phase at 37 ° C. An inducer, i.e. 3-β-indolacrylic acid (Signa Chemical Co., St. Louis, MO) is then added to a final concentration of 20 µg / ml from a freshly prepared stock solution of this material containing 20 mg / ml in 95% ethanol. . Cultures after induction are then grown at 37 ° C with aeration for 4-5 hours, pelleted by centrifugation and optionally frozen. The protein yield of pENV-5 is usually less than 1 mg / L.

Pelleted bacterial cells were disrupted using p-RIPA buffer (PBS containing 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, and 1% Aprotinin), which added to disrupt E. coli cells. The suspension can be further treated by sonication to cleave DNA and RNA followed by centrifugation to separate the solids. It may then be necessary to dilute or thicken the material to obtain a standard protein concentration.

The monoclonal antibody HIV-gp110-1 initially precipitates from the ascites liquid or cell culture supernatant at room temperature or cold using ammonium sulfate or sodium sulfate, the solutions are buffered to pH 7.3 and used to a final saturation of 33% v in the case of ammonium sulfate and 18% in the case of sodium sulfate. The precipitated protein is collected by centrifugation, redissolved in PBS and reprecipitated by the addition of 33% ammonium sulfate or 12 to 15% sodium sulfate. This procedure can be repeated if necessary. Then the protein is redissolved in PBS and excess salts are removed by gel filtration or dialysis against PBS.

The purified monoclonal antibody 110-1 can then bind to activated Sepharose

O cyanogen bromide. The required amount of gel is swelled in 10 M hydrochloric acid on a glass filter (1 g of lyophilized material provides approximately 3.5 ml of final gel volume), washed with the same solution for 15 minutes, and then the antibody is added immediately. however, in the range of 8 to 10, a lower pH may be used if necessary for the stability of the antibody. The antibody can be dissolved in PBS or in a carbonate, bicarbonate or borate buffer with 50 mM sodium chloride of high ionic strength. The activated sepharose and antibody in suspension are gently stirred for 2-4 hours at room temperature or overnight at 4 ° C and then washed on a glass frit with buffer. Any remaining active groups were blocked by treatment with 1.0 M ethanolamine at pH 8 for a total of 2 hours. The resulting antibody-bound sepharose is then washed alternately with a high and low pH buffer solution (borate buffer 0.1 M pH 8.5, 1 M NaCl and acetate buffer, 0.1 M pH 4.0 and 1 M NaCl). four or five times. This washing removes traces of non-covalently bound material. Then, the finished immunoaffinity separation matrix is stored at a temperature above 8 ° C in the presence of a suitable bacteriostatic agent, for example 0.01% azide.

Addition of a suspension protein to the above matrix results in selective removal of the gp110 antigen. The mixture is left in contact with the matrix for 2 to 24 hours, preferably 12 to 18 hours, with slow stirring or shaking. It is also possible to use a column in which the matrix is deposited and then the protein suspension is slowly passed through the column. Once the liquid is completely added to the column, the flow of liquid through the column should be stopped to maximize the formation of the immunological complex.

Unbound material is washed out by thoroughly washing the column with adsorption buffer. May be

GS 275 838 B6 also used suction. Then, the bound material is eluted using a low or high pH buffer, for example, an acetate buffer at pH 4.0 or a borate buffer at pH 8.56 or a chaotropic agent.

Example 6

Immunoaffinity purification of recombinant gp110 from a mammalian expression system

The monoclonal antibodies obtained by the method of the invention can be used in the purification of the recombinant gp110 composite protein expressed in mammalian cells. Mammalian cells are infected with recombinant varicella virus according to Mackett et al., J. Virol. 49: 857 (19S4), which comprises sequences that code for at least a portion of the gp110 protein that is antigenically effective and elicits neutralizing antibodies.

The recombinant chickenpox virus is obtained by the method of US Patent Application No. 842,984. The sequence encoded by the HIV envelope glycoprotein is inserted into the vector, which is the plasmid pGS20 downstream of the control site for the transcription of the chickenpox virus. . This chimeric gel has sequences at its edges that encode viral thymidine kinase (TK) kinase.

Chimeric vectors containing the varicella virus promoter linked to the LAV envelope gene were used to transform E. coli strain MCI000. The incorporation of the chimeric LAV-env sequence into the varicella virus genome was achieved by in vivo recombination and was made possible by the fact that the chimeric genes in the pv-env5 plasmids are flanked by the varicella virus sequence coding for TK. This plasmid is then inserted into cells pre-infected with wild-type varicella virus and recombination occurs between the TK sequence of the plasmid and the homologous sequence in the genome of the varicella virus, thereby incorporating the chimeric gene.

African monkey kidney cells (BSC-40 strain), a cell line derived from immortalized BSC-1 cells deposited with the ATCC under CCL26, were used as host cells for expression.

A continuous layer of BSC-40 cells is infected with ten recombinant varicella viruses. The infection lasts for 12 hours, then the cells are isolated, washed with PBS and collected by centrifugation. The cell pellets thus obtained are resuspended in a cell lysis buffer (1.0% NP 40)

2.5% sodium deoxycholate, 0.1 M sodium chloride, 0.01 M tris-HCl pH 7.4, 1 mM EDTA) and the disrupted cell material is then clarified by centrifugation. Immunoaffinity separation of the recombinant composite protein after expression is performed as described above using the monoclonal antibody gp-110-1. Proteins produced by this expression system are closer to naturally produced HIV gp110 because production and glycosylation occurred in mammalian cells.

Example 7

Inhibition of HIV blocking peptide infection

The efficacy of blocking peptides in inhibiting cell infection in LAV-ggu virus strain cultures was studied using a modification of the procedure for evaluating peptides T according to Pert et al., Supra. A preferred inhibition of HIV is to mix equal volumes of 2.5 x 10 6 blocking peptides and OEM cells in RPMI containing 10% FC3 and 2 mg / ml polybrene and incubate for 45 minutes at 37 ° C. The virus is then added at various doses, at 10, 50, or 500 TCID 50, incubated for 14 days at 37 ° C, and then the supernatant is assayed for viral antigen production, for example, p25.

Pre-incubation of OEM cells with peptides before addition of the virus to the culture assists the inhibitory effect. Inhibition is dose-dependent, strong enough at low and moderate doses, but apparently insufficient at the highest virus doses.

Approximately 60% to 90% Inhibition of viral antigen production was achieved at low doses of virus using T peptides. Further experiments with peptides X-XV with amidated carboxyl terminus and acetylated terminal amino group gave similar results

Wherein peptide XI was particularly effective over a wide dose range. Neutralizing antibodies can be added either during preincubation or while the virus is being added to produce additive or synergistic inhibition of viral antigen production.

From the foregoing, it is clear that monoclonal antibodies and peptides, including blocking peptides, can provide improved procedures for neutralizing and / or inhibiting HIV infections. This makes it easy to obtain prophylactic and therapeutic pharmaceutical compositions effective against most or all strains of HIV. In addition, new materials can be used for diagnostic and other known procedures.

While the invention has been described in connection with preferred embodiments for clarity and clarity, it will be apparent that various changes and modifications may be made within the scope of the invention.

The following table will give details of the microorganisms that have been used during the application.

Information on the storage of the micro-organism

The following microorganisms were deposited at ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852, USA.

description laboratory designation ATCC number date saved mouse hybridoma (balb / c / NS-2) HIV-gp 110-1 HB 9175 15. 8. 1986 II HIV-gp 110-2 HB 9176 15. 8. 1986 HIV-gp 110-3 HB 9177 15. 8. 1986 "AND HIV-gp 110-6 HB 9404 30. 4. 1987 II HIV-gp 110-4 HB 9405 30. 4 1987 II HIV-gp 110-5 HB 9406 30. 4. 1987 II HIV-β 25-2 HB 9407 30. 4. 1987 II HIV-β 25-3 HB 9408 30. 4. 1987 H HIV-β 25-6 HB 9409 30. 4. 1987 II HIV-β 25-7 HB 9410 30. 4. 1987 Hybridomas HB 9175) HB 9176 and HB 9177 were tested a were live August 26, 1986. The hybridomas were tested and were viable 4. 5. 1987.

Remaining-

Claims (1)

PATENT CLAIMS A method for producing hybridomas for the production of a monoclonal antibody against HIV, characterized in that the lysed virus extract binds to a carrier, for example a lens lectin, and the obtained material is used to immunize mice whose spleen B lymphocytes are then fused with NS -1 myelomatic cells to form hybridoma, the culture medium in which hybridoma cells are maintained is screened for the presence of antibodies and monoclonal antibody-producing cells capable of interacting with the determinant HIV envelope glycoprotein gpl10 antigen contained in the blocking peptide T or a derivative thereof or analog are further grown.